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

Abstract

Splicing abnormalities have profound impact in human cancer. Several splicing factors, including SAM68, have pro-oncogenic functions, and their increased expression often correlates with human cancer development and progression. Herein, we have identified using mass spectrometry proteins that interact with endogenous SAM68 in prostate cancer (PCa) cells. Among other interesting proteins, we have characterized the interaction of SAM68 with SND1, a transcriptional co-activator that binds spliceosome components, thus coupling transcription and splicing. We found that both SAM68 and SND1 are upregulated in PCa cells with respect to benign prostate cells. Upregulation of SND1 exerts a synergic effect with SAM68 on exon v5 inclusion in the CD44 mRNA. The effect of SND1 on CD44 splicing required SAM68, as it was compromised after knockdown of this protein or mutation of the SAM68-binding sites in the CD44 pre-mRNA. More generally, we found that SND1 promotes the inclusion of CD44 variable exons by recruiting SAM68 and spliceosomal components on CD44 pre-mRNA. Inclusion of the variable exons in CD44 correlates with increased proliferation, motility and invasiveness of cancer cells. Strikingly, we found that knockdown of SND1, or SAM68, reduced proliferation and migration of PCa cells. Thus, our findings strongly suggest that SND1 is a novel regulator of alternative splicing that promotes PCa cell growth and survival.Oncogene advance online publication, 2 September 2013; doi:10.1038/onc.2013.360Keywords: alternative splicing; SND1; SAM68; CD44; prostate cancer INTRODUCTION Nuclear processing of pre-mRNAs requires tightly regulated steps that ultimately yield a mature and functional mRNA. Splicing is the step that insures the removal of long non-coding sequences (introns) from the pre-mRNA and the joining of the exons. This phenomenon is driven by a large macromolecular complex, the spliceosome, composed of five small nuclear ribonucleoproteins (snRNPs) and over 200 auxiliary proteins. 1 In higher eukaryotes, a large number of exons can be alternatively spliced to yield different transcripts from a single gene, thereby increasing the coding potential of the genome. 2,3 Indeed, the large majority of multi-exon human genes undergo alternative splicing (AS) to produce at least two mRNA variants. 4,5 As regulation of AS profoundly influences physiological and pathological processes, 3,6 the full comprehension of the molecular mechanisms regulating this step of pre-mRNA processing is of fundamental importance.Splicing is physically and functionally coupled to transcription. 7-10 Two models have been proposed for how transcription might affect changes in AS patterns. The 'recruitment model' suggests that the transcription apparatus physically interacts with splicing regulators, thereby affecting splicing decisions. The C-terminal domain (CTD) of the largest subunit of the RNA polymerase II (RNAPII) has a central role in this coupling process by favoring the recruitment of RNA processing factors on the nascent transcripts. 9,10 ...

Tudor staphylococcal nuclease: biochemistry and functions

“…Mass spectrometry analysis of proteins interacting with splicing factor SAM68 in prostate cancer cells showed significant accumulation of both SAM68 and TSN. 31 Upregulation of these proteins exerted interdependent synergic effect on exon v5 inclusion in the CD44 mRNA, providing further evidence for TSN as a novel regulator of alternative splicing, promoting prostate cancer cell growth and survival.…”

“…Recent work has connected the function of TSN in splicing regulation to oncogenesis by demonstrating interaction of TSN with a splicing factor SAM68 in prostate cancer cells. 31 The authors have revealed that TSN can promote SAM68-dependent splicing of specific exons in DC44 mRNA, an event associated with tumor progression and metastasis. However, whether this phenomenon is specific for prostate cancer or is a more general event requires additional investigations.…”

“…24 It has recently been shown that human TSN interacts through its Tudor domain with core components of RNA splicing machinery, including Prp8 (human U5-220 kD Protein), two Sm proteins, SmB and SmD1/D3 and the splicing factor SAM68 (Src-associated in mitosis of 68 kDa). [30][31][32] The interaction of Tudor domain with SmD1, a Sm protein from Plasmodium falciparum suggests an evolutionary conserved role of TSN in splicing. 33 The interaction with PIWIL1/Miwi, a specific member of the Argonaute family, suggests a role for TSN in the biogenesis of noncoding RNAs in mammals.…”

Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay

“…Moreover, TSN is cleaved by caspases and facilitates programmed cell death during development and stress responses (Sundström et al, 2009). TSN also interacts with small ribonucleoproteins and Sm proteins involved in pre-mRNA splicing Gao et al, 2012;Cappellari et al, 2014). In plants, TSN is an RNA-binding protein involved in RNA transport and localization, which is essential for stress tolerance and has been linked to the formation of stress granules and processing bodies (Wang et al, 2008;Frei dit Frey et al, 2010;Gutierrez-Beltran et al, 2015).…”

Sam68 Regulates S6K1 Alternative Splicing during Adipogenesis

“…The binding of Sam68 near alternative splice junctions in pre-mRNAs has been shown to regulate splice site selection and regulate the usage of alternative exons (1). Sam68 promotes the inclusion of CD44 variable exon 5 (v5), and interaction of Sam68 with SND1 (staphylococcal nuclease domain 1) enhances v5 inclusion (5,6). The alternative splicing of Bcl-x is regulated by Sam68 and its interaction with hnRNPA1 and FBI-1, affecting prosurvival and apoptotic pathways (7,8).…”