B . HeLa cells were transfected with indicated siRNAs , followed by RL - 3xBulge construct . Immunoprecipitation ( IP ) was performed with control mouse IgG ( IgG IP ) or anti - AGO2 antibody ( AGO2 IP ) using lysates prepared from these cells . Bottom right panel ; AGO2 immunoprecipitates were analyzed by western blotting using AGO2 specific antibody . Vinculin was used as loading control . Data are presented as mean SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. HeLa cells were transfected with indicated siRNAs, followed by RL-3xBulge construct. Immunoprecipitation (IP) was performed with control mouse IgG (IgG IP) or anti-AGO2 antibody (AGO2 IP) using lysates prepared from these cells. Bottom right panel; AGO2 immunoprecipitates were analyzed by western blotting using AGO2 specific antibody. Vinculin was used as loading control. Data are presented as mean SD (n = 3).


C . HeLa cells were transfected with control ( siControl ) or Nup358 specific siRNA ( siNup358 ) for 96 h . Cells were lysed and subjected to immunoprecipitation with control IgG ( IgG IP ) or anti - AGO2 ( AGO2 IP ) . Total RNA was extracted from initial lysate and IP samples and analyzed by q - PCR for validated miRNA targets , Serbp1 and Dnajb11 . GAPDH was considered as negative control . Data expressed as the relative amount of target mRNA associated with AGO2 as compared to GAPDH mRNA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. HeLa cells were transfected with control (siControl) or Nup358 specific siRNA (siNup358) for 96 h. Cells were lysed and subjected to immunoprecipitation with control IgG (IgG IP) or anti-AGO2 (AGO2 IP). Total RNA was extracted from initial lysate and IP samples and analyzed by q-PCR for validated miRNA targets, Serbp1 and Dnajb11. GAPDH was considered as negative control. Data expressed as the relative amount of target mRNA associated with AGO2 as compared to GAPDH mRNA.


C . HeLa cells were transfected with control ( siControl ) or Nup358 specific siRNA ( siNup358 ) for 96 h . Western blots indicate the extent of Nup358 depletion and AGO2 immunoprecipitation in siControl and siNup358 samples . Vinculin was used as loading control . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. HeLa cells were transfected with control (siControl) or Nup358 specific siRNA (siNup358) for 96 h. Western blots indicate the extent of Nup358 depletion and AGO2 immunoprecipitation in siControl and siNup358 samples. Vinculin was used as loading control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


D . HeLa cells were treated with indicated siRNAs for 96 h . Cells were lysed and analyzed for extent of Nup358 depletion and for the levels of validated miRNA targets , Ras and c - Myc by western analysis . Vinculin was used as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. HeLa cells were treated with indicated siRNAs for 96 h. Cells were lysed and analyzed for extent of Nup358 depletion and for the levels of validated miRNA targets, Ras and c-Myc by western analysis. Vinculin was used as loading control.


E . HeLa cells were lysed and immunoprecipitation was performed with control ( IgG - IP ) or Nup358 ( Nup358 - IP , left panel ) or AGO2 ( AGO2 - IP , right panel ) using specific antibodies . The immunoprecipitates were washed with a buffer containing ( + ) or not containing ( - ) RNAse A and probed for the presence of indicated proteins by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. HeLa cells were lysed and immunoprecipitation was performed with control (IgG-IP) or Nup358 (Nup358-IP, left panel) or AGO2 (AGO2-IP, right panel) using specific antibodies. The immunoprecipitates were washed with a buffer containing (+) or not containing (-) RNAse A and probed for the presence of indicated proteins by western blotting.


F . HeLa cells were transfected with RL - 3xBulge construct and immunoprecipitation was performed using control ( IgG - IP ) or Nup358 ( Nup358 - IP ) antibodies . Total RNA was isolated from the immunoprecipitates and analyzed for let - 7a miRNA ( left panel ) , RL - 3xBulge mRNA and the endogenous miRNA target Dnajb11 using q - PCR ( right panel ) . Fold enrichment in Nup358 - IP as compared to IgG - control was calculated and the data are presented as mean SD ( n = 3 ) , P ‐ value was obtained using Student ' s t ‐ test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. HeLa cells were transfected with RL-3xBulge construct and immunoprecipitation was performed using control (IgG-IP) or Nup358 (Nup358-IP) antibodies. Total RNA was isolated from the immunoprecipitates and analyzed for let-7a miRNA (left panel), RL-3xBulge mRNA and the endogenous miRNA target Dnajb11 using q-PCR (right panel). Fold enrichment in Nup358-IP as compared to IgG-control was calculated and the data are presented as mean SD (n = 3), P‐value was obtained using Student's t‐test.


B . HEK293T cells were co - transfected with GFP - maltose binding protein ( MBP ) - control or GFP - tagged version of indicated Nup358 fragments along with HA - AGO2 . Immunoprecipitation ( IP ) was performed using GFP antibodies and the presence of AGO2 was detected by western blotting ( WB ) using HA antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. HEK293T cells were co-transfected with GFP-maltose binding protein (MBP)-control or GFP-tagged version of indicated Nup358 fragments along with HA-AGO2. Immunoprecipitation (IP) was performed using GFP antibodies and the presence of AGO2 was detected by western blotting (WB) using HA antibodies.


C . Cells were transfected with indicated constructs and IP and WB were performed as described for B .

C. Cells were transfected with indicated constructs and IP and WB were performed as described for B.


D . HEK293T cells transfected with GFP - MBP ( control ) , GFP - Nup358 - C or GFP - Nup358 - CΔIR ( a mutant devoid of IR region ) and HA - AGO2 . IP and WB analyses were performed as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. HEK293T cells transfected with GFP-MBP (control), GFP-Nup358-C or GFP-Nup358-CΔIR (a mutant devoid of IR region) and HA-AGO2. IP and WB analyses were performed as indicated.


E . Cells were transfected with HA - tagged version of indicated AGO subfamily member and GFP - Nup358 - IR . IP and WB analyses were performed using indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Cells were transfected with HA-tagged version of indicated AGO subfamily member and GFP-Nup358-IR. IP and WB analyses were performed using indicated antibodies.


F . Lysates from cells expressing GFP - control , GFP - human ( h ) Nup358 - IR or GFP - zebrafish ( z ) Nup358 - IR and HA - AGO2 were immunoprecipitated using GFP antibodies and the immunoprecipitates were analyzed for the presence of HA - AGO2 by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Lysates from cells expressing GFP-control, GFP-human (h) Nup358-IR or GFP-zebrafish (z) Nup358-IR and HA-AGO2 were immunoprecipitated using GFP antibodies and the immunoprecipitates were analyzed for the presence of HA-AGO2 by western blotting.


B . HEK293T cells were transfected with indicated constructs and immunoprecipitation ( IP ) was performed using GFP specific antibodies and probed for HA - AGO2 by western blotting ( WB ) using HA antibodies . Endogenous RanGAP and Ubc9 were probed with specific antibodies .

B. HEK293T cells were transfected with indicated constructs and immunoprecipitation (IP) was performed using GFP specific antibodies and probed for HA-AGO2 by western blotting (WB) using HA antibodies. Endogenous RanGAP and Ubc9 were probed with specific antibodies.


C . Lysates prepared from cells expressing the indicated constructs were subjected to IP and WB using indicated antibodies . Presence of endogenous RanGAP and Ubc9 was determined by WB .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Lysates prepared from cells expressing the indicated constructs were subjected to IP and WB using indicated antibodies. Presence of endogenous RanGAP and Ubc9 was determined by WB.


D . GFP - control , GFP - IR1 + 2 wild type or mutants were co - expressed with HA - AGO and IP and WB analysis were performed to detect the interaction using indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. GFP-control, GFP-IR1+2 wild type or mutants were co-expressed with HA-AGO and IP and WB analysis were performed to detect the interaction using indicated antibodies.


E . Lysates prepared from cells expressing the indicated proteins were immunoprecipitated with GFP specific antibodies and the presence of specific proteins in the immunoprecipitates was detected by WB with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Lysates prepared from cells expressing the indicated proteins were immunoprecipitated with GFP specific antibodies and the presence of specific proteins in the immunoprecipitates was detected by WB with indicated antibodies.


A . Top panel ; depicts the amino acid sequence corresponding to the Nup358 - SIM1 region and substitutions introduced in the SIM1 mutant . Bottom panel : HEK293T cells were co - transfected with GFP - control , GFP - SIM1 or GFP - SIM1 - mut along with HA - AGO2 and immunoprecipitation ( IP ) was performed with GFP specific antibodies and western analysis ( WB ) of the input lysate and immunoprecipitates were carried out using indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Top panel; depicts the amino acid sequence corresponding to the Nup358-SIM1 region and substitutions introduced in the SIM1 mutant. Bottom panel: HEK293T cells were co-transfected with GFP-control, GFP-SIM1 or GFP-SIM1-mut along with HA-AGO2 and immunoprecipitation (IP) was performed with GFP specific antibodies and western analysis (WB) of the input lysate and immunoprecipitates were carried out using indicated antibodies.


B . Top panel ; depiction of amino acid sequence corresponding to the SIMs in the indicated proteins used in this study . Hydrophobic residues involved in SUMO binding are indicated in red . Bottom panel ; constructs expressing GFP - control or GFP - fused SIMs of indicated proteins were co - transfected with HA - AGO2 in HEK293T cells and IP and WB were performed to determine the extent of interaction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Top panel; depiction of amino acid sequence corresponding to the SIMs in the indicated proteins used in this study. Hydrophobic residues involved in SUMO binding are indicated in red. Bottom panel; constructs expressing GFP-control or GFP-fused SIMs of indicated proteins were co-transfected with HA-AGO2 in HEK293T cells and IP and WB were performed to determine the extent of interaction.


C . HA - MBP - control , HA - AGO1 or HA - AGO2 was co - expressed with GFP - SIM1 and the cell lysates were subjected to IP with HA antibodies and WB analysis with indicated antibodies to detect the presence of proteins in the immunoprecipitates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. HA-MBP-control, HA-AGO1 or HA-AGO2 was co-expressed with GFP-SIM1 and the cell lysates were subjected to IP with HA antibodies and WB analysis with indicated antibodies to detect the presence of proteins in the immunoprecipitates.


D . SIM1 directly interacts with AGO2 . Bacterial lysates expressing MBP control or MBP - AGO2 and GST or GST - SIM1 were mixed and GST pull down assay was performed . The presence of proteins in the pull down samples was analyzed by WB using specific antibodies . The extent of GST pull down was monitored by coomassie staining of the membrane ( coomassie ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. SIM1 directly interacts with AGO2. Bacterial lysates expressing MBP control or MBP-AGO2 and GST or GST-SIM1 were mixed and GST pull down assay was performed. The presence of proteins in the pull down samples was analyzed by WB using specific antibodies. The extent of GST pull down was monitored by coomassie staining of the membrane (coomassie).


E . SIM1 interacts with PIWI subfamily of proteins . HEK293T cells were co - transfected with indicated constructs expressing HA - tagged version of PIWI proteins and GFP - SIM1 . IP was performed using HA antibodies . The immunoprecipitates were probed for the presence of GFP - SIM1 using GFP specific antibodies by WB .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. SIM1 interacts with PIWI subfamily of proteins. HEK293T cells were co-transfected with indicated constructs expressing HA-tagged version of PIWI proteins and GFP-SIM1. IP was performed using HA antibodies. The immunoprecipitates were probed for the presence of GFP-SIM1 using GFP specific antibodies by WB.


F . Putative SIM - binding regions in AGO2 . Three distinct superimpositions of AGO2 ( grey ) and SUMO ( blue ) , in complex with Nup358 - SIM1 ( red ) , are shown in cartoon representation rendered with Chimera [ 90 ] . The residue numbers on the AGO2 that could be the putative SIM - binding regions are labelled . To identify possible Nup358 binding sites on AGO2 , the 3D structure of AGO2 ( PDB ID : 4W5O ) [ 89 ] was compared with that of SUMO ( PDB ID : 1Z5S ) [ 47 ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Putative SIM-binding regions in AGO2. Three distinct superimpositions of AGO2 (grey) and SUMO (blue), in complex with Nup358-SIM1(red), are shown in cartoon representation rendered with Chimera [90]. The residue numbers on the AGO2 that could be the putative SIM-binding regions are labelled. To identify possible Nup358 binding sites on AGO2, the 3D structure of AGO2 (PDB ID: 4W5O) [89] was compared with that of SUMO (PDB ID: 1Z5S) [47].


A . Right panel ; Western blots showing the expression of transfected constructs using indicated antibodies . a - tubulin was used as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Right panel; Western blots showing the expression of transfected constructs using indicated antibodies. a-tubulin was used as loading control.


A . Left panel ; HeLa cells were co - transfected with GFP - MBP ( control ) , GFP - SIM1 or GFP - SIM1 - mut ( AGO interaction defective ) along with RL - control or RL - 3xbulge . Firefly luciferase was used as transfection control . Dual luciferase assay was performed . RL / FL luminescence ration was plotted after normalization with GFP - control . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Left panel; HeLa cells were co-transfected with GFP-MBP (control), GFP-SIM1 or GFP-SIM1-mut (AGO interaction defective) along with RL-control or RL-3xbulge. Firefly luciferase was used as transfection control. Dual luciferase assay was performed. RL/FL luminescence ration was plotted after normalization with GFP-control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


B . Bottom right panel ; Western blot for monitoring the expression levels of indicated proteins using antibodies . a - tubulin was used as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Bottom right panel; Western blot for monitoring the expression levels of indicated proteins using antibodies. a-tubulin was used as loading control.


B . Bottom left panel ; HEK293T cells were co - transfected with indicated expression constructs and the RL reporter . Firefly luciferase ( FL ) was used as transfection control . Dual luciferase assay was performed . The ratio of RL / FL luminescence was plotted after normalization with N - HA - MBP control . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Bottom left panel; HEK293T cells were co-transfected with indicated expression constructs and the RL reporter. Firefly luciferase (FL) was used as transfection control. Dual luciferase assay was performed. The ratio of RL/FL luminescence was plotted after normalization with N-HA-MBP control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


C . Ectopic expression of AGO2 restores P body formation , but fails to rescue the impairment in miRNA function in Nup358 depleted cells . HeLa cells were transfected with control ( siContriol ) or Nup358 ( siNup358 ) specific siRNA , followed by indicated constructs . Middle panel : HeLa cells were transfected with indicated siRNAs , followed by expression constructs and miRNA RL reporters . FL was used as transfection control . The ratio of RL / FL luminescence was calculated . Data are presented as mean SD ( n = 3 ) , P ‐ value was obtained using Student ' s t ‐ test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Middle panel: HeLa cells were transfected with indicated siRNAs, followed by expression constructs and miRNA RL reporters. FL was used as transfection control. The ratio of RL/FL luminescence was calculated. Data are presented as mean SD (n = 3), P‐value was obtained using Student's t‐test.


C . Ectopic expression of AGO2 restores P body formation , but fails to rescue the impairment in miRNA function in Nup358 depleted cells . HeLa cells were transfected with control ( siContriol ) or Nup358 ( siNup358 ) specific siRNA , followed by indicated constructs . Right panel : Western blot analysis for the relative expression of indicated constructs in HEK293T cells . a - tubulin is used as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Right panel: Western blot analysis for the relative expression of indicated constructs in HEK293T cells. a-tubulin is used as loading control.


C . Ectopic expression of AGO2 restores P body formation , but fails to rescue the impairment in miRNA function in Nup358 depleted cells . HeLa cells were transfected with control ( siContriol ) or Nup358 ( siNup358 ) specific siRNA , followed by indicated constructs . Left panel : Quantitative representation showing the number of cells showing microscopically distinct P bodies in different indicated conditions . The data was obtained from three independent experiments , and in each experiment , 100 cells were counted from different fields for the presence of intact P bodies and expressed as percentage .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Left panel: Quantitative representation showing the number of cells showing microscopically distinct P bodies in different indicated conditions. The data was obtained from three independent experiments, and in each experiment, 100 cells were counted from different fields for the presence of intact P bodies and expressed as percentage.


D . HeLa cells were transfected with siControl or siNup358 and subjected to membrane flotation assay . Fractions were collected and analyzed for the presence of indicated proteins by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. HeLa cells were transfected with siControl or siNup358 and subjected to membrane flotation assay. Fractions were collected and analyzed for the presence of indicated proteins by western blotting.


LC - MS / MS measurements of m6A levels in total RNA ( B ) or in poly ( A ) + RNA ( C ) upon KD of predicted methyltransferases in Drosophila S2R + cells . m6A abundance in total RNA is significantly reduced when Mettl5 is depleted , while its depletion has no effect on m6A level in mRNA . As expected , the KD of Mettl3 and Mettl14 reduce m6A levels in mRNA . Bar chart represents the mean ± standard deviation of three technical measurements from three biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( two - tailed t - test ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LC-MS/MS measurements of m6A levels in total RNA (B) or in poly(A)+ RNA (C) upon KD of predicted methyltransferases in Drosophila S2R+ cells. m6A abundance in total RNA is significantly reduced when Mettl5 is depleted, while its depletion has no effect on m6A level in mRNA. As expected, the KD of Mettl3 and Mettl14 reduce m6A levels in mRNA. Bar chart represents the mean ± standard deviation of three technical measurements from three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test).


Subcellular localization of Mettl5 - FLAG ( purple ) and Trmt112 - eGFP ( green ) expressed under the control of their own promoter in S2R + cells . 63X magnified merge of immunofluorescently - labelled S2R + cells . Scale bar : 4 . 47 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Subcellular localization of Mettl5-FLAG (purple) and Trmt112-eGFP (green) expressed under the control of their own promoter in S2R+ cells. 63X magnified merge of immunofluorescently-labelled S2R+ cells. Scale bar: 4.47 μm.


Developmental expression of Mettl5 transcript assayed by RT - qPCR analysis . The figure shows mean ± standard deviation of three technical measurements from three biological replicates .

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Developmental expression of Mettl5 transcript assayed by RT-qPCR analysis. The figure shows mean ± standard deviation of three technical measurements from three biological replicates.


In situ hybridization of Mettl5 transcript at different embryonic stages . The central nervous system ( CNS ) is highlighted in the schematics . Data retrieved from FlyExpress 7 . ( http : / / www . flyexpress . net / search . php ? type = image & search = FBgn0036856 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

In situ hybridization of Mettl5 transcript at different embryonic stages. The central nervous system (CNS) is highlighted in the schematics. Data retrieved from FlyExpress 7. (http://www.flyexpress.net/search.php?type=image&search=FBgn0036856).


LC - MS / MS measurements of m6A levels in total RNA of WT and Mettl5 mutant flies . Bars represent mean ± standard deviation of measurements of three biological replicates . * * * P < 0 . 001 ( two - tailed t - test ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LC-MS/MS measurements of m6A levels in total RNA of WT and Mettl5 mutant flies. Bars represent mean ± standard deviation of measurements of three biological replicates. ***P<0.001 (two-tailed t-test).


Purified 18S rRNA analyzed for its m6A content by quantitative HPLC . The 18S rRNA was extracted from 40S subunits isolated on sucrose gradients . The calibration control is a commercial source of m6A ( in grey ) . m6A elutes at 48 min .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O

Purified 18S rRNA analyzed for its m6A content by quantitative HPLC. The 18S rRNA was extracted from 40S subunits isolated on sucrose gradients. The calibration control is a commercial source of m6A (in grey). m6A elutes at 48 min.


Mature rRNA analysis on ethidium - stained denaturing agarose gels . The same amounts of total RNA extracted from the indicated flies , and from S2R + cells depleted or not of Mettl5 , were loaded . The 28S / 18S ratio was established by densitometry .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mature rRNA analysis on ethidium-stained denaturing agarose gels. The same amounts of total RNA extracted from the indicated flies, and from S2R+ cells depleted or not of Mettl5, were loaded. The 28S/18S ratio was established by densitometry.


Analysis of 18S rRNA maturation in WT and mutant flies ( Mettl5fs ) . Total RNA extracted from the indicated animals was resolved on denaturing agarose gels and processed for northern blotting with specific probes ( complementary to ITS1 or ITS2 sequences ) . The pre - rRNAs detected accumulate to normal levels indicating that processing is unaffected .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of 18S rRNA maturation in WT and mutant flies (Mettl5fs). Total RNA extracted from the indicated animals was resolved on denaturing agarose gels and processed for northern blotting with specific probes (complementary to ITS1 or ITS2 sequences). The pre-rRNAs detected accumulate to normal levels indicating that processing is unaffected.


Western blot validation of the co - immunoprecipitated proteins Mettl5 - FLAG - Myc and Trmt112 , bearing either a N - terminal HA tag ( top ) or a C - terminal GFP tag ( bottom ) , in S2R + cells . The arrows point at the indicated proteins . HA - Trmt12 migrates below the antibody light chains ( top ) . Trmt112 - eGFP migrates above the heavy chains indicated by an asterisk ( bottom ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot validation of the co-immunoprecipitated proteins Mettl5-FLAG-Myc and Trmt112, bearing either a N-terminal HA tag (top) or a C-terminal GFP tag (bottom), in S2R+ cells. The arrows point at the indicated proteins. HA-Trmt12 migrates below the antibody light chains (top). Trmt112-eGFP migrates above the heavy chains indicated by an asterisk (bottom).


Trmt112 depletion reduces m6A levels in total RNA from Drosophila S2R + cells . Mean ± standard deviation of three technical measurements from three biological replicates . * P < 0 . 05 ( two - tailed t - test ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Trmt112 depletion reduces m6A levels in total RNA from Drosophila S2R+ cells. Mean ± standard deviation of three technical measurements from three biological replicates. *P < 0.05 (two-tailed t-test).


Representative trajectories of WT and Mettl5 mutant flies analysed by the Buridan ' s Paradigm . The blue lines indicate when the fly stops and changes direction . Orientation evaluated with the help of the Buridan ' s paradigm for Mettl5 mutant flies . Orientation was measured as the angular deviation from the straight path needed to move from one landmark to another in the arena . Number of flies tested per genotype : 30 . Mean ± standard deviation . Shapiro - Wilk test was used to test for normal distribution in each group . Normally distributed groups were tested by t - test . Due to multiple comparison Bonferroni correction was applied . ( p < 0 . 05 = * ; p < 0 . 0001 = * * * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative trajectories of WT and Mettl5 mutant flies analysed by the Buridan's Paradigm. The blue lines indicate when the fly stops and changes direction. Orientation evaluated with the help of the Buridan's paradigm for Mettl5 mutant flies. Orientation was measured as the angular deviation from the straight path needed to move from one landmark to another in the arena. Number of flies tested per genotype: 30. Mean ± standard deviation. Shapiro-Wilk test was used to test for normal distribution in each group. Normally distributed groups were tested by t-test. Due to multiple comparison Bonferroni correction was applied. (p<0.05 = *; p<0.0001=****).


( A ) smFISH targeting Bmal1 , Cry1 and Nr1d1 in wild - type NIH 3T3 cells at 17h , 29h and 25h after synchronisation with Dex , respectively . Nuclei are stained with DAPI ( blue ) . Each fluorescent dot ( white ) corresponds to a single transcript . Segmented cell boundaries are delineated in grey . The blue and white channels represent maximum z - projections . Scale bar : 20μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) smFISH targeting Bmal1, Cry1 and Nr1d1 in wild-type NIH 3T3 cells at 17h, 29h and 25h after synchronisation with Dex, respectively. Nuclei are stained with DAPI (blue). Each fluorescent dot (white) corresponds to a single transcript. Segmented cell boundaries are delineated in grey. The blue and white channels represent maximum z-projections. Scale bar: 20μm.


( B ) Number of Bmal1 , Cry1 , and Nr1d1 transcripts per cell as a function of time after treatment with Dex . These data combine all smFISH hybridizations from the 4h sampled Nr1d1 / Cry1 and Bmal1 / Cry1 experiment . Each dot shows the average over one replicate from an independent slide ( Methods ) . The solid lines represent fits using a two - harmonic cosinor model ( Equation ( 1 ) , Methods ) for each gene individually .

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(B) Number of Bmal1, Cry1, and Nr1d1 transcripts per cell as a function of time after treatment with Dex. These data combine all smFISH hybridizations from the 4h sampled Nr1d1/Cry1 and Bmal1/Cry1 experiment. Each dot shows the average over one replicate from an independent slide (Methods). The solid lines represent fits using a two-harmonic cosinor model (Equation (1), Methods) for each gene individually.


( D ) Distributions of Nr1d1 , Cry1 and Bmal1 transcripts at 21h , 29h and 37h after synchronisation with Dex . μ represents the mean of the distribution , CV represent the coefficient of variation ( standard deviation / mean ) , and N is the number of cells at the given time point . Total number of cells analysed for the Nr1d1 / Cry1 pair : 21h - 449 ; 25h - 414 ; 29h - 521 ; 33h - 463 ; 37h - 477 ; 41h - 429 . Total number of cells analysed for the Bmal1 / Cry1 pair : 17h - 465 ; 21h - 490 ; 25h - 504 ; 29h - 436 ; 33h - 407 ; 37h - 454 ; 41h - 404 .

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(D) Distributions of Nr1d1, Cry1 and Bmal1 transcripts at 21h, 29h and 37h after synchronisation with Dex. μ represents the mean of the distribution, CV represent the coefficient of variation (standard deviation / mean), and N is the number of cells at the given time point. Total number of cells analysed for the Nr1d1/Cry1 pair: 21h - 449; 25h - 414; 29h - 521; 33h - 463; 37h - 477; 41h - 429. Total number of cells analysed for the Bmal1/Cry1 pair: 17h - 465; 21h - 490; 25h - 504; 29h - 436; 33h - 407; 37h - 454; 41h - 404.


( A ) Log conditional mean mRNA count for a given area as a function of the log area . Each cell is sorted by area by placing it in one of 20 bins that range from the 5th to the 95th percentile . The conditional mean given the area E [ m | A ] is then the mean mRNA count of each bin . The area is normalised such that the average area is equal to one . Solid lines represent a least - squares linear fit to the data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Log conditional mean mRNA count for a given area as a function of the log area. Each cell is sorted by area by placing it in one of 20 bins that range from the 5th to the 95th percentile. The conditional mean given the area E[m|A] is then the mean mRNA count of each bin. The area is normalised such that the average area is equal to one. Solid lines represent a least-squares linear fit to the data.


( B ) Top image : Dual - channel smFISH targeting Bmal1 and Cry1 simultaneously , taken at 17h after Dex synchronisation . Blue , nuclei stained with DAPI ; green dots , Bmal1 transcripts ; red , Cry1 transcripts . Bottom image : Dual - channel smFISH targeting Nr1d1 and Cry1 simultaneously , taken at 25h after Dex synchronisation . Blue , nuclei stained with DAPI ; green dots , Nr1d1 transcripts ; red , Cry1 transcripts . Scale bar : 20μm .

(B) Top image: Dual-channel smFISH targeting Bmal1 and Cry1 simultaneously, taken at 17h after Dex synchronisation. Blue, nuclei stained with DAPI; green dots, Bmal1 transcripts; red, Cry1 transcripts. Bottom image: Dual-channel smFISH targeting Nr1d1 and Cry1 simultaneously, taken at 25h after Dex synchronisation. Blue, nuclei stained with DAPI; green dots, Nr1d1 transcripts; red, Cry1 transcripts. Scale bar: 20μm.


( C ) Bivariate distributions of mRNA counts per cell for dual - channel smFISH targeting either Bmal1 and Cry1 or Nr1d1 and Cry1 . Each dot corresponds to a single cell , and the contours represent KDE estimates of the density . Time represents the number of hours after Dex synchronisation , and R represents the Pearson correlation coefficient .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Bivariate distributions of mRNA counts per cell for dual-channel smFISH targeting either Bmal1 and Cry1 or Nr1d1 and Cry1. Each dot corresponds to a single cell, and the contours represent KDE estimates of the density. Time represents the number of hours after Dex synchronisation, and R represents the Pearson correlation coefficient.


( C ) Posterior parameter estimates . Burst frequency is plotted as a function of time , where the solid line represents the posterior mean and the shaded area represents the 90 % confidence interval . Posterior probability densities are shown for the average burst size , β ( which controls the dependence between burst size and cell area ) and ρ ( representing the correlation in burst size between genes ( in log space ) ) , where β is inferred from model M2 and the average burst size and ρ are calculated from model M4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Posterior parameter estimates. Burst frequency is plotted as a function of time, where the solid line represents the posterior mean and the shaded area represents the 90% confidence interval. Posterior probability densities are shown for the average burst size, β (which controls the dependence between burst size and cell area) and ρ (representing the correlation in burst size between genes (in log space)), where β is inferred from model M2 and the average burst size and ρ are calculated from model M4.


( D ) Comparison of the probability density of the data ( kernel density estimates ) with the model ( using model M4 ) . To estimate the probability from the model , the data set was simulated 15 times using the posterior mean parameter values combined with the measured cell areas .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Comparison of the probability density of the data (kernel density estimates) with the model (using model M4). To estimate the probability from the model, the data set was simulated 15 times using the posterior mean parameter values combined with the measured cell areas.


( a ) FLAG - AMBRA1 was transfected into HEK293 cells together with ULK1WT or ULK1K46I Myc - tagged proteins . Protein extracts were immunoprecipitated ( IP ) using anti - FLAG antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) FLAG-AMBRA1 was transfected into HEK293 cells together with ULK1WT or ULK1K46I Myc-tagged proteins. Protein extracts were immunoprecipitated (IP) using anti-FLAG antibody.


( b ) HEK293 cells were grown either in normal or in EBS medium for 1 h ; protein extracts were immunoprecipitated using anti - ULK1 antibody or rabbit immunoglobulin as a negative control ( IgG ) . Autophagy induction is shown by p62 degradation .

(b) HEK293 cells were grown either in normal or in EBS medium for 1 h; protein extracts were immunoprecipitated using anti-ULK1 antibody or rabbit immunoglobulin as a negative control (IgG). Autophagy induction is shown by p62 degradation.


( c ) HEK293 cells were treated as in b , in the presence of leupeptin and stained by anti - AMBRA1 ( red ) and anti - ULK1 ( green ) antibodies . The merge of the two signals and DAPI ( blue ) for nuclear staining is shown in the right panels . Scale bar , 6 μm . The outlined areas are shown at a higher magnification in the lower panels . White arrows point to the co - localization puncta . Measurements by Metamorph indicate a co - localization ratio of 46 % between ULK1 and AMBRA1 on autophagy induction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) HEK293 cells were treated as in b, in the presence of leupeptin and stained by anti-AMBRA1 (red) and anti-ULK1 (green) antibodies. The merge of the two signals and DAPI (blue) for nuclear staining is shown in the right panels. Scale bar, 6 μm. The outlined areas are shown at a higher magnification in the lower panels. White arrows point to the co-localization puncta. Measurements by Metamorph indicate a co-localization ratio of 46% between ULK1 and AMBRA1 on autophagy induction.


( d , e ) ULK1 and AMBRA1 constructs are illustrated . Asterisks mark nonspecific bands corresponding to immunoglobulins . ( d ) HEK293 cells were co - transfected with vectors encoding AMBRA1 together with ULK1 - Myc FL ( full length ) or its deletion mutants encoding the kinase and the proline - rich domains ( Δ829 - 1 , 051 ) , and the ULK1 carboxy - terminal domain ( Δ1 - 828 ) . Protein extracts were immunoprecipitated using anti - Myc antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d,e) ULK1 and AMBRA1 constructs are illustrated. Asterisks mark nonspecific bands corresponding to immunoglobulins. (d) HEK293 cells were co-transfected with vectors encoding AMBRA1 together with ULK1-Myc FL (full length) or its deletion mutants encoding the kinase and the proline-rich domains (Δ829-1,051), and the ULK1 carboxy-terminal domain (Δ1-828). Protein extracts were immunoprecipitated using anti-Myc antibody.


( d , e ) ULK1 and AMBRA1 constructs are illustrated . Asterisks mark nonspecific bands corresponding to immunoglobulins . ( e ) HEK293 cells were co - transfected with vectors encoding ULK1 - HA together with β - galactosidase - Myc ( βgal ) , AMBRA1 full - length ( FL ) , and F1 , F2 or F3 Myc - tagged proteins . Protein extracts were immunoprecipitated using anti - HA antibody . The relative amounts of co - immunoprecipitatedULK1 and F1 or F3 , respectively , are the same ( see upper and lower left panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d,e) ULK1 and AMBRA1 constructs are illustrated. Asterisks mark nonspecific bands corresponding to immunoglobulins. (e) HEK293 cells were co-transfected with vectors encoding ULK1-HA together with β-galactosidase-Myc (βgal), AMBRA1 full-length (FL), and F1, F2 or F3 Myc-tagged proteins. Protein extracts were immunoprecipitated using anti-HA antibody. The relative amounts of co-immunoprecipitatedULK1 and F1 or F3, respectively, are the same (see upper and lower left panel).


( f ) HEK293 cells were co - transfected with vectors encoding AMBRA1 , ULK1 - Myc and ATG13 - FLAG . Protein extracts were immunoprecipitated using anti - FLAG antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) HEK293 cells were co-transfected with vectors encoding AMBRA1, ULK1-Myc and ATG13-FLAG. Protein extracts were immunoprecipitated using anti-FLAG antibody.


( g ) HEK293 cells were transfected with scramble siRNA or ATG13 siRNA . Protein extracts were immunoprecipitated using ULK1 antibody or IgG . The band density ratio of immunoprecipitated AMBRA1 relative to immunoprecipitated ULK1 is analysed , with the control ratio arbitrarily defined as 1 . 00 . n = 3 extracts prepared from independent experiments ; data are presented as means ± s . d . and significance is P = 0 . 04 . Uncropped images of blots / gels are shown in Supplementary Fig . S6 . Source data of statistical analysis are shown in Supplementary Table S1 .

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(g) HEK293 cells were transfected with scramble siRNA or ATG13 siRNA. Protein extracts were immunoprecipitated using ULK1 antibody or IgG. The band density ratio of immunoprecipitated AMBRA1 relative to immunoprecipitated ULK1 is analysed, with the control ratio arbitrarily defined as 1.00. n = 3 extracts prepared from independent experiments; data are presented as means±s.d. and significance is P = 0.04. Uncropped images of blots/gels are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.


( a ) HEK293 cells were transfected with AMBRA1 or the empty vector , as a control . ULK1 , AMBRA1 and actin were analysed by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) HEK293 cells were transfected with AMBRA1 or the empty vector, as a control. ULK1, AMBRA1 and actin were analysed by western blotting.


( b ) Protein extracts from wild - type ( Ambra1 + / + ) and homozygous ( Ambra1gt / gt ) embryos for the Ambra1 gene - trap mutation at developmental day 14 . 5 were analysed by western blotting using anti - Ulk1 or anti - Hsp90 ( loading control ) antibodies . Quantification is shown on the right . n = 6 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 037 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) Protein extracts from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos for the Ambra1 gene-trap mutation at developmental day 14.5 were analysed by western blotting using anti-Ulk1 or anti-Hsp90 (loading control) antibodies. Quantification is shown on the right. n = 6 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.037.


( c ) AMBRA1 , ULK1 and actin ( as a protein loading control ) were analysed by western blotting in HeLa cells transiently transfected with scramble siRNA or AMBRA1 siRNA . Quantification is shown on the right . n = 5 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 007 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) AMBRA1, ULK1 and actin (as a protein loading control) were analysed by western blotting in HeLa cells transiently transfected with scramble siRNA or AMBRA1 siRNA. Quantification is shown on the right. n = 5 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.007.


( d ) HeLa cells stably interfered for AMBRA1 were transiently transfected with empty vector or AMBRA1 vector ; AMBRA1 , ULK1 , ATG13 , p - ATG13 and actin levels were analysed by western blotting . Quantification of ULK1 and pATG13 bands is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 006 for ULK1 and P = 0 . 044 and P = 0 . 011 for pATG13 .

(d) HeLa cells stably interfered for AMBRA1 were transiently transfected with empty vector or AMBRA1 vector; AMBRA1, ULK1, ATG13, p-ATG13 and actin levels were analysed by western blotting. Quantification of ULK1 and pATG13 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.006 for ULK1 and P = 0.044 and P = 0.011 for pATG13.


( e ) HeLa cells were transfected or not with vector encoding AMBRA1 and treated with 35 μM cycloheximide ( CHX ) for 6 and 8 h before extraction . ULK1 , actin and AMBRA1 were analysed by western blotting . Quantification is shown below . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 049 and P = 0 . 02 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) HeLa cells were transfected or not with vector encoding AMBRA1 and treated with 35 μM cycloheximide (CHX) for 6 and 8 h before extraction. ULK1, actin and AMBRA1 were analysed by western blotting. Quantification is shown below. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.049 and P = 0.02.


( f ) HEK293 cells were transfected as in a ; ATG13 , pATG13 , ULK1 , AMBRA1 and actin were analysed by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) HEK293 cells were transfected as in a; ATG13, pATG13, ULK1, AMBRA1 and actin were analysed by western blotting.


( g ) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected with β - galactosidase or AMBRA1 plasmid respectively using an anti - ULK1 antibody . As a control , rabbit IgG was used . The resulting precipitates were subjected to ULK1kinase assay using MBP as the substrate . Purified complexes were analysed by autoradiography and by western blotting using an anti - ULK1 antibody . Total extracts were analysed using an anti - AMBRA1 antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected with β-galactosidase or AMBRA1 plasmid respectively using an anti-ULK1 antibody. As a control, rabbit IgG was used. The resulting precipitates were subjected to ULK1kinase assay using MBP as the substrate. Purified complexes were analysed by autoradiography and by western blotting using an anti-ULK1 antibody. Total extracts were analysed using an anti-AMBRA1 antibody.


( a ) Protein extracts from HeLa cells were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control and analysed by western blotting using anti - Lys63 specific ubiquitin and anti - ULK1 antibodies .

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(a) Protein extracts from HeLa cells were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control and analysed by western blotting using anti-Lys63 specific ubiquitin and anti-ULK1 antibodies.


( b ) HeLa cells were co - transfected with ULK1 - Myc alone or in combination with wild - type ubiquitin or its mutant ( K48R or K63R ) HA - tagged constructs . Protein extracts were immunoprecipitated using an anti - Myc antibody . Purified complexes and total extracts were analysed by western blotting using anti - Myc and anti - HA antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) HeLa cells were co-transfected with ULK1-Myc alone or in combination with wild-type ubiquitin or its mutant (K48R or K63R) HA-tagged constructs. Protein extracts were immunoprecipitated using an anti-Myc antibody. Purified complexes and total extracts were analysed by western blotting using anti-Myc and anti-HA antibodies.


( c ) In HeLa cells , AMBRA1 was downregulated by RNAi . Protein extracts were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control . Purified complexes and total extracts were analysed by western blotting using anti - ULK1 , anti - Lys - 63 ubiquitin and anti - AMBRA1 antibodies . Of note , ULK1 levels in the co - immunoprecipitation experiment do not change in this case in the AMBRA1 siRNA lane , owing to saturation of the anti - ULK1 antibody ( see also Fig . 4h ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) In HeLa cells, AMBRA1 was downregulated by RNAi. Protein extracts were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control. Purified complexes and total extracts were analysed by western blotting using anti-ULK1, anti-Lys-63 ubiquitin and anti-AMBRA1 antibodies. Of note, ULK1 levels in the co-immunoprecipitation experiment do not change in this case in the AMBRA1 siRNA lane, owing to saturation of the anti-ULK1 antibody (see also Fig. 4h).


( d ) HeLa cells were co - transfected with vectors encoding for ubiquitin Lys - 63 - HA alone or in combination with AMBRA1 . Protein extracts were immunoprecipitated using an anti - ULK1 antibody or with IgG as a negative control and Ub - Lys - 63 - HA , ULK1 and AMBRA1 were analysed by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) HeLa cells were co-transfected with vectors encoding for ubiquitin Lys-63-HA alone or in combination with AMBRA1. Protein extracts were immunoprecipitated using an anti-ULK1 antibody or with IgG as a negative control and Ub-Lys-63-HA, ULK1 and AMBRA1 were analysed by western blotting.


( e , f ) HeLa cells were cultured in complete or starvation medium for 30 min or in the presence of 100 nM of rapamycin for 3 h and then protein extracts were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control . Purified complexes and total extracts were analysed by western blotting as in a . Quantifications are shown on the right . In both panels , n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 003 and P = 0 . 0015 , respectively .

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(e,f) HeLa cells were cultured in complete or starvation medium for 30 min or in the presence of 100 nM of rapamycin for 3 h and then protein extracts were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control. Purified complexes and total extracts were analysed by western blotting as in a. Quantifications are shown on the right. In both panels, n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.003 and P = 0.0015, respectively.


( a ) HeLa cells were transfected with FLAG - TRAF6 or grown in starvation medium for 1 h . Some of them were treated with chloroquine . LC3 , p62 and actin were analysed by western blotting . Quantification of LC3II and p62 bands is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 017 for LC3 and P = 0 . 01 and P = 0 . 04 for p62 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) HeLa cells were transfected with FLAG-TRAF6 or grown in starvation medium for 1 h. Some of them were treated with chloroquine. LC3, p62 and actin were analysed by western blotting. Quantification of LC3II and p62 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.017 for LC3 and P = 0.01 and P = 0.04 for p62.


( b ) HeLa cells were transduced with AMBRA1 lentiviral shRNA and transfected or not with FLAG - TRAF6 . Quantification of LC3II and p62 bands is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 001 and P = 0 . 0018 for LC3 and P = 0 . 004 for p62 .

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(b) HeLa cells were transduced with AMBRA1 lentiviral shRNA and transfected or not with FLAG-TRAF6. Quantification of LC3II and p62 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.001 and P = 0.0018 for LC3 and P = 0.004 for p62.


( c ) Count of LC3 puncta in HeLa cells transduced as in b . Five different confocal microscopy images for each experiment were randomly chosen and GFP - positive dots were examined on the images with identical brightness and contrast settings . The results are represented as the average number of LC3 dots with 100 cells analysed per sample . Confocal images are shown in Supplementary Fig . S3b .

(c) Count of LC3 puncta in HeLa cells transduced as in b. Five different confocal microscopy images for each experiment were randomly chosen and GFP-positive dots were examined on the images with identical brightness and contrast settings. The results are represented as the average number of LC3 dots with 100 cells analysed per sample. Confocal images are shown in Supplementary Fig. S3b.


( d ) HEK293 cells were transfected with AMBRA1 and FLAG - TRAF6 as indicated . Protein extracts were immunoprecipitated using an anti - FLAG antibody .

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(d) HEK293 cells were transfected with AMBRA1 and FLAG-TRAF6 as indicated. Protein extracts were immunoprecipitated using an anti-FLAG antibody.


( e ) HeLa cells were transfected as in b and FLAG - TRAF6 was purified as in d . Quantification of immunoprecipitated TRAF6 is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 0003 .

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(e) HeLa cells were transfected as in b and FLAG-TRAF6 was purified as in d. Quantification of immunoprecipitated TRAF6 is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.0003.


( f ) In HeLa cells , TRAF6 was downregulated by RNAi . AMBRA1 , ULK1 , TRAF6 , ATG13 and actin were analysed by western blotting .

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(f) In HeLa cells, TRAF6 was downregulated by RNAi. AMBRA1, ULK1, TRAF6, ATG13 and actin were analysed by western blotting.


( g ) HeLa cells were co - transfected with vectors encoding ULK1 - Myc , FLAG - TRAF6 and Ub - Lys - 63 - HA , as indicated . Protein extracts were immunoprecipitated using an anti - Myc antibody .

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(g) HeLa cells were co-transfected with vectors encoding ULK1-Myc, FLAG-TRAF6 and Ub-Lys-63-HA, as indicated. Protein extracts were immunoprecipitated using an anti-Myc antibody.


( h ) HeLa cells stably interfered for AMBRA1 were transfected with FLAG - TRAF6 and Ub - Lys - 63 - HA as indicated . Protein extracts were immunoprecipitated using anti - ULK1 antibody .

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(h) HeLa cells stably interfered for AMBRA1 were transfected with FLAG-TRAF6 and Ub-Lys-63-HA as indicated. Protein extracts were immunoprecipitated using anti-ULK1 antibody.


( i ) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected as in b . The resulting precipitates were subjected to a ULK1 kinase assay using MBP as the substrate . Purified complexes were analysed by autoradiography and by western blotting using an anti - ULK1 antibody .

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(i) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected as in b. The resulting precipitates were subjected to a ULK1 kinase assay using MBP as the substrate. Purified complexes were analysed by autoradiography and by western blotting using an anti-ULK1 antibody.


( a - c ) Two canonical TRAF6 - binding sites in AMBRA1 are essential for its interaction with TRAF6 . HEK293 cells were co - transfected with vectors encoding FLAG - TRAF6 together with AMBRA1WT and mutant constructs . Protein extracts were immunoprecipitated using an anti - FLAG antibody . AMBRA1 and FLAG - TRAF6 were analysed by western blotting .

(a-c) Two canonical TRAF6-binding sites in AMBRA1 are essential for its interaction with TRAF6. HEK293 cells were co-transfected with vectors encoding FLAG-TRAF6 together with AMBRA1WT and mutant constructs. Protein extracts were immunoprecipitated using an anti-FLAG antibody. AMBRA1 and FLAG-TRAF6 were analysed by western blotting.


( d ) HeLa cells were transfected with AMBRA1 RNAi oligonucleotide ( siRNA AMBRA1 ) . Then , cells were co - transfected with vectors encoding ubiquitin - HA and AMBRA1WT or AMBRA1AA , respectively . Protein extracts were immunoprecipitated using anti - ULK1 antibody ; ubiquitin - HA , ULK1 and AMBRA1 were analysed by western blotting .

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(d) HeLa cells were transfected with AMBRA1 RNAi oligonucleotide (siRNA AMBRA1). Then, cells were co-transfected with vectors encoding ubiquitin-HA and AMBRA1WT or AMBRA1AA, respectively. Protein extracts were immunoprecipitated using anti-ULK1 antibody; ubiquitin-HA, ULK1 and AMBRA1 were analysed by western blotting.


( e ) HeLa cells stably interfered for AMBRA1 were transduced with lentiviral vectors encoding β - galactosidase ( negative control ) AMBRA1WT or AMBRA1AA respectively ; protein extracts were analysed by western blotting using anti - ULK1 , anti - AMBRA1 anti - ATG13 , anti - pATG13 and anti - actin antibodies .

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(e) HeLa cells stably interfered for AMBRA1 were transduced with lentiviral vectors encoding β-galactosidase (negative control) AMBRA1WT or AMBRA1AA respectively; protein extracts were analysed by western blotting using anti-ULK1, anti-AMBRA1 anti-ATG13, anti-pATG13 and anti-actin antibodies.


( f ) HeLa cells were transfected as in e and some of them were treated with chloroquine for 1 h . LC3 , p62 , AMBRA1 and actin were analysed by western blotting . Quantification of LC3II and p62 are shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 03 for LC3 and P = 0 . 045 for p62 .

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(f) HeLa cells were transfected as in e and some of them were treated with chloroquine for 1 h. LC3, p62, AMBRA1 and actin were analysed by western blotting. Quantification of LC3II and p62 are shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.03 for LC3 and P = 0.045 for p62.


( g ) GFP - LC3 MEF cells from wild - type ( Ambra1 + / + ) and homozygous ( Ambra1gt / gt ) embryos were transduced with the indicated lentiviral vectors and quantification of LC3 puncta per cells is shown on the right . Bars represent mean ± s . e . m . of triplicate samples with 50 cells analysed per sample ; n = 3 independent experiments . Significance is P = 0 . 001 and P = 0 . 003 ( n = 3 ) . Scale bar , 10 μm .

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(g) GFP-LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with the indicated lentiviral vectors and quantification of LC3 puncta per cells is shown on the right. Bars represent mean±s.e.m. of triplicate samples with 50 cells analysed per sample; n = 3 independent experiments. Significance is P = 0.001 and P = 0.003 (n = 3). Scale bar, 10 μm.


( a ) HEK293 cells were co - transfected with vectors encoding ULK1 - HA and ULK1 - Myc and then grown either in normal ( Ctrl ) or in starvation ( EBS ) medium for 1 h . Protein extracts were immunoprecipitated using an anti - Myc antibody , and analysed by western blotting using anti - HA , and anti - Myc antibodies . Quantification is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 001 .

(a) HEK293 cells were co-transfected with vectors encoding ULK1-HA and ULK1-Myc and then grown either in normal (Ctrl) or in starvation (EBS) medium for 1 h. Protein extracts were immunoprecipitated using an anti-Myc antibody, and analysed by western blotting using anti-HA, and anti-Myc antibodies. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.001.


( b ) HEK293 cells were transfected with TRAF6 RNAi oligonucleotide ( TRAF6 siRNA ) or unrelated oligonucleotides as a control ( Ctrl siRNA ) . Then , cells were co - transfected with vectors encoding ULK1 - HA and ULK1 - Myc and protein complexes were immunoprecipitated using anti - Myc antibody . Protein extracts were analysed by western blotting using anti - HA , anti - Myc and anti - TRAF6 antibodies . Quantification of ULK1 - HA relative to ULK1 - Myc is reported on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 037 .

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(b) HEK293 cells were transfected with TRAF6 RNAi oligonucleotide (TRAF6 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected with vectors encoding ULK1-HA and ULK1-Myc and protein complexes were immunoprecipitated using anti-Myc antibody. Protein extracts were analysed by western blotting using anti-HA, anti-Myc and anti-TRAF6 antibodies. Quantification of ULK1-HA relative to ULK1-Myc is reported on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.037.


( c ) HEK293 cells were transfected with AMBRA1 RNAi oligonucleotide ( AMBRA1 siRNA ) or unrelated oligonucleotides as a control ( Ctrl siRNA ) . Then , cells were co - transfected and analysed as in a . Quantification is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 044 .

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(c) HEK293 cells were transfected with AMBRA1 RNAi oligonucleotide (AMBRA1 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected and analysed as in a. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.044.


( d ) HEK293 cells were co - transfected with vectors encoding for ULK1 - HA , ULK1 - Myc and AMBRA1WT or AMBRA1AA respectively . Protein complexes were immunoprecipitated and analysed as in a . Quantification is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 014 .

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(d) HEK293 cells were co-transfected with vectors encoding for ULK1-HA, ULK1-Myc and AMBRA1WT or AMBRA1AA respectively. Protein complexes were immunoprecipitated and analysed as in a. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.014.


( a ) Highly purified mTOR was incubated with radiolabelled ATP without substrate , with purified FLAG - AMBRA1 and purified FLAG - AMBRA1S52A and analysed by autoradiography . The lower panels show a FLAG and mTOR western blot ( WB ) .

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(a) Highly purified mTOR was incubated with radiolabelled ATP without substrate, with purified FLAG-AMBRA1 and purified FLAG-AMBRA1S52A and analysed by autoradiography. The lower panels show a FLAG and mTOR western blot (WB).


( b ) HEK293 cells expressing FLAG - RAPTOR were subjected to FLAG immunoprecipitation . Protein complexes were analysed by western blotting using anti - FLAG and anti - AMBRA1 antibodies .

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(b) HEK293 cells expressing FLAG-RAPTOR were subjected to FLAG immunoprecipitation. Protein complexes were analysed by western blotting using anti-FLAG and anti-AMBRA1 antibodies.


( c ) HEK293 cells were treated with Torin1 for 3 h or cultured in starvation medium for 2 h and protein extracts were analysed by western blotting using anti - pSer - 52 and anti - AMBRA1 antibodies .

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(c) HEK293 cells were treated with Torin1 for 3 h or cultured in starvation medium for 2 h and protein extracts were analysed by western blotting using anti-pSer-52 and anti-AMBRA1 antibodies.


( d ) HeLa cells stably interfered for AMBRA1 were transduced with the indicated lentiviral vectors encoding for β - galactosidase , AMBRA1WT or AMBRA1S52A respectively and some of them were treated with chloroquine for 1 h . Protein extracts were analysed by western blotting using anti - LC3 , anti - p62 and anti - actin antibodies . Quantification of LC3II and p62 bands is shown below . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 038 .

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(d) HeLa cells stably interfered for AMBRA1 were transduced with the indicated lentiviral vectors encoding for β-galactosidase, AMBRA1WT or AMBRA1S52A respectively and some of them were treated with chloroquine for 1 h. Protein extracts were analysed by western blotting using anti-LC3, anti-p62 and anti-actin antibodies. Quantification of LC3II and p62 bands is shown below. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.038.


( e ) GFP - LC3 MEF cells from wild - type ( Ambra1 + / + ) and homozygous ( Ambra1gt / gt ) embryos were transduced with indicated lentiviral vectors and quantification of LC3 puncta per cell is shown on the right . Bars represent mean ± s . e . m . of triplicate samples and 50 cells analysed per sample . n = 3 independent experiments . Significance is P = 0 . 001 and P = 0 . 003 . Scale bar , 10

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(e) GFP-LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with indicated lentiviral vectors and quantification of LC3 puncta per cell is shown on the right. Bars represent mean±s.e.m. of triplicate samples and 50 cells analysed per sample. n = 3 independent experiments. Significance is P = 0.001 and P = 0.003. Scale bar, 10 μm


( f ) HeLa cells stably interfered for AMBRA1 were co - transfected with vectors encoding Ub - Lys - 63 - HA and AMBRA1WT or AMBRA1S52A , respectively . Protein extracts were immunoprecipitated using an anti - ULK1 antibody and ubiquitin Lys - 63 - HA , ULK1 , AMBRA1 and actin were analysed by western blotting .

(f) HeLa cells stably interfered for AMBRA1 were co-transfected with vectors encoding Ub-Lys-63-HA and AMBRA1WT or AMBRA1S52A, respectively. Protein extracts were immunoprecipitated using an anti-ULK1 antibody and ubiquitin Lys-63-HA, ULK1, AMBRA1 and actin were analysed by western blotting.


( g ) HeLa cells stably interfered for AMBRA1 were transduced as in d ; protein extracts were analysed by western blotting using anti - AMBRA1 , anti - ATG13 , anti - pATG13 and anti - actin antibodies . Quantification of the p - ATG13 band is shown below .

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(g) HeLa cells stably interfered for AMBRA1 were transduced as in d; protein extracts were analysed by western blotting using anti-AMBRA1, anti-ATG13, anti-pATG13 and anti-actin antibodies. Quantification of the p-ATG13 band is shown below.


( h ) HeLa cells transfected with ULK1 - HA , ULK1 - Myc and different AMBRA1 constructs , respectively . Protein complexes were immunoprecipitated using anti - HA antibody and analysed by western blotting using anti - HA , anti - Myc and anti - AMBRA1 antibodies .

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(h) HeLa cells transfected with ULK1-HA, ULK1-Myc and different AMBRA1 constructs, respectively. Protein complexes were immunoprecipitated using anti-HA antibody and analysed by western blotting using anti-HA, anti-Myc and anti-AMBRA1 antibodies.


( B ) Phase - contrast images of wild - type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA - mediated knockdown . Scale bar , 100 μm . ( C ) Total numbers of viable cells in ( B ) were quantified with acridine orange and propidium iodide staining and graphed as a percentage of wild - type HeLa cells . Error bars represent the SEM of three biological replicates .

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(B) Phase-contrast images of wild-type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA-mediated knockdown. Scale bar, 100 μm. (C) Total numbers of viable cells in (B) were quantified with acridine orange and propidium iodide staining and graphed as a percentage of wild-type HeLa cells. Error bars represent the SEM of three biological replicates.


( C ) Phase - contrast images of PABPDHFR cells , or PABPDHFR cells transfected with a plasmid coding for wild - type PABPC1 , 48 hours post - TMP removal . Scale bar , 100 μm .

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(C) Phase-contrast images of PABPDHFR cells, or PABPDHFR cells transfected with a plasmid coding for wild-type PABPC1, 48 hours post-TMP removal. Scale bar, 100 μm.


( D ) Western blot analysis of lysates derived from cells imaged in ( C ) .

(D) Western blot analysis of lysates derived from cells imaged in (C).


( B ) SUnSET assay of PABPDHFR cells grown in the presence or absence of TMP for 12 hours to maintain or deplete PABP . Cells were subsequently pulsed with either puromycin or puromycin and cycloheximide ( control ) , lysed and equal protein amounts were resolved by SDS - PAGE . Western blot analysis was performed using a monoclonal antibody against puromycin or actin ( loading control ) .

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(B) SUnSET assay of PABPDHFR cells grown in the presence or absence of TMP for 12 hours to maintain or deplete PABP. Cells were subsequently pulsed with either puromycin or puromycin and cycloheximide (control), lysed and equal protein amounts were resolved by SDS-PAGE. Western blot analysis was performed using a monoclonal antibody against puromycin or actin (loading control).


( D ) Scatter plot comparing log2 fold - changes [ ( - ) PABP vs ( + ) PABP ] in polysome - associated mRNA ( y - axis ) to corresponding changes in total cytoplasmic mRNA . Transcripts identified as regulated via altered translation efficiency ( orange and red ) or abundance ( light and dark green ) according to anota2seq are visualized together with non - regulated transcripts ( grey ) .

(D) Scatter plot comparing log2 fold-changes [(-) PABP vs (+) PABP] in polysome-associated mRNA (y-axis) to corresponding changes in total cytoplasmic mRNA. Transcripts identified as regulated via altered translation efficiency (orange and red) or abundance (light and dark green) according to anota2seq are visualized together with non-regulated transcripts (grey).


Boxplots of 5 ´ UTR ( A ) , 3 ´ UTR ( B ) lengths for mRNAs whose abundance decreased ( ' DOWN ' , dark green ) or increased ( ' UP ' , light green ) in PABP - depleted cells as compared to cells expressing PABP . Solid horizonal middle lines in box plots denote the median ; lower and upper box limits correspond to first and third quartiles ; whiskers extend from box limits to the most extreme values . Wilcoxon - Mann - Whitney tests ( two - sided ) were used to determine differences between upregulated mRNAs and downregulated mRNAs relative to each other and to the background ( mRNAs whose abundance did not change in the absence of PABP relative to PABP - expressing cells ) .

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Boxplots of 5´UTR (A), 3´UTR (B) lengths for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP. Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values. Wilcoxon-Mann-Whitney tests (two-sided) were used to determine differences between upregulated mRNAs and downregulated mRNAs relative to each other and to the background (mRNAs whose abundance did not change in the absence of PABP relative to PABP-expressing cells).


( A ) Boxplots of mRNA half - lives ( hrs ) assessed by metabolic labeling of normal human umbilical vein endothelial cells for mRNAs whose abundance decreased ( ' DOWN ' , dark green ) or increased ( ' UP ' , light green ) in PABP - depleted cells as compared to cells expressing PABP comparing ( Tiana et al . , 2020 ) . Solid horizonal middle lines in box plots denote the median ; lower and upper box limits correspond to first and third quartiles ; whiskers extend from box limits to the most extreme values but not further than 1 . 5 × the inter - quartile range ( IQR ) . ( B - H ) PABPDHFR cells were cultured in the presence or absence of TMP for 12 hours . The stabilities of select mRNAs was assessed by using actinomycin D ( 5 μg / ml ) for the indicated amounts of time . Total RNA was isolated , reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR . mRNA decay rates were normalized to an in vitro synthesized spike - in RNA with the zero time point set at 1 . Error bars represent the SEM of three independent experiments . First order exponential decay trend lines were generated by non - linear regression analysis .

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(A) Boxplots of mRNA half-lives (hrs) assessed by metabolic labeling of normal human umbilical vein endothelial cells for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP comparing (Tiana et al., 2020). Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values but not further than 1.5× the inter-quartile range (IQR). (B- H) PABPDHFR cells were cultured in the presence or absence of TMP for 12 hours. The stabilities of select mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.


( B ) PABPDHFR cells were transfected with plasmids encoding RL - 6xB or RL - 6xBMUT . 24 hours after transfections , the cells were split and cultured for an additional 12 hours in the presence or absence of TMP . The stabilities of reporter mRNAs was assessed by using actinomycin D ( 5 μg / ml ) for the indicated amounts of time . Total RNA was isolated , reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR . mRNA decay rates were normalized to an in vitro synthesized spike - in RNA with the zero time point set at 1 . Error bars represent the SEM of three independent experiments . First order exponential decay trend lines were generated by non - linear regression analysis .

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(B) PABPDHFR cells were transfected with plasmids encoding RL-6xB or RL-6xBMUT. 24 hours after transfections, the cells were split and cultured for an additional 12 hours in the presence or absence of TMP. The stabilities of reporter mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.


( A ) Western blot analysis PABPDHFR cells depleted of LSM1 , DCP2 or XRN1 by siRNA - mediated knockdown . siGFP represents a negative control .

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(A) Western blot analysis PABPDHFR cells depleted of LSM1, DCP2 or XRN1 by siRNA-mediated knockdown. siGFP represents a negative control.


( B ) Phase - contrast images of PABPDHFR cells previously transfected with siRNAs targeting GFP ( control ) , LSM1 , DCP2 or XRN1 and cultured in the presence or absence of TMP for 48 hours . Total numbers of viable cells , denoted in the upper right hand corner of images , were quantified with acridine orange and propidium iodide and calculated as a percentage of PABPDHFR cells grown in the presence of TMP , along with the SEM . Scale bar , 100 μm .

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(B) Phase-contrast images of PABPDHFR cells previously transfected with siRNAs targeting GFP (control), LSM1, DCP2 or XRN1 and cultured in the presence or absence of TMP for 48 hours. Total numbers of viable cells, denoted in the upper right hand corner of images, were quantified with acridine orange and propidium iodide and calculated as a percentage of PABPDHFR cells grown in the presence of TMP, along with the SEM. Scale bar, 100 μm.


( A ) Western blot analysis PABPDHFR cells depleted of LSM1 by siRNA - mediated knockdown . Transfected cells were subsequently cultured in the presence or absence of TMP to maintain or deplete endogenous PABP .

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(A) Western blot analysis PABPDHFR cells depleted of LSM1 by siRNA-mediated knockdown. Transfected cells were subsequently cultured in the presence or absence of TMP to maintain or deplete endogenous PABP.


( C and D ) Poly ( A ) tail analysis of GAPDH and ACTB mRNAs from total RNA isolated from PABPDHFR cells described in ( B ) was determined by extension poly ( A ) tail ( ePAT ) . PCR products were run on high - resolution agarose gels . ' A12 ' represents the size of ePAT RT - PCR amplicons derived from a mRNA with a fixed ( A12 ) - tail .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C and D) Poly(A) tail analysis of GAPDH and ACTB mRNAs from total RNA isolated from PABPDHFR cells described in (B) was determined by extension poly(A) tail (ePAT). PCR products were run on high-resolution agarose gels. 'A12' represents the size of ePAT RT-PCR amplicons derived from a mRNA with a fixed (A12)-tail.


( D - G ) Cell death mechanisms of C3H BMDMs transfected with miR - 342 - 3p mimic ( D ) , or B6 BMDMs transfected with miR - 342 - 3p inhibitor ( F ) , followed by Mtb infection for 36 hours . Cell viabilities of C3H BMDMs transfected with miR - 342 - 3p mimic ( E ) , or B6 BMDMs transfected with miR - 342 - 3p inhibitor ( G ) , followed by stimulation with Mtb or z - VAD ( 20 μM ) for 24 hours . Data are shown as the mean ± s . e . m . of n = 3 biological replicates .

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(D-G) Cell death mechanisms of C3H BMDMs transfected with miR-342-3p mimic (D), or B6 BMDMs transfected with miR-342-3p inhibitor (F), followed by Mtb infection for 36 hours. Cell viabilities of C3H BMDMs transfected with miR-342-3p mimic (E), or B6 BMDMs transfected with miR-342-3p inhibitor (G), followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( A , B ) Survival of Mir342 + / + C3H mice and their wild - type littermate controls ( C3H ) ( n = 10 ) ( A ) , or Mir342 - / - B6 mice and their wild - type littermate controls ( B6 ) ( n = 10 ) ( B ) after aerosol infection with around 400 CFU Mtb . Data are shown as the Kaplan - Meier curves .

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(A, B) Survival of Mir342+/+C3H mice and their wild-type littermate controls (C3H) (n=10) (A), or Mir342-/-B6 mice and their wild-type littermate controls (B6) (n=10) (B) after aerosol infection with around 400 CFU Mtb. Data are shown as the Kaplan-Meier curves.


( G , H ) Secretion of cytokines TNF - α , IL - 1 , IL - 6 and CXCL15 in BMDMs obtained from Mir342 + / + C3H and their littermates ( C3H ) ( G ) , or from Mir342 - / - B6 and their littermates ( B6 ) ( H ) after Mtb stimulation , was detected by ELISA . Data are shown as the mean ± s . e . m . of n = 3 biological replicates .

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(G, H) Secretion of cytokines TNF-α, IL-1, IL-6 and CXCL15 in BMDMs obtained from Mir342+/+C3H and their littermates (C3H) (G), or from Mir342-/-B6 and their littermates (B6) (H) after Mtb stimulation, was detected by ELISA. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( C , D ) MiR - 342 - 3p mimic or inhibitor was transfected to NIH - 3T3 fibroblasts . After 48 hours , cells were collected for qRT - PCR ( C , data are shown as the mean ± s . e . m . of n = 3 biological replicates ) and western blotting ( D , representative blots from n = 3 biological replicates are shown ) to detect SOCS6 expression .

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(C, D) MiR-342-3p mimic or inhibitor was transfected to NIH-3T3 fibroblasts. After 48 hours, cells were collected for qRT-PCR (C, data are shown as the mean ± s.e.m. of n = 3 biological replicates) and western blotting (D, representative blots from n = 3 biological replicates are shown) to detect SOCS6 expression.


( F - H ) C3H BMDMs were transfected with miR - 342 - 3p mimic or SOCS6 - overexpressing lentivirus , followed by stimulation with Mtb or z - VAD ( 20 μM ) for 24 hours . Cell death mechanisms ( F ) , cell viabilities ( G ) and Mtb growth rates ( H ) were analyzed respectively . Data are shown as the mean ± s . e . m . of n = 3 biological replicates .

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(F-H) C3H BMDMs were transfected with miR-342-3p mimic or SOCS6-overexpressing lentivirus, followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Cell death mechanisms (F), cell viabilities (G) and Mtb growth rates (H) were analyzed respectively. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( A , B ) Survival of Mir342 - / - B6 and Mir342 - / - B6 mice supplemented with Socs6 siRNA ( n = 10 ) ( A ) , or Mir342 + / + C3H and Mir342 + / + C3H mice supplemented with SOCS6 - overexpressing vector ( n = 10 ) ( B ) , after aerosol infection with around 400 CFU Mtb . Data are shown as the Kaplan - Meier curves .

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(A, B) Survival of Mir342-/-B6 and Mir342-/-B6 mice supplemented with Socs6 siRNA (n=10) (A), or Mir342+/+C3H and Mir342+/+C3H mice supplemented with SOCS6-overexpressing vector (n=10) (B), after aerosol infection with around 400 CFU Mtb. Data are shown as the Kaplan-Meier curves.


( C - F ) Relative expressions of chemokines CCL5 , CXCL10 , ICAM1 ( C ) , and Caspase 3 , Caspase 7 , Caspase 8 ( F ) in Mtb - stimulated Socs6 + / + or Socs6 - / - BMDMs were detected by qRT - PCR . Data are shown as the mean ± s . e . m . of n = 3 biological replicates . Cluster analysis of clinical samples .

(C-F) Relative expressions of chemokines CCL5, CXCL10, ICAM1 (C), and Caspase 3, Caspase 7, Caspase 8 (F) in Mtb-stimulated Socs6+/+ or Socs6-/- BMDMs were detected by qRT-PCR. Data are shown as the mean ± s.e.m. of n = 3 biological replicates. Cluster analysis of clinical samples.


( C ) Socs6 - / - BMDMs were transfected with plasmids expressing Myc - RIPK3 or Myc - RIPK3 K51A mutant for 24 hours . Afterwards , transfected cells were stimulated with Mtb for 12 hours , cell lysates were collected and immunoprecipitated using an anti - RIPK1 antibody . The recruitment of Caspase 8 , RIPK3 , FADD and MLKL was analyzed by immunoblot . The lower panel represents the immunoblot analysis of whole cell lysates . Representative blots from n = 3 biological replicates are shown .

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(C) Socs6-/- BMDMs were transfected with plasmids expressing Myc-RIPK3 or Myc-RIPK3 K51A mutant for 24 hours. Afterwards, transfected cells were stimulated with Mtb for 12 hours, cell lysates were collected and immunoprecipitated using an anti-RIPK1 antibody. The recruitment of Caspase 8, RIPK3, FADD and MLKL was analyzed by immunoblot. The lower panel represents the immunoblot analysis of whole cell lysates. Representative blots from n = 3 biological replicates are shown.


( D - F ) Cell viabilities ( D ) , cell death mechanisms ( E ) , or Mtb growth rates ( F ) of Mtb - infected Socs6 - / - BMDMs that were transfected with plasmids expressing Myc - RIPK3 or Myc - RIPK3 K51A mutant as indicated . Z - VAD treatment concentration was 20 μM and the treatment time was 24 hours . Data are shown as the mean ± s . e . m . of n = 3 biological replicates .

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(D-F) Cell viabilities (D), cell death mechanisms (E), or Mtb growth rates (F) of Mtb-infected Socs6-/- BMDMs that were transfected with plasmids expressing Myc-RIPK3 or Myc-RIPK3 K51A mutant as indicated. Z-VAD treatment concentration was 20 μM and the treatment time was 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( E - G ) RAW264 . 7 cells were transfected with plasmids expressing A20 and RIPK3 ( E ) , or A20 and RIPK3 mutants ( G ) as indicated , cell lysates were collected for immunoprecipitation and immunoblot . Myc - A20 or Flag - RIPK3 purified from transfected HEK - 293T cells was incubated with ATP , E1 , E2 and ubiquitin as indicated . The in vitro ubiquitination of RIPK3 was analyzed by immunoblot using an anti - Ub antibody ( F ) . Representative blots from n = 3 biological replicates are shown .

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(E-G) RAW264.7 cells were transfected with plasmids expressing A20 and RIPK3 (E), or A20 and RIPK3 mutants (G) as indicated, cell lysates were collected for immunoprecipitation and immunoblot. Myc-A20 or Flag-RIPK3 purified from transfected HEK-293T cells was incubated with ATP, E1, E2 and ubiquitin as indicated. The in vitro ubiquitination of RIPK3 was analyzed by immunoblot using an anti-Ub antibody (F). Representative blots from n = 3 biological replicates are shown.


( A , B ) Production of cytokines TNF - α , IL - 1 , IL - 6 , CXCL15 ( A ) , or chemokines Ccl5 , Cxcl10 , Icam1 ( B ) in the A20 - / - Socs6 + / + , A20 + / + Socs6 + / + , A20 - / - Socs6 - / - and A20 + / + Socs6 - / - BMDMs after Mtb stimulation . Data are shown as the mean ± s . e . m . of n = 3 biological replicates .

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(A, B) Production of cytokines TNF-α, IL-1, IL-6, CXCL15 (A), or chemokines Ccl5, Cxcl10, Icam1 (B) in the A20-/-Socs6+/+, A20+/+Socs6+/+, A20-/-Socs6-/- and A20+/+Socs6-/- BMDMs after Mtb stimulation. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( B ) Percentage infectivity of mouse tail fibroblasts ( MTFs ) infected with WT , RH , RHEV and EV CHIKV ( MOI 10 , n = 3 per group ) harvested at 12 hpi . Data are presented as the means ± SEM .

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(B) Percentage infectivity of mouse tail fibroblasts (MTFs) infected with WT, RH, RHEV and EV CHIKV (MOI 10, n=3 per group) harvested at 12 hpi. Data are presented as the means ± SEM.


( C ) Viremia progression in virus - infected mice was monitored over 2 weeks ( n = 11 per group ) . Viremia is detected with CHIKV nsP1 probe via qRT - PCR .

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(C) Viremia progression in virus-infected mice was monitored over 2 weeks (n=11 per group). Viremia is detected with CHIKV nsP1 probe via qRT-PCR. Statistical


( D ) joint inflammation in virus - infected mice was monitored over 2 weeks ( n = 11 per group ) .

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(D) joint inflammation in virus-infected mice was monitored over 2 weeks (n=11 per group).


( E ) Compiled percentages of CHIKV - infected ( ZsGreen positive ) leukocytes at 2 days post infection ( dpi ) for the respective groups ( n = 5 per group ) . Background ZsGreen signals are indicated by the dotted line .

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(E) Compiled percentages of CHIKV-infected (ZsGreen positive) leukocytes at 2 days post infection (dpi) for the respective groups (n=5 per group). Background ZsGreen signals are indicated by the dotted line.


( F ) Dot - plots of the localized type - I interferon ( IFN - α and IFN - β ) concentrations at 15 hpi . Data are expressed as the means ± SD .

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(F) Dot-plots of the localized type-I interferon (IFN-α and IFN-β) concentrations at 15 hpi. Data are expressed as the means ± SD.


Cell lysates were collected from the joints of mice infected with WT CHIKV or CHIKV nsP - mutants at 15 hpi , 2 dpi or 6 dpi ( n = 5 per group ) . The concentrations of 36 immune mediators were quantified using a 36 - plex microbead - based immunoassay . Immune mediators are grouped based on function . Each color represents the relative concentration of a particular analyte . Blue and red indicates low and high concentration , respectively .

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Cell lysates were collected from the joints of mice infected with WT CHIKV or CHIKV nsP-mutants at 15 hpi, 2 dpi or 6 dpi (n=5 per group). The concentrations of 36 immune mediators were quantified using a 36-plex microbead-based immunoassay. Immune mediators are grouped based on function. Each color represents the relative concentration of a particular analyte. Blue and red indicates low and high concentration, respectively.