The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes

Änkö, Minna‐Liisa; Müller-McNicoll, Michaela; Brandl, Holger; Curk, Tomaž; Gorup, Črtomir; Henry, Ian; Ule, Jernej; Neugebauer, Karla M.

doi:10.1186/gb-2012-13-3-r17

Cited by 240 publications

(239 citation statements)

References 51 publications

(96 reference statements)

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“…However, we unexpectedly found that both SRSF1 and SRSF2 increase CD44 exon v5 exclusion in a concentration-dependent manner, suggesting that SRSF1 is not always specific for exon inclusion but may also be involved in exon exclusion. In agreement with this, a recent genome-wide analysis has revealed that SR proteins (SRSF1-4) cause similar numbers of exon inclusion and exon inclusion events (31)(32)(33). This finding suggests that the combinational contents of the splicing machinery complex are more important for alternative splicing than the specific mRNA interaction tendency of a single SR protein.…”

Section: Discussionsupporting

confidence: 66%

Nuclear Speckle-related Protein 70 Binds to Serine/Arginine-rich Splicing Factors 1 and 2 via an Arginine/Serine-like Region and Counteracts Their Alternative Splicing Activity

Kim

Choi

et al. 2016

Journal of Biological Chemistry

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Nuclear speckles are subnuclear storage sites containing pre-mRNA splicing machinery. Proteins assembled in nuclear speckles are known to modulate transcription and pre-mRNA processing. We have previously identified nuclear speckle-related protein 70 (NSrp70) as a novel serine/arginine (SR)-related protein that co-localizes with classical SR proteins such as serine/arginine-rich splicing factor 1 (SRSF1 or ASF/SF2) and SRSF2 (SC35). NSrp70 mediates alternative splice site selection, targeting several pre-mRNAs, including CD44 exon v5. Here we demonstrated that NSrp70 interacts physically with two SR proteins, SRSF1 and SRSF2, and reverses their splicing activity in terms of CD44 exon v5 as exon exclusion. The NSrp70 RS-like region was subdivided into three areas. Deletion of the first arginine/serine-rich-like region (RS1) completely abrogated binding to the SR proteins and to target mRNA and also failed to induce splicing of CD44 exon v5, suggesting that RS1 is critical for NSrp70 functioning. Interestingly, RS1 deletion also resulted in the loss of NSrp70 and SR protein speckle positioning, implying a potential scaffolding role for NSrp70 in nuclear speckles. NSrp70 contains an N-terminal coiled-coil domain that is critical not only for self-oligomerization but also for splicing activity. Consistently, deletion of the coiled-coil domain resulted in indefinite formation of nuclear speckles. Collectively, these results demonstrate that NSrp70 acts as a new molecular counterpart for alternative splicing of target RNA, counteracting SRSF1 and SRSF2 splicing activity.The mammalian cell nucleus contains non-membranous subnuclear bodies such as the nucleolus, Cajal bodies, promyelocytic leukemia bodies, and nuclear speckles. These bodies are generally classified by the presence of a distinct group of proteins and RNAs within them (1, 2). Nuclear speckles are irregular, punctuate structures that vary in size and shape at the immunofluorescence microscope level and are located in interchromatin regions of the nucleoplasm of mammalian cells (3,4). Several reports have demonstrated that nuclear speckles serve as storage/assembly sites for pre-mRNA splicing machinery and, therefore, elaborately regulate gene expression (2). Nuclear speckles are known to be functional centers that spatially organize transcriptional and posttranscriptional mechanisms of gene regulation. RNA-binding proteins involved in transcription and pre-mRNA processing, such as small nuclear ribonucleoprotein and arginine/serine-rich family factors (SR protein, SR-related protein), are associated with nuclear speckles (5, 6). Remarkably, two classical SR proteins, SRSF1 and SRSF2, are well known components of nuclear speckles. These SR proteins modulate both constitutive and alternative splicing by assembly of the mature spliceosome (7,8).In a previous report, we identified a novel SR-related protein, nuclear speckle-related protein 70 (NSrp70), that modulates alternative splicing site selection of several mRNAs in vivo, including Tra2␤1 v2, Fas v6, and...

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Section: Discussionsupporting

confidence: 66%

Nuclear Speckle-related Protein 70 Binds to Serine/Arginine-rich Splicing Factors 1 and 2 via an Arginine/Serine-like Region and Counteracts Their Alternative Splicing Activity

Kim

Choi

et al. 2016

Journal of Biological Chemistry

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“…1E). In contrast, motif 2, which is similar to the consensus for a strong exonic splicing enhancer sequence, and the binding site of several serine/arginine-rich (SR) proteins (Tacke et al 1998;Sanford et al 2008Sanford et al , 2009Änkö et al 2012;Pandit et al 2013; for review, see Long and Caceres 2009;Howard and Sanford 2015), was preferentially found in exons (Fig. 1E).…”

Section: Iclip Annotation and Binding Motifsmentioning

confidence: 95%

The RNA-binding profile of Acinus, a peripheral component of the exon junction complex, reveals its role in splicing regulation

Rodor

Pan

Blencowe

et al. 2016

RNA

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Acinus (apoptotic chromatin condensation inducer in the nucleus) is an RNA-binding protein (RBP) originally identified for its role in apoptosis. It was later found to be an auxiliary component of the exon junction complex (EJC), which is deposited at exon junctions as a consequence of pre-mRNA splicing. To uncover the cellular functions of Acinus and investigate its role in splicing, we mapped its endogenous RNA targets using the cross-linking immunoprecipitation protocol (iCLIP). We observed that Acinus binds to pre-mRNAs, associating specifically to a subset of suboptimal introns, but also to spliced mRNAs. We also confirmed the presence of Acinus as a peripheral factor of the EJC. RNA-seq was used to investigate changes in gene expression and alternative splicing following siRNA-mediated depletion of Acinus in HeLa cells. This analysis revealed that Acinus is preferentially required for the inclusion of specific alternative cassette exons and also controls the faithful splicing of a subset of introns. Moreover, a large number of splicing changes can be related to Acinus binding, suggesting a direct role of Acinus in exon and intron definition. In particular, Acinus regulates the splicing of DFFA/ICAD transcript, a major regulator of DNA fragmentation. Globally, the genome-wide identification of RNA targets of Acinus revealed its role in splicing regulation as well as its involvement in other cellular pathways, including cell cycle progression. Altogether, this study uncovers new cellular functions of an RBP transiently associated with the EJC.

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“…Another, perhaps more likely, function involves the binding of additional splicing factors. Several recent studies suggest that cross-regulation by multiple splicing factors is a common mechanism for regulating AS-NMD in splicing factor transcripts (Dreumont et al 2010;Änkö et al 2012;Huelga et al 2012). It is likely that the conserved region within the U11-48K transcript similarly provides yet additional binding sites for other regulatory factors, such as SR proteins and other hnRNP proteins.…”

Section: Discussionmentioning

confidence: 99%

“…This arrangement leads to a self-regulating negative feedback loop that maintains constant protein levels. However, AS-NMD events in the transcripts of splicing factors are also subject to regulation by other splicing factors, giving rise to finely tuned regulatory networks (Änkö et al 2012;Huelga et al 2012). …”

Section: Introductionmentioning

confidence: 99%

HnRNPH1/H2, U1 snRNP, and U11 snRNP cooperate to regulate the stability of the U11-48K pre-mRNA

Turunen

Verma

Nyman

et al. 2013

RNA

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Alternative splicing (AS) is a major contributor to proteome diversity, but it also regulates gene expression by introducing premature termination codons (PTCs) that destabilize transcripts, typically via the nonsense-mediated decay (NMD) pathway. Such AS events often take place within long, conserved sequence elements, particularly in genes encoding various RNA binding proteins. AS-NMD is often activated by the protein encoded by the same gene, leading to a self-regulating feedback loop that maintains constant protein levels. However, cross-regulation between different RNA binding proteins is also common, giving rise to finely tuned regulatory networks. Recently, we described a feedback mechanism regulating two protein components of the U12-dependent spliceosome (U11-48K and U11/U12-65K) through a highly conserved sequence element. These elements contain a U11 snRNP-binding splicing enhancer (USSE), which, through the U11 snRNP, activates an upstream U2-type 3 ′ ss, resulting in the degradation of the U11-48K mRNA by AS-NMD. Through phylogenetic analysis, we now identify a G-rich sequence element that is conserved in fishes as well as mammals. We show that this element binds hnRNPF/H proteins in vitro. Knockdown of hnRNPH1/H2 or mutations in the G-run both lead to enhanced activation of the 3 ′ ss in vivo, suggesting that hnRNPH1/H2 proteins counteract the 3 ′ ss activation. Furthermore, we provide evidence that U1 binding immediately downstream from the G-run similarly counteracts the U11-mediated activation of the alternative 3 ′ ss. Thus, our results elucidate the mechanism in which snRNPs from both spliceosomes together with hnRNPH1/H2 proteins regulate the recognition and activation of the highly conserved alternative splice sites within the U11-48K pre-mRNA.

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The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes

Cited by 240 publications

References 51 publications

Nuclear Speckle-related Protein 70 Binds to Serine/Arginine-rich Splicing Factors 1 and 2 via an Arginine/Serine-like Region and Counteracts Their Alternative Splicing Activity

Nuclear Speckle-related Protein 70 Binds to Serine/Arginine-rich Splicing Factors 1 and 2 via an Arginine/Serine-like Region and Counteracts Their Alternative Splicing Activity

The RNA-binding profile of Acinus, a peripheral component of the exon junction complex, reveals its role in splicing regulation

HnRNPH1/H2, U1 snRNP, and U11 snRNP cooperate to regulate the stability of the U11-48K pre-mRNA

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