SFmap: a web server for motif analysis and prediction of splicing factor binding sites

Paz, Inbal; Akerman, Martin; Dror, Iris; Kosti, Idit; Mandel-Gutfreund, Yael

doi:10.1093/nar/gkq444

Cited by 93 publications

(86 citation statements)

References 22 publications

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“…predicted within ESS1 and ESS2 using Human Splicing Finder, SFmap, and Splicing Rainbow (52,54,55). The interactions between SRSF1 and ESS1 and between PTBP1 and ESS1 were observed in the previous pulldown assays (Fig.…”

Section: Resultsmentioning

confidence: 66%

Characterization of the Regulation of CD46 RNA Alternative Splicing

Tang

Luo

et al. 2016

Journal of Biological Chemistry

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Here we present a detailed analysis of the alternative splicing regulation of human CD46, which generates different isoforms with distinct functions. CD46 is a ubiquitous membrane protein that protects host cells from complement and plays other roles in immunity, autophagy, and cell adhesion. CD46 deficiency causes an autoimmune disorder, and this protein is also involved in pathogen infection and cancer. Before this study, the mechanisms of CD46 alternative splicing remained unexplored even though dysregulation of this process has been associated with autoimmune diseases. We proved that the 5 splice sites of CD46 cassette exons 7 and 8 encoding extracellular domains are defined by noncanonical mechanisms of base pairing to U1 small nuclear RNA. Next we characterized the regulation of CD46 cassette exon 13, whose inclusion or skipping generates different cytoplasmic tails with distinct functions. Using splicing minigenes, we identified multiple exonic and intronic splicing enhancers and silencers that regulate exon 13 inclusion via trans-acting splicing factors like PTBP1 and TIAL1. Interestingly, a common splicing activator such as SRSF1 appears to repress CD46 exon 13 inclusion. We also report that expression of CD46 mRNA isoforms is further regulated by non-sense-mediated mRNA decay and transcription speed. Finally, we successfully manipulated CD46 exon 13 inclusion using antisense oligonucleotides, opening up opportunities for functional studies of the isoforms as well as for therapeutics for autoimmune diseases. This study provides insight into CD46 alternative splicing regulation with implications for its function in the immune system and for genetic disease.CD46 is a ubiquitously expressed type I membrane-bound protein with a main function of protecting human host cells from complement (1). CD46 exerts such function by acting as a cofactor for Factor I-mediated cleavage of C3b and C4b (1). In addition, CD46 acts as a co-stimulator of T and other immune cells (2-6) and plays important roles in epithelial and sperm cells (7-11). Human CD46 deficiency results in a genetic disorder called atypical hemolytic uremic syndrome (12), its overexpression is used by cancer cells to evade the immune system (13,14), and its expression is often altered in autoimmune disorders like multiple sclerosis, rheumatoid arthritis, and asthma (15)(16)(17)(18)(19). Finally, CD46 is used as an entry receptor for several bacteria and viruses (20 -23). All of these studies underline the multiple connections between CD46 and human disease and the relevance of studying the regulation of CD46 expression.The joining of exons in different combinations, by means of alternative splicing, gives rise to multiple mRNA and protein isoforms (24). The human CD46 gene consists of 14 exons, and four of them are alternatively spliced to generate several CD46 isoforms (25). These four exons fall in the category of cassette exons, which can be either included or skipped from the mature messenger RNA (25-27). Cassette exons 7, 8, and 9 encode the extra...

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Section: Resultsmentioning

confidence: 66%

Characterization of the Regulation of CD46 RNA Alternative Splicing

Tang

Luo

et al. 2016

Journal of Biological Chemistry

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“…The samples were divided in two independent sets, one for discovery of AS events and one for validation. The discovery set consisted of 24 RNA Extraction-Sample tissue composition was evaluated by hematoxylin and eosin staining, and macroscopic trimming was used when necessary to enrich the fraction of tumor cells. A minimum of 60% neoplastic cells was required before total RNA was purified from serial cryo-sections.…”

Section: Methodsmentioning

confidence: 99%

“…Splicing Factor Binding Site Predictions-To predict splicing factor binding sites, we used the SFmap software with default settings (24). Briefly, SFmap implements the COS(WR) algorithm (25), which computes similarity scores for a given regulatory motif on the basis of information derived from its sequence environment and its evolutionary conservation.…”

Section: Methodsmentioning

confidence: 99%

Alternative Splicing of SLC39A14 in Colorectal Cancer is Regulated by the Wnt Pathway

Thorsen

Mansilla

Schepeler

et al. 2011

Molecular & Cellular Proteomics

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Alternative splicing is a crucial step in the generation of protein diversity and its misregulation is observed in many human cancer types. By analyzing 143 colorectal samples using exon arrays, SLC39A14, a divalent cation transporter, was identified as being aberrantly spliced in tumor samples. SLC39A14 contains two mutually exclusive exons 4A and 4B and the exon 4A/4B ratio was significantly altered in adenomas (p ‫؍‬ 3.6 ؋ 10 ؊10 ) and cancers (p ‫؍‬ 9.4 ؋ 10 ؊11 ), independent of microsatellite stability status. The findings were validated in independent exon array data sets and by quantitative real-time reverse-transcription PCR (qRT-PCR). Aberrant Wnt signaling is a hallmark of colorectal tumorigenesis and is characterized by nuclear ␤-catenin. Experimental inactivation of Wnt signaling in DLD1 and Ls174T cells by knockdown of ␤-catenin or overexpression of dominant negative TCFs (TCF1 and TCF4) altered the 4A/4B ratio, indicating that SLC39A14 splicing is regulated by the Wnt pathway. An altered 4A/4B ratio was also observed in gastric and lung cancer where Wnt signaling is also known to be aberrantly activated. The splicing factor SRSF1 and its regulator, the kinase SRPK1, were found to be deregulated upon Wnt inactivation in colorectal carcinoma cells. SRPK1 was also found up-regulated in both adenoma samples (p ‫؍‬ 1.5 ؋ 10 ؊5 ) and cancer samples (p ‫؍‬ 5 ؋ 10 ؊4 ). In silico splicing factor binding analysis predicted SRSF1 to bind predominantly to the cancer associated exon 4B, hence, it was hypothesized that SRPK1 activates SRSF1 through phosphorylation, followed by SRSF1 binding to exon 4B and regulation of SLC39A14 splicing. Indeed, siRNA-mediated knockdown of SRPK1 and SRSF1 in DLD1 and SW480 colorectal cancer cells led to a change in the 4A/4B isoform ratio, supporting a role of these factors in the regulation of SLC39A14 splicing. In conclusion, alternative splicing of SLC39A14 was identified in colorectal tumors and found to be regulated by the Wnt pathway, most likely through regulation of SRPK1 and SRSF1. Molecular &

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“…To seek for an alternative to in cellulo and in vitro analyses, we analyzed hidden scenarios of the branch point [48], the 3' splice site [49], and the 5' splice site [50] by exploiting in silico analysis methods. Tools to predict RNA-binding proteins for specific cis-elements are also available online [29,42], and further development of such tools will facilitate identification of splicing mutations and their underlying mechanisms.…”

Section: Resultsmentioning

confidence: 99%

“…Representative tools to predict splicing mutations at the 5' and 3' splice sites and the branch point are Human Splicing Finder [29], SD score [30], MaxEntScan [31], Analyzer Splice Tool (AST) [31,32], DBASS5 [33], DBASS3 [34]. Similarly, representative tools to predict splicing cis-elements and their associated RNAbinding proteins are SpliceAid [35], SpliceAid2 [36], ESEfinder [37,38], Rescue-ESE [39,40], FAS-ESS [41], and SF map [42].The details of these tools as well as the other tools and databases are discussed in detail in our recent review article [43]. Among these tools, we developed the SD score algorithm to predict the splicing effect of mutations affecting three nucleotides at the 3' end of an exon and six nucleotides at the 5' end of an intron [30].…”

Section: In Silico Programs To Analyze Splicing Aberrationsmentioning

confidence: 99%

Splicing Aberrations in Congenital Myasthenic Syndromes

Ohno¹

2015

JIG

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SFmap: a web server for motif analysis and prediction of splicing factor binding sites

Cited by 93 publications

References 22 publications

Characterization of the Regulation of CD46 RNA Alternative Splicing

Characterization of the Regulation of CD46 RNA Alternative Splicing

Alternative Splicing of SLC39A14 in Colorectal Cancer is Regulated by the Wnt Pathway

Splicing Aberrations in Congenital Myasthenic Syndromes

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