ABSTRACT: A functional complex between XRN2 and Sam68 orchestrates an alternative polyadenylation program involved in prostate cancer cell cycle progression
Project description:A functional complex between XRN2 and Sam68 orchestrates an alternative polyadenylation program involved in prostate cancer cell cycle progression
Project description:Alternative cleavage and polyadenylation (APA) of pre-mRNAs yields multiple transcripts differing in their 3' end and its regulation is often aberrant in human cancers, including prostate cancer (PC). In this study, we have uncovered a novel mechanism of APA regulation which impinges on the functional interaction between the 5'-3' exonuclease XRN2 and the A/U-rich motif RNA binding protein Sam68 in PC cells. XRN2 promotes recruitment of Sam68 to its target transcripts, where it competes with the cleavage and polyadenylation specificity factor (CPSF) for the recognition of the AAUAAA core polyadenylation signal (PAS) motif. In particular, the Sam68/XRN2 complex suppresses the usage of strong canonical PASs at distal ends of genes and, consequently, promotes the usage of suboptimal proximal PASs.
Project description:Male germ cells express the widest repertoire of transcript variants in mammalian tissues. Nevertheless, factors and mechanisms underlying such pronounced diversity are largely unknown. The splicing regulator Sam68 is highly expressed in meiotic cells and its ablation results in defective spermatogenesis. Herein, we uncover an extensive splicing program operated by Sam68 across meiosis, primarily characterized by alternative last exon (ALE) regulation in genes of functional relevance for spermatogenesis. Lack of Sam68 preferentially causes premature transcript termination at internal polyadenylation sites.
Project description:Neuronal alternative splicing is dynamically regulated in a spatiotemporal fashion. We previously found that STAR family proteins (SAM68, SLM1, SLM2) regulate spatiotemporal alternative splicing in the nervous system. However, the whole aspect of alternative splicing programs governed by STARs remains unclear. We deciphered the alternative splicing programs of SAM68 and SLM1 proteins using transcriptomics. We reveal that SAM68 and SLM1 encode distinct alternative splicing programs; SAM68 preferentially controls alternative last exon (ALE) splicing. Interleukin 1-receptor accessory protein (Il1rap) is a novel target for SAM68. The usage of Il1rap ALEs results in mainly two variants encoding two functionally different isoforms, a membrane-bound (mIL1RAcP) and a soluble (sIL1RAcP) type. The brain exclusively expresses mIL1RAcP. SAM68 knockout results in remarkable conversion into sIL1RAcP in the brain, which significantly disturbs IL1RAcP neuronal function. Thus, we uncover the critical role of proper neuronal isoform selection through ALE choice by the SAM68-specific splicing program.
Project description:Our previous studies of proteomic-coupled-network analysis of AR protein interaction complexes (Paliouras et al., Integrative Biology, 2011) identified a number of proteins involved in RNA metabolism, specifically alternative RNA splicing. We selected two interacting RNA splicing proteins, SAM68 and DDX5 to examine RNA splicing events in prostate cancer (PCa). This analysis suggests a much more robust effect on RNA splicing with AR dictating either an exon-inclusion or -exclusion pathway. To establish the true physiological roles of AR in alternative RNA splicing, we opted to further examine the changes in global splicing profiles of LNCaP PCa cells, stimulated with and without androgens in conjunction with overexpression studies of SAM68 and DDX5.
Project description:The metabolic conversion of oxidative phosphorylation to glycolysis provides tumor cells with energy and biosynthetic substrates, thereby promoting tumorigenesis and malignant progression. However, the mechanisms controlling the tumor metabolic switch is still not entirely clear. Here we demonstrate that SAM68 (gene name: KHDRBS1) as a splicing regulatory factor is frequently overexpressed in Lung adenocarcinoma (LUAD) and negatively correlated with the prognosis of LUAD patients. we find SAM68 promotes LUAD cells tumorigenesis and metastasis both in vitro and in vivo by regulating cancer metabolic switch. SAM68 drives cancer metabolism by mediating alternative splicing of Pyruvate kinase (PKM) pre-mRNAs, finally promoting the formation of PKM2. Mechanically, Sam68 interacted with the splicing repressor hnRNP A1, and depletion of hnRNP A1 or mutations that impair this interaction attenuated the PKM splicing regulation. Together, our work demonstrates key roles of SAM68 in the cancer metabolic conversion by regulating alternative splicing and SAM68 may be a promising therapeutic target for treating LUAD.This project looks into how SAM68 levels affect cancer cell phenotype in vitro
Project description:Sam68 is a member of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins and its role is modulated by post-translational modifications, including serine/threonine phosphorylation, that differ at various stages of the cell cycle. However, the molecular basis and mechanisms of these modulations remain largely unknown. Here, we combined mass spectrometry, NMR spectroscopy, and cell biology techniques to provide a comprehensive post-translational modification (PTM) mapping of Sam68 at different stages of the cell cycle in HEK293 and HCT116 cells. We established that Sam68 is specifically phosphorylated at T33 and T317 by Cdk1, and demonstrated that these phosphorylation events reduce the binding of Sam68 to RNA, control its cellular localization, and reduce its alternative splicing activity, leading to a reduction in the induction of apoptosis and an increase in the proliferation of HCT116 cells.
2023-03-11 | PXD032716 | Pride
Project description:A Sam68-dependent alternative splicing program shapes postsynaptic protein complexes
Project description:Termination is a ubiquitous phase in every transcription cycle but is incompletely understood and a subject of debate. We have used gene editing as a new approach to address its mechanism through engineered conditional depletion of the 5’-3’ exonuclease, Xrn2, or the polyadenylation signal (PAS) endonuclease, CPSF73. The ability to rapidly control Xrn2 reveals a clear and general role for it in co-transcriptional degradation of 3’ flanking region RNA and transcriptional termination. This defect is characterised genome-wide, at high resolution, using native elongating transcript sequencing (mNET-seq). An Xrn2 effect on termination requires prior RNA cleavage and we provide evidence for this by showing that catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73. Notably, Xrn2 plays no significant role in either Histone or snRNA gene termination even though both RNA classes undergo 3’ end cleavage. In sum, efficient termination on most protein-coding genes involves CPSF73 mediated RNA cleavage and co-transcriptional degradation of polymerase-associated RNA by Xrn2. However, as CPSF73 loss caused more extensive read-through transcription than Xrn2 elimination, it likely plays a more underpinning role in termination.