Fig 2: In vitro Purified SAM68 associated with U1 snRNP in an RNA-independent manner. (A) In vitro purified hSAM68-Flag was added to shSAM68 HEK-293T cell lysates for 1 h at 4°C, in the presence or absence of 50 µg/ml RNaseA. hSAM68-Flag and associated proteins were immunoprecipitated using Flag-M2 affinity beads and treated further with RNaseA at 37°C for 30 min. Bound proteins were eluted with Laemmli and immunoblotted with antibodies specific to U1–70K, U1A and U1C. To assess RNaseA treatment efficiency, total RNA from shSAM68 HEK-293T was treated with either Mock or RNaseA for 30 min at 37°C, and the remaining total RNA was assessed on agarose gel. (B) RNA-binding defective mutant hSAM68I184N interacts with U1 snRNP. shSAM68 HEK-293T were transiently transfected with Flag-hSAM68, Flag-hSAM68I184N and Flag-YFP (negative control). The Flag-tagged proteins were immunoprecipitated using anti-Flag M2 agarose beads and immunoblotted with antibodies directed against U1–70K, U1A and U1C. (C) Association of hSAM68-Flag with U1 snRNP withstands high salt washes. Purified in vitro produced hSAM68-Flag was added to cell lysates of shSAM68 HEK-293T for 1 h at 4°C. Flag-M2 affinity beads were added to the reaction and left for 1 h at 4°C. The washes were done, by increasing salt concentration, from 150 to 500 mM of NaCl. Bound proteins were eluted with Laemmli and immunoblotted with antibodies directed against U1–70K, U1A and U1C. (D) SAM68 interacts with U1A in vitro. About 300 ng of purified hSAM68-Flag was incubated with 100 ng of glutathione-agarose bound GST-U170k-His, GST-U1A-His, GST-U1C-His and GST-His. Following washes, the beads were washed five times in binding buffer and the bound proteins eluted with Laemmli and immunoblotted using anti-Flag or anti-His antibodies.

Fig 3: Both SAM68 and intronic enhancer sequences in mTor intron 5 are required for U1A recruitment to 5'SS in vitro. (A) Schematic representation of the various in vitro transcribed mTor minigene baits with the 5' splice site. As shown, the baits span from last 7 nucleotides of exon5 to the poly-adenylation signal in intron 5. WT refers to the wild-type intronic SAM68-binding sequences of SB-1 (UUUUAU) and SB-A (UAAAA), the latter is embedded in the cryptic poly-adenylation signal (AAUAAA). The ‘mut’ denotes the combined mutations of SB-1 (UUUUAU to UUUCAU) and SB-A (AAUAAAA to AAUAACC). (B) SAM68 recruits U1A to 5' splice site in vitro. Recombinant in vitro purified hSAM68-Flag was tested for its ability to recruit U1A to mTor intron 5 baits with either WT or mutated SAM68-binding sites. GST-Flag was used as negative control. (C) Schematic representation of the various in vitro transcribed mTor minigene baits that are deleted for the 5' splice site. As shown, the baits span 18 nucleotides downstream of the 5' splice site to the poly-adenylation signal of intron 5. WT refers to the wild-type intronic SAM68-binding sequences, SB-1 (UUUUAU) and SB-A (UAAAA). The ‘mut’ denotes the combined mutations of SB-1 (UUUUAU to UUUCAU) and SB-A (AAUAAAA to AAUAACC). (D) SAM68 recruits U1A in the absence of 5' splice site in vitro. Recombinant in vitro purified hSAM68-Flag was tested for its ability to recruit U1A to mTor intron 5 baits lacking 5'SSs with either WT or mutated SAM68-binding sites. GST-Flag was used as negative control. (E) Schematic representation of the in vitro transcribed mTor minigene bait and the primers used for the RppH/Xrn1 protection assays. (F) Assessment of the processivity of RppH and Xrn1 enzyme on the naked mRNA bait, showing that RppH treatment is necessary for Xrn1-mediated degradation of the mRNA bait. (G) RppH and Xrn1 protection assays in vitro produced mRNA bait incubated with either WT MEFs cell lysate (lane 1), Sam68-/- MEFs cell lysate (lane 2), in vitro produced mSAM68(WT) + Sam68-/- MEFs cell lysate (lane 3) or in vitro produced mSAM68(WT) + Sam68-/- MEFs cell lysate + U1 nRNAs antisense oligo (lane 4). U1snRNP components (U1A, U1C) and mSAM68 levels were assessed by western blot, while U1 snRNA levels was assessed by RT-PCR. GAPDH served as loading control for the western blot. (H) SAM68 protects the mTor RNA bait from Xrn1 degradation. Biotinylated RNA baits were incubated with buffer (lane 1), 100 ng of GST-Flag (lane 2) or 100 ng of mSAM68-Flag (lane 3) for 30 min on ice. Sam68 levels were assessed by western blotting using anti-Flag, while baits levels were measured by semi-quantitative RT-PCR using FSS-RSB primers for the full-length RNA and FSB-RSB for the SAM68 protected fragment.

Fig 4: RBM39 interactions with spliceosome components. (A) RBM39 interacts with U1 snRNP and U2AF. Interaction of RBM39 with the U1-specific protein U1-70K, the U2-specific protein U2A’ and the small subunit of U2AF was assayed by immunoprecipitations. HeLa cells were transiently transfected with U1-70K-GFP, U2A'-GFP or U2AF35-GFP, immunoprecipitated with anti-GFP antibodies and probed with antibodies shown to the right. U1C and SF3B4 served as positive controls for immunoprecipitations for U1-70K-GFP and U2A’-GFP, respectively. Asterisks denote a partially degraded U2A’-GFP. (B, C) RBM39 interactions monitored by FRET. Cells were transiently co-transfected with RBM39-CFP and C-terminally YFP-tagged U1-70K. (B) YFP was bleached in a small region comprising the nucleoplasm and nuclear speckles; CFP fluorescence was measured before and after bleaching. Fluorescence of RBM39 increased after bleaching of U1-70K-YFP [cf. CFP fluorescence in the bleached region (rectangles) before (top panel) and after (bottom panel) bleaching]. A, acceptor; D, donor; scale bar, 5 µm. (C) Quantification of individual donor-acceptor FRET efficiencies upon the inhibition of RNA polymerase II by DRB. Columns indicate means; errors bars SEMs. Interaction between RBM39-CFP and U2AF35-YFP (22) served as a positive control and interaction between RBM39-CFP and YFP as a negative control. Significantly different means are denoted by an asterisk (P< 0.01; t-test).

Fig 5: In vivo association of SAM68 with U1 snRNP. (A) Schematic representation of a portion of mTor pre-mRNA spanning from exon4 to exon6 (upper panel), with a close-up of the 5' splice site and the subsequent SAM68-binding site (SB-1), as well as the cryptic polyadenylation signal that harbor SAM68-binding site (SB-A). (B) Co-immunoprecipitation of U1 snRNP with Flag-hSAM68. HEK-293T cells depleted of endogenous SAM68 (shSAM68 HEK-293T) were transiently transfected with Flag-hSAM68 or Flag-YFP (yellow-fluorescent protein), the latter serving as negative control. Flag-tagged proteins were immunoprecipitated using anti-Flag M2 agarose beads and immunoprecipitated proteins were detected with antibodies specific to U1–70K, U1A and U1C. ß-Actin was used as negative control. Portion of the Flag-immunoprecipitates was used for RNA isolation and RT-PCR using U1 snRNA specific primers. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) RNA was used as negative control of the RT-PCR made from the RNA immunoprecipitation. (C) Co-immunoprecipitation of endogenous hSAM68 with U1–70K. Immunoprecipitated proteins were detected with antibodies directed against SAM68 and U1–70K. ß-Actin was used as negative control of immunoprecipitated proteins. (D) Co-immunoprecipitation of endogenous hSAM68 with U1A. Immunoprecipitated proteins were detected with antibodies directed against SAM68 and U1A. ß-Actin was used as negative control of immunoprecipitated proteins. (E) Coomassie staining of purified human SAM68 and U1A. (F) RNA binding assay with purified SAM68 and labeled U1snRNA. Reactions contained 10 nM ?-p32 labeled U1snRNA in buffer with no protein (lane 1) or with purified SAM68 (lanes 2–5). Bottom panel: quantification from three independent binding experiments. Error bars represent the corresponding standard error. Unpaired two-tailed t-tests were used to compare the different concentrations of purified protein to the RNA only control. SAM68 P-values are 0.0014, 0.0005, <0.0001, <0.0001 in increasing order of SAM68 concentration. (G) RNA binding assay with purified U1A and labeled U1snRNA. Reactions contained 10 nM ?-p32 labeled U1snRNA in buffer with no protein (lane 1) or with purified U1A (lanes 2–5). Bottom panel: U1snRNA P-values = 0.0008, <0.0001, <0.0001, <0.0001 in increasing order of U1A concentration. **P-value < 0.005, ***P-value < 0.001.