Combined Use of MS2 and PP7 Coat Fusions Shows that TIA-1 Dominates hnRNP A1 for K-SAM Exon Splicing Control

Splicing of the FGFR2 K-SAM exon is repressed by hnRNP A1 bound to the exon and activated by TIA-1 bound to the downstream intron. Both proteins are expressed similarly by cells whether they splice the exon or not, so it is important to know which one is dominant. To answer this question, we used bacteriophage PP7 and bacteriophage MS2 coat fusions to tether hnRNP A1 and TIA-1 to distinct sites on the same pre-mRNA molecule. hnRNP A1 fused to one coat protein was tethered to a K-SAM exon containing the corresponding coat protein's binding site. TIA-1 fused to the other coat protein was tethered to the downstream intron containing that coat protein's binding site. This led to efficient K-SAM exon splicing. Our results show that TIA-1 is dominant for K-SAM exon splicing control and validate the combined use of PP7 and MS2 coat proteins for studying posttranscriptional events

Determinants of Nam8-dependent splicing of meiotic pre-mRNAs

Nam8, a component of yeast U1 snRNP, is optional for mitotic growth but required during meiosis, because Nam8 collaborates with Mer1 to promote splicing of essential meiotic mRNAs AMA1, MER2 and MER3. Here, we identify SPO22 and PCH2 as novel targets of Nam8-dependent meiotic splicing. Whereas SPO22 splicing is co-dependent on Mer1, PCH2 is not. The SPO22 intron has a non-consensus 5′ splice site (5′SS) that dictates its Nam8/Mer1-dependence. SPO22 splicing relies on Mer1 recognition, via its KH domain, of an intronic enhancer 5′-AYACCCUY. Mutagenesis of KH and the enhancer highlights Arg214 and Gln243 and the CCC triplet as essential for Mer1 activity. The Nam8-dependent PCH2 pre-mRNA has a consensus 5′SS and lacks a Mer1 enhancer. For PCH2, a long 5′ exon and a non-consensus intron branchpoint dictate Nam8-dependence. Our results implicate Nam8 in two distinct meiotic splicing regulons. Nam8 is composed of three RRM domains, flanked by N-terminal leader and C-terminal tail segments. The leader, tail and RRM1 are dispensable for splicing meiotic targets and unnecessary for vegetative Nam8 function in multiple synthetic lethal genetic backgrounds. Nam8 activity is enfeebled by alanine mutations in the putative RNA binding sites of the RRM2 and RRM3 domains

Splicing Reporter Mice Revealed the Evolutionally Conserved Switching Mechanism of Tissue-Specific Alternative Exon Selection

Since alternative splicing of pre-mRNAs is essential for generating tissue-specific diversity in proteome, elucidating its regulatory mechanism is indispensable to understand developmental process or tissue-specific functions. We have been focusing on tissue-specific regulation of mutually exclusive selection of alternative exons because this implies the typical molecular mechanism of alternative splicing regulation and also can be good examples to elicit general rule of “splice code”. So far, mutually exclusive splicing regulation has been explained by the outcome from the balance of multiple regulators that enhance or repress either of alternative exons discretely. However, this “balance” model is open to questions of how to ensure the selection of only one appropriate exon out of several candidates and how to switch them. To answer these questions, we generated an original bichromatic fluorescent splicing reporter system for mammals using fibroblast growth factor-receptor 2 (FGFR2) gene as model. By using this splicing reporter, we demonstrated that FGFR2 gene is regulated by the “switch-like” mechanism, in which key regulators modify the ordered splice-site recognition of two mutually exclusive exons, eventually ensure single exon selection and their distinct switching. Also this finding elucidated the evolutionally conserved “splice code,” in which combination of tissue-specific and broadly expressed RNA binding proteins regulate alternative splicing of specific gene in a tissue-specific manner. These findings provide the significant cue to understand how a number of spliced genes are regulated in various tissue-specific manners by a limited number of regulators, eventually to understand developmental process or tissue-specific functions

Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance

© 2014 The Authors. The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A 165 b. Whereas flTIA-1 selectively bound VEGF-A 165 mRNA and increased translation of VEGF-A 165 b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy

The Splicing Factor RBM4 Controls Apoptosis, Proliferation, and Migration to Suppress Tumor Progression

Splicing dysregulation is one of the molecular hallmarks of cancer. However, the underlying molecular mechanisms remain poorly defined. Here we report the splicing factor RBM4 suppresses proliferation and migration of various cancer cells by specifically controlling cancer-related splicing. Particularly, RBM4 regulates Bcl-x splicing to induce apoptosis, and co-expression of Bcl-xL partially reverses the RBM4-mediated tumor suppression. Moreover, RBM4 antagonizes an oncogenic splicing factor, SRSF1, to inhibit mTOR activation. Strikingly, RBM4 expression is dramatically decreased in cancer patients, and RBM4 level is positively correlated with improved survival. In addition to providing mechanistic insights of cancer-related splicing dysregulation, this study establishes RBM4 as a tumor suppressor with therapeutic potentials and clinical values as a prognostic factor

iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions

Transcriptome-wide analysis of protein-RNA interactions predicts the dual splicing effects of TIA proteins, showing that their local enhancing function is associated with diverse distal splicing silencing effects

Intronic splicing enhancers, cognate splicing factors and context-dependent regulation rules

SummaryMost human genes produce multiple splicing isoforms with distinct functions. To systematically understand splicing regulation, we conducted an unbiased screen and identified >100 intronic splicing enhancers (ISEs) that were clustered by sequence similarity into six groups. All ISEs functioned in another cell type and heterologous introns, and their distribution and conservation patterns in different pre-mRNA regions are similar to exonic splicing silencers. Consistently all ISEs inhibited use of splice sites from exonic locations. The putative trans-factors of each ISE group were identified and validated. Five distinct ISE motifs were recognized by hnRNP H and F whose C-terminal domains were sufficient to render context-dependent activities of ISEs. The sixth group was controlled by factors that either activate or suppress splicing. This work provided a comprehensive picture of general ISE activities and provided new models of how a single element can function oppositely depending on its locations and binding factors