The PTB interacting protein raver1 regulates alpha-tropomyosin alternative splicing

Regulated switching of the mutually exclusive exons 2 and 3 of alpha-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. Here we show that raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induced very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor.     

RBM10 regulates alternative splicing

RBM10, originally called S1-1, is a nuclear RNA-binding protein with domains characteristic of RNA processing proteins. It has been reported that RBM10 constitutes spliceosome complexes and that RBM5, a close homologue of RBM10, regulates alternative splicing of apoptosis-related genes, Fas and cFLIP. In this study, we examined whether RBM10 has a regulatory function in splicing similar to RBM5, and determined that it indeed regulates alternative splicing of Fas and Bcl-x genes. RBM10 promotes exon skipping of Fas pre-mRNA as well as selection of an internal 5′-splice site in Bcl-x pre-mRNA. We propose a consensus RBM10-binding sequence at 5′-splice sites of target exons and a mechanistic model of RBM10 action in the alternative splicing.     

PTB regulates the processing of a 3'-terminal exon by repressing both splicing and polyadenylation

The polypyrimidine tract binding protein (PTB) has been described as a global repressor of regulated exons. To investigate PTB functions in a physiological context, we used a combination of morpholino-mediated knockdown and transgenic overexpression strategies in Xenopus laevis embryos. We show that embryonic endoderm and skin deficient in PTB displayed a switch of the alpha-tropomyosin pre-mRNA 3' end processing to the somite-specific pattern that results from the utilization of an upstream 3'-terminal exon designed exon 9A9'. Conversely, somitic targeted overexpression of PTB resulted in the repression of the somite-specific exon 9A9' and a switch towards the nonmuscle pattern. These results validate PTB as a key physiological regulator of the 3' end processing of the alpha-tropomyosin pre-mRNA. Moreover, using a minigene strategy in the Xenopus oocyte, we show that in addition to repressing the splicing of exon 9A9', PTB regulates the cleavage/polyadenylation of this 3'-terminal exon.     

Chromatin-mediated alternative splicing regulates cocaine-reward behavior

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Modification of Akt by SUMO conjugation regulates alternative splicing and cell cycle

Akt/PKB is a key signaling molecule in higher eukaryotes and a crucial protein kinase in human health and disease. Phosphorylation, acetylation, and ubiquitylation have been reported as important regulatory post-translational modifications of this kinase. We describe here that Akt is modified by SUMO conjugation, and show that lysine residues 276 and 301 are the major SUMO attachment sites within this protein. We found that phosphorylation and SUMOylation of Akt appear as independent events. However, decreasing Akt SUMOylation levels severely affects the role of this kinase as a regulator of fibronectin and Bcl-x alternative splicing. Moreover, we observed that the Akt mutant (Akt E17K) found in several human tumors displays increased levels of SUMOylation and also an enhanced capacity to regulate fibronectin splicing patterns. This splicing regulatory activity is completely abolished by decreasing Akt E17K SUMO conjugation levels. Additionally, we found that SUMOylation controls Akt regulatory function at G?/S transition during cell cycle progression. These findings reveal SUMO conjugation as a novel level of regulation for Akt activity, opening new areas of exploration related to the molecular mechanisms involved in the diverse cellular functions of this kinase.     

Mammary epithelial-mesenchymal interaction regulates fibronectin alternative splicing via phosphatidylinositol 3-kinase

The way alternative splicing is regulated within tissues is not understood. A relevant model of this process is provided by fibronectin, an important extracellular matrix protein that plays a key role in cell adhesion and migration and contains three alternatively spliced regions known as EDI, EDII, and IIICS. We used a cell culture system to simulate mammary epithelial-stromal communication, a process that is crucial for patterning and function of the mammary gland, and studied the effects of extracellular signals on the regulation of fibronectin pre-mRNA alternative splicing. We found that soluble factors from a mammary mesenchymal cell-conditioned medium, as well as the growth factors HGF/SF (hepatocyte growth factor/scatter factor), KGF (keratinocyte growth factor), and aFGF (acidic fibroblast growth factor), stimulate EDI and IIICS but not EDII inclusion into fibronectin mRNA in the mammary epithelial cell line SCp2, favoring fibronectin isoforms associated with proliferation, migration, and tissue remodeling. We explored the signaling pathways involved in this regulation and found that the mammary mesenchymal cell-conditioned medium and HGF/SF act through a phosphatidylinositol 3-kinase-dependent cascade to alter fibronectin alternative splicing. This splicing regulation is independent from promoter structure and de novo protein synthesis but does require two exonic elements within EDI. These results shed light on how extracellular stimuli are converted into changes in splicing patterns.     

Rbm20 regulates titin alternative splicing as a splicing repressor

Titin, a sarcomeric protein expressed primarily in striated muscles, is responsible for maintaining the structure and biomechanical properties of muscle cells. Cardiac titin undergoes developmental size reduction from 3.7 megadaltons in neonates to primarily 2.97 megadaltons in the adult. This size reduction results from gradually increased exon skipping between exons 50 and 219 of titin mRNA. Our previous study reported that Rbm20 is the splicing factor responsible for this process. In this work, we investigated its molecular mechanism. We demonstrate that Rbm20 mediates exon skipping by binding to titin pre-mRNA to repress the splicing of some regions; the exons/introns in these Rbm20-repressed regions are ultimately skipped. Rbm20 was also found to mediate intron retention and exon shuffling. The two Rbm20 speckles found in nuclei from muscle tissues were identified as aggregates of Rbm20 protein on the partially processed titin pre-mRNAs. Cooperative repression and alternative 3′ splice site selection were found to be used by Rbm20 to skip different subsets of titin exons, and the splicing pathway selected depended on the ratio of Rbm20 to other splicing factors that vary with tissue type and developmental age.     

A splicing silencer that regulates smooth muscle specific alternative splicing is active in multiple cell types

Alternative splicing of α-tropomyosin (α-TM) involves mutually exclusive selection of exons 2 and 3. Selection of exon 2 in smooth muscle (SM) cells is due to inhibition of exon 3, which requires both binding sites for polypyrimidine tract-binding protein as well as UGC (or CUG) repeat elements on both sides of exon 3. Point mutations or substitutions of the UGC-containing upstream regulatory element (URE) with other UGC elements disrupted the α-TM splicing pattern in transfected cells. Multimerisation of the URE caused enhanced exon skipping in SM and various non-SM cells. In the presence of multiple UREs the degree of splicing regulation was decreased due to the high levels of exon skipping in non-SM cell lines. These results suggest that the URE is not an intrinsically SM- specific element, but that its functional strength is fine tuned to exploit differences in the activities of regulatory factors between SM and other cell types. Co-transfection of tropomyosin reporters with members of the CUG-binding protein family, which are candidate URE-binding proteins, indicated that these factors do not mediate repression of tropomyosin exon 3.     

A bidirectional antagonism between aPKC and Yurt regulates epithelial cell polarity

During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells.     

Differential alternative splicing activity of isoforms of polypyrimidine tract binding protein (PTB).

Polypyrimidine tract binding protein (PTB) is an RNA-binding protein that regulates splicing by repressing specific splicing events. It also has roles in 3'-end processing, internal initiation of translation, and RNA localization. PTB exists in three alternatively spliced isoforms, PTB1, PTB2, and PTB4, which differ by the insertion of 19 or 26 amino acids, respectively, between the second and third RNA recognition motif domains. Here we show that the PTB isoforms have distinct activities upon alpha-tropomyosin (TM) alternative splicing. PTB1 reduced the repression of TM exon 3 in transfected smooth muscle cells, whereas PTB4 enhanced TM exon 3 skipping in vivo and in vitro. PTB2 had an intermediate effect. The PTB4 > PTB2 > PTB1 repressive hierarchy was observed in all in vivo and in vitro assays with TM, but the isoforms were equally active in inducing skipping of alpha-actinin exons and showed the opposite hierarchy of activity when tested for activation of IRES-driven translation. These findings establish that the ratio of PTB isoforms could form part of a cellular code that in turn controls the splicing of various other pre-mRNAs.