Fig 2: Tudor domains of TDRD3 and SMN recognize methylated Aven.(A) Recombinant Tudor domains of TDRD3, SPF30, and SMN were fused GST and used in ‘pull-down’ assays with HEK293T lysates expressing pcDNA3.1 (control), FLAG-Aven, or FLAG-Aven?RGG. The bound proteins were separated by SDS-PAGE and immunoblotted with anti-FLAG antibodies. (B, C) Lysates from HEK293T lysates expressing pcDNA3.1 (control), FLAG-Aven, or FLAG-Aven?RGG were IP with anti-FLAG antibodies. Co-immunoprecipitation of endogenous TDRD3 and SMN was detected by immunoblotting. (D) Aven interaction with TDRD3 and SMN was reduced in cells deficient for PRMT1 using siRNAs. FLAG-Aven was co-expressed with either siControl or siPRMT1 in U2OS cells. Anti-FLAG antibody immunoprecipitations were performed and the presence of endogenous TDRD3 and SMN monitored by immunoblotting following separation by SDS-PAGE. (E) PRMT1FL/-;CreERT MEFs treated with OHT for 6 days or left untreated were lysed and IP with anti-Aven antibodies. Co-immunoprecipitation of endogenous TDRD3 and SMN was detected by immunoblotting (upper panels).DOI: http://dx.doi.org/10.7554/eLife.06234.007

Fig 3: Aven binds G4 RNA sequences in an arginine methylation independent manner.(A) U2OS cells transfected with pcDNA3.1, or expression vectors encoding FLAG-Aven or FLAG-Aven?RGG were IP with anti-FLAG antibodies and immunoblotted with ASYM25b or anti-FLAG antibodies as indicated. The molecular mass markers are shown on the left in kDa and the migration of FLAG-Aven and FLAG-Aven?RGG is shown. The asterisks (*) denote unknown arginine methylated proteins. (B) Biotinylated sc1 G4 bound to Streptavidin were incubated with HEK293T cell lysates. The bound proteins were washed with increasing NaCl (mM) as indicated and visualized by SDS-PAGE followed by immunoblotting with anti-Aven antibodies. (C) Biotinylated sc1 G4 or (G4m) bound to Streptavidin was incubated with cellular lysates expressing FLAG-Aven and FLAG-Aven?RGG and detected as in panel B. (D) Biotinylated sc1 G4 RNA bound to Streptavidin beads was used to pull-down Aven from PRMT1-depleted HEK293T cells. Aven binding was performed as in panel B. PRMT1 depletion was confirmed by immunoblotting. (E) Biotinylated methylated and unmethylated Aven TriRG peptides were pre-bound on Streptavidin plates and were incubated with fluorescein-labeled sc1 G4 RNA. The bound RNA was quantified by measuring fluorescence at 521 nm. The experiment was performed twice in triplicates.DOI: http://dx.doi.org/10.7554/eLife.06234.005

Fig 4: PRMT1 and Aven RGG/RG motif required for optimal translation of MLL4 G4 sequence.(A) Schematic of the luciferase reporter plasmid pGL3, as well as the chimeric pGL3-MLL4-G4 and pGL3-MLL4-G4mutant. pGL3-MLL4-G4 harbors the human MLL4 G4 sequence nucleotide 262 to 318 inserted in-frame at the N-terminus of luciferase, while pGL3-MLL4-G4mutant contains glycine to alanine mutations that disrupts the G4 structure. (B) Aven-/- HEK293T cells were transfected with the following reporter genes pGL3, pGL3-MLL4-G4, or pGL3-MLL4-G4mutant and pRenilla as well as pcDNA3.1, FLAG-Aven, FLAG-Aven?RGG, or FLAG-AvenR-K. The cells were harvested 24 hr post-transfection and dual luciferase assays were performed. The relative luciferase/Renilla ratio was normalized to 1.0 in pGL3 pcDNA3.1 transfected cells. Extracts were immunoblotted with anti-FLAG antibodies to confirm Aven, Aven?RGG, or AvenR-K expression. Error bars represent standard deviation values. The experiments were performed three independent times (n = 3) and each independent experiment was performed in technical triplicates. The significance was measured by ANOVA followed by post hoc comparison using Tukey test. *p < 0.05. (C) HEK293T cells were co-transfected with FLAG-Aven and either siGFP (siCTRL) or siPRMT1 along with the following reporter genes pGL3, pGL3-MLL4-G4, or pGL3-MLL4-G4mutant and pRenilla. The cells were harvested 24 hr post-transfection and dual luciferase assays were performed. The relative luciferase/Renilla ratio was normalized to 1.0 in pGL3 siCTRL transfected cells. Extracts were immunoblotted with anti-PRMT1 or anti-ß-actin antibodies, as indicated. Error bars represent standard deviation values. The experiments were performed three independent times (n = 3) and each independent experiment was performed in technical triplicates. The significance was measured by ANOVA followed by post hoc comparison using Tukey test. *p < 0.05, n.s. non-significant. (D) HEK293T cells were co-transfected with FLAG-Aven and either siGFP (siCTRL) or siDHX36 along with the following reporter genes pGL3, pGL3-MLL4-G4, or pGL3-MLL4-G4mutant and pRenilla. The cells were harvested 24 hr post-transfection and dual luciferase assays were performed. The relative luciferase/Renilla ratio was normalized to 1.0 in pGL3 siCTRL transfected cells. Extracts were immunoblotted with anti-DHX36 or anti-ß-actin antibodies, as indicated. The error bars represent ± the standard deviation. Experiments were performed three times (n = 3) and each experiment was analyzed in triplicates. The significance was measured by ANOVA followed by post hoc comparison using Tukey test. *p < 0.05, n.s. non-significant.DOI: http://dx.doi.org/10.7554/eLife.06234.018

Fig 5: Methylation of Aven and its association with TDRD3 and SMN is required for polysomal localization.(A) Cytoplasmic extracts from HEK293T cells were sedimented by centrifugation on a 5–50% sucrose gradient. Polysome profiles were obtained by continuous monitoring of UV absorbance at 254 nm. 40S, 60S, and 80S indicate the positions of the respective ribosomal subunits and the monomer on the gradient. (B) The distribution of endogenous Aven and PRMT1 across the gradient of panel A was monitored by immunoblotting. Ribosomal protein rpS6 was used as a loading control, whereas ß-tubulin served as a cytoplasmic marker. (C–H) The distribution of FLAG-Aven or FLAG-Aven?RGG across the gradient was monitored by immunoblotting as well as FLAG-Aven in siControl, siPRMT1, siSMNsiTDRD3, or with puromycin treatment. Both short (5 s, panels C–H) and long exposures (30 s, panels C–H) are shown. rpS6 was used as a loading control. The exposure time was determined using a standard curve with increasing amounts of lysates expressing FLAG-Aven immunoblotted with anti-FLAG antibodies for various times. Each polysomal profile experiment was performed independently three times.DOI: http://dx.doi.org/10.7554/eLife.06234.00810.7554/eLife.06234.009Figure 4—source data 1.Quantitative mass spectrometry of Aven and Aven?RGG interacting proteins.DOI: http://dx.doi.org/10.7554/eLife.06234.009