hnRNP A1 contacts exon 5 to promote exon 6 inclusion of apoptotic Fas gene

Oh, Hyun Kyung; Lee, Eunkyung; Jang, Ha Na; Lee, Jae‐Hoon; Moon, Heegyum; Sheng, Zhi; Jun, Youngsoo; Loh, Tiing Jen; Cho, Sunghee; Zhou, Jianhua; Green, Michael R.; Zheng, Xuexiu; Shen, Haihong

doi:10.1007/s10495-013-0824-8

Cited by 32 publications

(28 citation statements)

References 35 publications

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“…The changes were expressed as percentages of exon inclusion, using empty vector as a control. (63). Alternatively, the SRSF1 pseudo-RRM also could compete with the proofreading activity of hnRNP A1 at the 3′ splice site of FAS introns 5 (64).…”

Section: Discussionmentioning

confidence: 99%

Isolated pseudo–RNA-recognition motifs of SR proteins can regulate splicing using a noncanonical mode of RNA recognition

Cléry

Sinha

Anczuków

et al. 2013

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

118

166

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Serine/arginine (SR) proteins, one of the major families of alternativesplicing regulators in Eukarya, have two types of RNA-recognition motifs (RRMs): a canonical RRM and a pseudo-RRM. Although pseudo-RRMs are crucial for activity of SR proteins, their mode of action was unknown. By solving the structure of the human SRSF1 pseudo-RRM bound to RNA, we discovered a very unusual and sequence-specific RNA-binding mode that is centered on one α-helix and does not involve the β-sheet surface, which typically mediates RNA binding by RRMs. Remarkably, this mode of binding is conserved in all pseudo-RRMs tested. Furthermore, the isolated pseudo-RRM is sufficient to regulate splicing of about half of the SRSF1 target genes tested, and the bound α-helix is a pivotal element for this function. Our results strongly suggest that SR proteins with a pseudo-RRM frequently regulate splicing by competing with, rather than recruiting, spliceosome components, using solely this unusual RRM.NMR | protein-RNA complex | splicing factor S erine/arginine (SR) proteins are highly conserved in Eukarya and are key regulators of gene expression. These proteins are required for pre-mRNA splicing, regulate alternative splicing events, and play crucial roles in genomic stability, mRNA transcription, nuclear export, nonsense-mediated mRNA decay, and translation (1-5). Several studies have revealed links between these proteins and diseases (6), making the proteins potential therapeutic targets (7). For the last two decades, SR proteins have been studied intensively as regulators of alternative splicing, a mechanism used to modulate the expression of more than 95% of human genes (8, 9). Importantly, it is estimated that 15-50% of human disease-causing mutations affect splicing (10). Alternative splicing consists of the alternative selection of splice sites present within pre-mRNA, leading to different versions of mature mRNAs from a single gene (11). As a result, in some cases, proteins with opposite functions can be generated. In the context of cancer, alternative splicing can generate pro-or antiapoptotic isoforms (12). As an example, alternatively spliced variants of FAS/CD95 can be generated by inclusion or skipping of exon 6, depending on competition between antagonistic splicing factors (13-16). In the absence of Fas exon 6, the apoptotic receptor lacks the transmembrane domain and becomes soluble and antiapoptotic (17).Each SR protein has a modular structure with one or two RNA-recognition motifs (RRMs) at its N-terminal part, followed by a C-terminal arginine-serine-rich (RS) domain containing multiple RS dipeptide repeats. In many cases, SR proteins have been shown to interact with purine-rich exonic splicing enhancers (ESEs) and to promote inclusion of the targeted exon in mRNAs (18). Two main models have been proposed to explain the mode of action of ESE-bound SR proteins in splicing regulation. In the recruitment model SR proteins recruit U1-70K and/or U2AF35 to 5′ and 3′ splice sites, respectively, to promote spliceosome assembly (4)....

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Section: Discussionmentioning

confidence: 99%

Isolated pseudo–RNA-recognition motifs of SR proteins can regulate splicing using a noncanonical mode of RNA recognition

Cléry

Sinha

Anczuków

et al. 2013

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

118

166

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“…RNA binding analysis of SRp20 were performed as described previously [33]. Briefly, chemically synthesized 5’ biotin labeled wild type (UUCUUCAUCC) and mutant (UGAAGCGUCC) RNA were covalently linked with Streptavidin agarose conjugate (Millipore).…”

Section: Methodsmentioning

confidence: 99%

Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8

Jang

Loh

Choi

et al. 2014

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and proapoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRp20 with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRp20 promoted exon 9 skipping. Thus we conclude that SRp20 promotes exon 9 skipping. In order to understand the functional target of SRp20 on caspase-2 premRNA, we performed substitution and deletion mutagenesis on the potential SRp20 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRp20 binding site on exon 8 severely disrupted the effects of SRp20 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRp20 interacts with the potential SRp20 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26 nt RNA from 5’ end of exon 8, a 33 nt RNA from 3’ end of exon 10 and a 2225 nt RNA from intron 9 did not compromise the function of SRp20 on exon 9 splicing. Therefore we conclude that SRp20 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of Caspase-2 pre-mRNA splicing.

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“…Mechanistically, hnRNPA1 has different modes of action, including ( a ) binding to exonic or intronic splicing silencer elements to repress exon inclusion by steric action (31), as is also the case for hrp48 and the Drosophila P element exonic splicing silencer (see below); ( b ) binding of hnRNPA1 to a higher-affinity binding site that promotes cooperative binding and “spreading” hnRNPA1 proteins to adjacent lower-affinity binding sites (140); and ( c ) interaction hnRNPA1 proteins bound to intronic silencer elements on both sides of an alternative exon, resulting in loop formation and exclusion of the exon (143). In some cases, hnRNPA1 can act as a splicing activator (133, 136, 144, 145). …”

Section: Alternative Splicing: Mechanismsmentioning

confidence: 99%

Mechanisms and Regulation of Alternative Pre-mRNA Splicing

Lee

Rio

2015

Annu. Rev. Biochem.

1,037

958

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Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA–protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA–RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions.

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hnRNP A1 contacts exon 5 to promote exon 6 inclusion of apoptotic Fas gene

Cited by 32 publications

References 35 publications

Isolated pseudo–RNA-recognition motifs of SR proteins can regulate splicing using a noncanonical mode of RNA recognition

Isolated pseudo–RNA-recognition motifs of SR proteins can regulate splicing using a noncanonical mode of RNA recognition

Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8

Mechanisms and Regulation of Alternative Pre-mRNA Splicing

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