The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x

Abstract

The RNA-binding protein Sam68 is involved in apoptosis, but its cellular mRNA targets and its mechanism of action remain unknown. We demonstrate that Sam68 binds the mRNA for Bcl-x and affects its alternative splicing. Depletion of Sam68 by RNA interference caused accumulation of antiapoptotic Bcl-x(L), whereas its up-regulation increased the levels of proapoptotic Bcl-x(s). Tyrosine phosphorylation of Sam68 by Fyn inverted this effect and favored the Bcl-x(L) splice site selection. A point mutation in the RNA-binding domain of Sam68 influenced its splicing activity and subnuclear localization. Moreover, coexpression of ASF/SF2 with Sam68, or fusion with an RS domain, counteracted Sam68 splicing activity toward Bcl-x. Finally, Sam68 interacted with heterogenous nuclear RNP (hnRNP) A1, and depletion of hnRNP A1 or mutations that impair this interaction attenuated Bcl-x(s) splicing. Our results indicate that Sam68 plays a role in the regulation of Bcl-x alternative splicing and that tyrosine phosphorylation of Sam68 by Src-like kinases can switch its role from proapoptotic to antiapoptotic in live cells.

The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells

“…The bound proteins were subjected to SDS-PAGE, visualized with Silver Staining and identified using mass spectrometry (Figure 1a). This approach confirmed some previously described SAM68 interactions, such as hnRNP A1 23 and K, 44 nucleolin, heat shock 70-kDa protein 5 and eIF4B; 45 however, it also yielded many new interacting proteins that are potentially interesting for the function of SAM68 (Figure 1b, Supplementary Table 1). PSF, for example, is a splicing regulator that associates with the androgen receptor and regulates its transcriptional activity, 46 similar to SAM68, 21 whereas the translation initiation factor eIF3 and the ribosomal proteins L7, S3, S4 and S7 might be involved in the mechanism of SAM68-dependent regulation of mRNA translation.…”

“…Cell extracts, cytosol/nuclear fractionations, 23,58 sucrose gradient fractionation of the nuclear compartment 59 and western blot analysis 23 were performed as previously described. Primary antibodies used (1:1000 dilution; overnight at 4 1C) were the following: rabbit anti-ERK2, mouse antiMyc, rabbit anti-SAM68 and goat anti-lamin B (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-tubulin, mouse anti-FLAG (Sigma-Aldrich) and rabbit anti-GFP (Molecular Probes, Eugene, OR, USA), mouse anti-RNAPII (CTD4H8; Millipore, Billerica, MA, USA) and mouse anti-SND1.…”

“…[14][15][16] An example of RBP that is upregulated in several human tumors is SAM68 (Src-associated in mitosis of 68 kDa), a member of the STAR (Signal Transduction Activator of RNA metabolism) family of RBPs. 17 SAM68 is involved in regulating several aspects of RNA processing, such as transcription, [18][19][20][21] AS [22][23][24][25] and translation of cellular mRNAs. [26][27][28] Moreover, SAM68 displays pro-oncogenic functions and is frequently upregulated in human cancer types, 17 including prostate cancer (PCa), wherein it supports cell proliferation and survival to genotoxic stresses.…”

Alternative splicing of human papillomavirus type-16 E6/E6* early mRNA is coupled to EGF signaling via Erk1/2 activation

“…Using HiPerfect (Qiagen) transfection reagent and following manufacturer's instructions, 7 × 10 5 cells were plated in 6-cm well plates and transfected with 100 nM siRNA (Dharmacon). siRNA sequences were described previously for Brm (33), ASF, hnRNPA1, hnRNPA2 (28), Sam68 (43), and siScramble (44). Twenty-four hours posttransfection, fresh media was added and, where indicated, EGF was depleted for an additional 24 h. Seventy-two hours posttransfection, cells were harvested.…”

Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged

“…Prompted by reports that Sam68 overexpression can result in apoptosis in NIH-3T3 cells (Babic et al 2006), and their observation that Sam68 interacts with the Bcl-x mRNA, Sette and colleagues (Paronetto et al 2007) investigated a possible role for this protein in Bcl-x splicing. Overexpression of Sam68 in 293 cells resulted in an increase in Bcl-x(s) isoform, consistent with the proapoptotic effects observed upon Sam68 overexpression (Taylor et al 2004;Paronetto et al 2007). Interestingly, Sam68 phosphorylation by the Src-like tyrosine kinase Fyn reversed the effects of Sam68 overexpression, switching splicing of Bcl-x back to the long isoform.…”