Interaction between the RNA binding domains of Ser-Arg splicing factor 1 and U1-70K snRNP protein determines early spliceosome assembly

Cho, Suhyung; Hoang, Amy; Sinha, Rahul; Zhong, Xiang-Yang; Fu, Xiang‐Dong; Krainer, Adrian R.; Ghosh, Gourisankar

doi:10.1073/pnas.1017700108

Cited by 196 publications

(236 citation statements)

References 32 publications

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“…Another prominent FUS target is the conserved intron of the gene coding for the small nuclear ribonucleoprotein 70 kDa polypeptide (snRNP70, U1-70K) (Fig. 3A), which is an essential component of the U1 snRNP that is required for recognition of the 5 ′ splice site during spliceosome assembly (Kohtz et al 1994;Cho et al 2011).…”

Section: Resultsmentioning

confidence: 99%

FUS regulates genes coding for RNA-binding proteins in neurons by binding to their highly conserved introns

Nakaya¹,

Alexiou²,

Maragkakis³

et al. 2013

RNA

128

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Dominant mutations and mislocalization or aggregation of Fused in Sarcoma (FUS), an RNA-binding protein (RBP), cause neuronal degeneration in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), two incurable neurological diseases. However, the function of FUS in neurons is not well understood. To uncover the impact of FUS in the neuronal transcriptome, we used high-throughput sequencing of immunoprecipitated and cross-linked RNA (HITS-CLIP) of FUS in human brains and mouse neurons differentiated from embryonic stem cells, coupled with RNA-seq and FUS knockdowns. We report conserved neuronal RNA targets and networks that are regulated by FUS. We find that FUS regulates splicing of genes coding for RBPs by binding to their highly conserved introns. Our findings have important implications for understanding the impact of FUS in neurodegenerative diseases and suggest that perturbations of FUS can impact the neuronal transcriptome via perturbations of RBP transcripts.

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Section: Resultsmentioning

confidence: 99%

FUS regulates genes coding for RNA-binding proteins in neurons by binding to their highly conserved introns

Nakaya¹,

Alexiou²,

Maragkakis³

et al. 2013

RNA

128

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“…additional splicing factors (31). In addition, phosphorylation of the RS region has been shown to stabilize its structure and decrease its conformational entropy (32).…”

Section: Discussionmentioning

confidence: 99%

Molecular mechanisms for the regulation of histone mRNA stem-loop–binding protein by phosphorylation

Zhang

Tan

DeRose

et al. 2014

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

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Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop-binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA.X-ray crystallography | NMR | intrinsically disordered protein H istone synthesis increases at the beginning of S-phase to package newly replicated DNA with histone proteins, but synthesis must be shut down rapidly and histone mRNA degraded at the end of DNA replication because of the toxicity of surplus histone proteins (1, 2). This cyclic demand for histones requires strict regulation, which is achieved mainly by controlling the synthesis and degradation of histone mRNA (3). Replication-dependent histone mRNAs are the only known cellular mRNAs that are not polyadenylated and instead end with a conserved stem loop (4). Histone mRNAs are generated from longer histone pre-mRNAs as a result of an endonucleolytic cleavage between the stem loop and a purine-rich downstream sequence termed the "histone downstream element" (HDE) (5).Stem-loop-binding protein (SLBP), also known as "hairpinbinding protein" (6), binds to the histone mRNA stem loop, and U7 small nuclear ribonucleoprotein binds to the HDE (7). Other factors, including the endonuclease CPSF-73, are involved in both polyadenylation and histone mRNA 3′-end processing (8-11). In mammalian nuclear extracts, SLBP is not absolutely required for the biochemical reaction of processing (12). In contrast, cleavage of histone pre-mRNA in Drosophila cells and nuclear extracts requires the binding of SLBP to the stem loop (10, 13).The minimal histone mRNA processing domain of Drosophila SLBP contains a 72-aa RNA-binding domain (RBD) unique to SLBPs and an 18-aa C-terminal region (Fig. 1A) (14). This RNAprocessing domain (RPD) is necessary and sufficient for histone mRNA 3′-end processing in vitro (15). The RBDs of human SLBP (hSLBP) and Drosophila SLBP (dSLBP) are phosphorylated at a Thr residue in a conserved TPNK motif (16,17). The recent crystal structure of hSLBP RBD in complex with histone mRNA stem loop and 3′ hExo, a 3′-5′ exonuclease required for histone mRNA degradation, provided the first molecular insights into the architecture of this complex, and revealed how the hSLBP RBD forms a new ...

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“…Because of the relatively small size of the domain, it is unlikely that it would act on splicing by recruiting spliceosome components. Indeed, it was reported recently that both RRMs of SRSF1 are required to interact with U1-70K (32). A more tempting hypothesis could be that the pseudo-RRM regulates splicing by competing for RNA binding with one or several splicing factors.…”

Section: Srsf1 (Sf2/asf)mentioning

confidence: 99%

“…However, in some cases, this domain also can be dispensable for constitutive and enhancer-dependent splicing (31), indicating a key function of the RRMs in this process. Although previously it was shown that the RRMs of SRSF1 can contact the spliceosomal component U1-70K directly (32), the main role of the RRMs seems to be to provide RNAbinding specificity and to dictate the position of SR proteins on pre-mRNAs (18).…”

mentioning

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.

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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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Interaction between the RNA binding domains of Ser-Arg splicing factor 1 and U1-70K snRNP protein determines early spliceosome assembly

Cited by 196 publications

References 32 publications

FUS regulates genes coding for RNA-binding proteins in neurons by binding to their highly conserved introns

FUS regulates genes coding for RNA-binding proteins in neurons by binding to their highly conserved introns

Molecular mechanisms for the regulation of histone mRNA stem-loop–binding protein by phosphorylation

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

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