Mechanisms for U2AF to define 3′ splice sites and regulate alternative splicing in the human genome

Shao, Chunli; Yang, Bo; Wu, Taihu; Huang, Jinfeng; Tang, Peng; Zhou, Yu; Zhou, Jie; Qiu, Jinsong; Jiang, Li; Li, Hairi; Chen, Geng; Sun, Hui; Zhang, Yi; Denise, Alain; Zhang, Dong‐Er; Fu, Xiang‐Dong

doi:10.1038/nsmb.2906

Cited by 162 publications

(222 citation statements)

References 46 publications

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“…A recent report has demonstrated that upstream intronic binding of U2AF 65 interferes with the immediate downstream 3′ splice-site of alternative exon or constitutive exon, causing exon skipping or inclusion (32). We found that intron 6 of SMN1/2 that includes uucuuuuuuuuuuuuuuuuuuuuuugag sequence contains pseudoexon with a potential U2AF 65 binding sequence; thus, according to the report, this sequence interferes with the 3′ splice-site of exon 7, thereby promoting an exon 7 skipping event (Fig.…”

Section: U2af 65 Promotes Its Own Binding Only On the Weaker Py Tractmentioning

confidence: 81%

“…We deduce that the stimulatory effects of U2AF 65 on alternative exon exclusion are induced by the splicing inhibitory effects of U2AF 65 . of alternative or constitutive exons to cause exon skipping or inclusion (32). In the SMN pre-mRNA, it was demonstrated that U2AF 65 interacts more strongly with the SMN1 Py tract than the SMN2 Py tract (33).…”

Section: Significancementioning

confidence: 99%

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Splicing inhibition of U2AF ⁶⁵ leads to alternative exon skipping

Cho

Moon

Loh

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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U2 snRNP auxiliary factor 65 kDa (U2AF 65 ) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF 65 stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (SMN) pre-mRNA. A stronger 5′ splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF 65 . U2AF 65 overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF 65 inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF 65 effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF 65 even inhibits general splicing of adenovirus major late (Ad ML) or β-globin pre-mRNA. Thus, we conclude that U2AF 65 possesses a splicing Inhibitory function that leads to alternative exon skipping.U2AF 65 | pre-mRNA splicing | splicing inhibition | exon exclusion | SMN P re-mRNA splicing is a process in which noncoding intron sequences are removed and exon sequences are then ligated together (1, 2). Pre-mRNA splicing is carried out by spliceosome, a large RNA-protein complex that contains five small nuclear ribonucleoproteins (U snRNPs) and more than 100 additional proteins (3). Pre-mRNA splicing occurs in the consensus sequences at the 5′ splice-site, 3′ splice-site, and branch point that are necessary for splicing. The sequence between 3′ AG dinucleotide and branch point is the polypyrimidine (Py) tract that directs spliceosome assembly on the 3′ splice-site. Alternative splicing provides an important regulatory mechanism in higher eukaryotes for multiple proteins produced from a single gene (4, 5).The U2 snRNP auxiliary factor 65 kDa (U2AF 65 ) exists as a heterodimer with U2AF 35 (6). U2AF 65 contains three C-terminal RNA recognition motifs (RRMs) and an N-terminal arginine/ serine-rich (RS) domain (7,8). Using U2AF 65 depletion/adding back technology with in vitro HeLa nuclear extract, it was demonstrated that U2AF 65 is an essential splicing factor (9). Whereas U2AF 65 binds to Py tract to promote prespliceosome assembly and branchpoint/U2 snRNA base pairing, U2AF 35 plays a role in the 3′ splice-site (10, 11). As U2AF 65 prefers high C/U-rich sequences in the Py tract, a stronger interaction between U2AF 65 and Py tract promotes prespliceosome assembly (12). U2AF 65 is also essential in vertebrate development (13,14). Its expression level is related to myotonic dystrophy, cystic fibrosis, and cancers (15, 16).Proximal spinal muscular atrophy (SMA) is an autosomal recessive genetic disease (17) and a leading cause of infant mortality. The motor neurons in the anterior horn of spinal cord are severely damaged in patients with type 1 SMA, usually leading to death before age 2 y as a result of a lack of respiratory support (18,19). In patients with SMA, the SMN1 gene is deleted or mutated, whereas the SMN2 gene, a duplicate of the SMN1 gene, is included (20). SMN2 genomic DNA...

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Section: U2af 65 Promotes Its Own Binding Only On the Weaker Py Tractmentioning

confidence: 81%

Section: Significancementioning

confidence: 99%

Splicing inhibition of U2AF ⁶⁵ leads to alternative exon skipping

Cho

Moon

Loh

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…4). Considering that U2AF1 mutations are reported in B10% of patients with MDS and associated with partial functional impairment in regulated splicing 2,19 , we tested two MDS samples each harbouring one of the two U2AF1 hotspot mutations, S34F and Q157P. Neither of these MDS samples presented any increase of the AG 0 /AG index of DPH5, demonstrating that U2AF1 and SF3B1 hotspot mutations do not lead to the same aberrant splicing phenotype (Fig.…”

Section: Sf3b1 Mutations Promote Upstream Alternative Acceptorsmentioning

confidence: 99%

Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage

Alsafadi¹,

Houy²,

Battistella³

et al. 2016

European Journal of Cancer

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Hotspot mutations in the spliceosome gene SF3B1 are reported in B20% of uveal melanomas. SF3B1 is involved in 3 0 -splice site (3 0 ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear. Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1 R625/K666 mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3 0 ss. Modelling the differential junctions in SF3B1 WT and SF3B1 R625/K666 cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3'ss-sequence context. SF3B1 WT knockdown or overexpression do not reproduce the SF3B1 R625/K666 splice pattern, qualifying SF3B1 R625/K666 as change-of-function mutants. Mutagenesis of predicted branchpoints reveals that the SF3B1 R625/K666 -promoted splice pattern is a direct result of alternative branchpoint usage. Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease.

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“…The insertion was identical to the U2snRNP binding region (20), and this finding may provide insight into why the ratio of FLIP isoforms is skewed toward cFLIP R in MSM mice. Indeed, genomewide RNA sequencing revealed a relative abundance of the long FLIP transcript in MSM mice and significantly more FLIP R mRNA in B6 mice, thus suggesting that splicing of FLIP in MSM/Ms is preferentially skewed toward spliced FLIP L mRNA.…”

Section: Significancementioning

confidence: 92%

“…In MSM/Ms mice, there is also the presence of a U2AF putative binding site in close proximity to the introduced U2 binding site (Fig. S2)-suggesting that, compared with B6, MSM/Ms may recruit the spliceosome complex more efficiently in this region, either leading to more efficient splicing of the intronic region between exons 5 and 6-producing more cFLIP L transcript-or creating an exon-skipping event of Exon 6 (20) leading to an ineffective transcript, or possibly making it physically difficult for the transcription of cFLIP R , either way decreasing the total amount of cFLIP R transcript that is produced. Although the 21-bp insertion is not conserved in humans, human data from the 1000 Genomes Project showed that there are SNPs within the 3′ UTR of the terminal exon for cFLIR R that may alter the binding sequence for U2 as well as for U2AF (Fig.…”

Section: Msm Micementioning

confidence: 99%

Balance between short and long isoforms of cFLIP regulates Fas-mediated apoptosis in vivo

Ram

Ilyukha

Volkova

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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cFLIP, an inhibitor of apoptosis, is a crucial regulator of cellular death by apoptosis and necroptosis; its importance in development is exemplified by the embryonic lethality in cFLIP-deficient animals. A homolog of caspase 8 (CASP8), cFLIP exists in two main isoforms: cFLIP L (long) and cFLIP R (short). Although both splice variants regulate death receptor (DR)-induced apoptosis by CASP8, the specific role of each isoform is poorly understood. Here, we report a previously unidentified model of resistance to Fas receptor-mediated liver failure in the wild-derived MSM strain, compared with susceptibility in C57BL/6 (B6) mice. Linkage analysis in F2 intercross (B6 x MSM) progeny identified several MSM loci controlling resistance to Fas-mediated death, including the caspase 8-and FADD-like apoptosis regulator (Cflar) locus encoding cFLIP. Furthermore, we identified a 21-bp insertion in the 3′ UTR of the fifth exon of Cflar in MSM that influences differential splicing of cFLIP mRNA. Intriguingly, we observed that MSM liver cells predominantly express the FLIP L variant, in contrast to B6 liver cells, which have higher levels of cFLIP R. In keeping with this finding, genome-wide RNA sequencing revealed a relative abundance of FLIP L transcripts in MSM hepatocytes whereas B6 liver cells had significantly more FLIP R mRNA. Importantly, we show that, in the MSM liver, CASP8 is present exclusively as its cleaved p43 product, bound to cFLIP L . Because of partial enzymatic activity of the heterodimer, it might prevent necroptosis. On the other hand, it prevents cleavage of CASP8 to p10/20 necessary for cleavage of caspase 3 and, thus, apoptosis induction. Therefore, MSM hepatocytes are predisposed for protection from DR-mediated cell death.T he Fas receptor [also called cluster of differentiation 95 (CD95), APO-1, or TNFRSF6] is a death receptor family member constitutively expressed by most tissues, including the liver (1), where ligation of ubiquitously expressed CD95 leads to potentially lethal hepatitis and liver failure. Although CD95L (Fas ligand) is the only known physiological ligand of CD95 (2), the agonistic antibody Jo2 has been used extensively to ligate CD95 and model CD95-mediated hepatotoxicity and mortality in mice (3). In contrast to the ability of tumor necrosis factor receptor (TNFR)-mediated signaling to lead to profound inflammatory responses in addition to cell death (4), CD95 is predominantly used in apoptosis and necrosis and therefore engages a limited number of downstream components (5). Specifically, ligand binding induces CD95 oligomerization and binding of Fas-associated death domain (FADD) via its death domain (DD), which then recruits caspase 8 (CASP8) via a death effector domain (DED), forming the death-inducing signaling complex (DISK) comprising receptor interacting protein kinase 1 (RIP1), FADD, and CASP8 (6).Interactions of caspase 8 (CASP8) with its enzymatically inactive homolog cFLIP L further complicate the regulation of CD95-mediated signaling (7): CASP8 forms partially enzymatic...

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Mechanisms for U2AF to define 3′ splice sites and regulate alternative splicing in the human genome

Cited by 162 publications

References 46 publications

Splicing inhibition of U2AF ⁶⁵ leads to alternative exon skipping

Splicing inhibition of U2AF ⁶⁵ leads to alternative exon skipping

Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage

Balance between short and long isoforms of cFLIP regulates Fas-mediated apoptosis in vivo

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Mechanisms for U2AF to define 3′ splice sites and regulate alternative splicing in the human genome

Cited by 162 publications

References 46 publications

Splicing inhibition of U2AF 65 leads to alternative exon skipping

Splicing inhibition of U2AF 65 leads to alternative exon skipping

Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage

Balance between short and long isoforms of cFLIP regulates Fas-mediated apoptosis in vivo

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Splicing inhibition of U2AF ⁶⁵ leads to alternative exon skipping

Splicing inhibition of U2AF ⁶⁵ leads to alternative exon skipping