The 5′-terminal sequence of U1 RNA complementary to the consensus 5′ splice site of hnRNA is single-stranded in intact U1 snRNP particles

Rinke, Jutta; Appel, Bernd; Blöcker, Helmut; Frank, Ronald; Lührmann, Reinhard

doi:10.1093/nar/12.10.4111

Cited by 37 publications

(15 citation statements)

References 31 publications

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“…We adapted an RNase-H mapping assay (34)(35)(36) to probe sites on roX2 available to hybridization in the context of cross-linked chromatin extracts. RNase-H specifically hydrolyzes the RNA strand of a DNA-RNA hybrid (37).…”

Section: Resultsmentioning

confidence: 99%

The genomic binding sites of a noncoding RNA

Simon

Wang

Kharchenko

et al. 2011

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

391

370

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Long noncoding RNAs (lncRNAs) have important regulatory roles and can function at the level of chromatin. To determine where lncRNAs bind to chromatin, we developed capture hybridization analysis of RNA targets (CHART), a hybridization-based technique that specifically enriches endogenous RNAs along with their targets from reversibly cross-linked chromatin extracts. CHART was used to enrich the DNA and protein targets of endogenous lncRNAs from flies and humans. This analysis was extended to genomewide mapping of roX2, a well-studied ncRNA involved in dosage compensation in Drosophila. CHART revealed that roX2 binds at specific genomic sites that coincide with the binding sites of proteins from the male-specific lethal complex that affects dosage compensation. These results reveal the genomic targets of roX2 and demonstrate how CHART can be used to study RNAs in a manner analogous to chromatin immunoprecipitation for proteins.chromatin-associated RNAs | chromatin-modifying complexes | RNase H mapping G enerating cellular diversity from genetic information requires the regulatory interplay between cis-acting elements encoded at specific loci in chromatin and trans-acting factors that bind them (1). Although the importance of trans-acting proteins (e.g., transcription factors) has long been appreciated, there is growing interest in the role of long noncoding RNAs (lncRNAs) (2) as factors that can regulate specific chromatin loci. This interest is enhanced by the recent discovery that the majority of eukaryotic genomes are transcribed (3) and that many of the resulting transcripts are developmentally regulated (4) but do not encode proteins. Although the functional scope of these RNAs remains unknown (5-7), several lncRNAs play important regulatory roles at the level of chromatin (8). Determining where these ncRNAs bind on the genome is central to determining their function.Examples of lncRNAs that influence chromatin include the roX ncRNAs in flies and Xist in mammals, both having wellestablished roles in dosage compensation (8, 9); Kcnq1ot1 and Air ncRNAs, which are expressed from genomically imprinted loci and affect chromatin silencing (10-13); Evf2, HSR1, and other ncRNAs that positively regulate transcription (14-16); lncRNAs that target the dihydrofolate reductase promoter and the rDNA promoters through triplex formation (17, 18); and the human HOTAIR and HOTTIP lncRNAs, which regulate polycomb-repressed and trithorax-activated chromatin, respectively (19,20). Dysregulation of several of these lncRNAs has been associated with disease (21, 22). Our understanding of the biochemical roles of these RNAs comes largely from their interactions with specific proteins-insights gained from classical biochemical techniques developed for studying translation and RNA-processing complexes and also more recent technological advances using RNA immunoprecipitation (23) and cross-linking and immunoprecipitation (24)(25)(26). These experiments suggest that several lncRNAs specifically interact with chromatin-m odifying machine...

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Section: Resultsmentioning

confidence: 99%

The genomic binding sites of a noncoding RNA

Simon

Wang

Kharchenko

et al. 2011

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

391

370

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“…We explored the possibility that RBM25 associates with the U1 snRNP complex and plays a role in recruiting U1 snRNP to the weak 5Ј ss. U1 snRNP is composed of a 165-nt RNA (U1 snRNA), seven different Sm proteins common to other snRNP, and three U1-specific proteins (U1-70K, U1-A, and U1-C) (38). To probe the interactions between RBM25 and U1 snRNP, RBM25 was immunoprecipitated from HeLa cell nuclear extracts and analyzed by Western blotting for the presence of U1-specific proteins.…”

Section: Resultsmentioning

confidence: 99%

Novel Splicing Factor RBM25 Modulates Bcl-x Pre-mRNA 5′ Splice Site Selection

Zhou

Cho

et al. 2008

Molecular and Cellular Biology

111

109

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RBM25 has been shown to associate with splicing cofactors SRm160/300 and assembled splicing complexes, but little is known about its splicing regulation. Here, we characterize the functional role of RBM25 in alternative pre-mRNA splicing. Increased RBM25 expression correlated with increased apoptosis and specifically affected the expression of Bcl-x isoforms. RBM25 stimulated proapoptotic Bcl-x S 5 splice site (5 ss) selection in a dose-dependent manner, whereas its depletion caused the accumulation of antiapoptotic Bcl-x L . Furthermore, RBM25 specifically bound to Bcl-x RNA through a CGGGCA sequence located within exon 2. Mutation in this element abolished the ability of RBM25 to enhance Bcl-x S 5 ss selection, leading to decreased Bcl-x S isoform expression. Binding of RBM25 was shown to promote the recruitment of the U1 small nuclear ribonucleoprotein particle (snRNP) to the weak 5 ss; however, it was not required when a strong consensus 5 ss was present. In support of a role for RBM25 in modulating the selection of a 5 ss, we demonstrated that RBM25 associated selectively with the human homolog of yeast U1 snRNP-associated factor hLuc7A. These data suggest a novel mode for Bcl-x S 5 ss activation in which binding of RBM25 with exonic element CGGGCA may stabilize the pre-mRNA-U1 snRNP through interactions with hLuc7A.Alternative splicing is a regulatory mechanism that allows eukaryotes to generate numerous protein isoforms, often with diverse biological functions, from a single gene (2,26,49). Pre-mRNA splicing takes place within the spliceosome, which is assembled stepwise by the addition of small nuclear ribonucleoprotein particles (snRNP) and numerous accessory nonsnRNP splicing factors (23, 31). The excision of introns and the joining of exons depend on the recognition and usage of 5Ј splice sites (5Ј ss) and 3Ј ss by the splicing machinery (21, 33). The commitment complex forms when the 5Ј ss is recognized by U1 snRNA base pairing and stabilized by U1 snRNP while the 3Ј ss is recognized by the U2 auxiliary factor (U2AF) through U2 snRNA base pairing with the branch point. Subsequently, the U4/5/6 tri-snRNP is incorporated into the complex and the U1 snRNA base paired at the 5Ј ss is replaced by U6 snRNA. These processes result in a fully assembled spliceosome that supports a series of rearrangements via RNA-RNA and RNA-protein interactions and activates the catalytic steps of cleavage, exon joining, and intron release (2, 26).However, the splice site signals that define the 5Ј ss and 3Ј ss are often degenerate. How and when they are used are believed to be modulated by a combinational interplay of positive (splicing enhancers) and negative (splicing silencers) cis elements and trans-acting factors (2, 26), forming the basis of alternative splicing. The splicing regulatory (SR) proteins (18, 41) and the heterogeneous nuclear ribonucleoproteins (hnRNPs) (11,46) bind with specificity to pre-mRNA (20).The SR proteins generally bind to enhancer elements through the RNA-binding domain and activate splicing at...

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“…In addition to U1 snRNA, U1 snRNP is composed of seven different Sm proteins common to other snRNPs and three U1-specific proteins: U1 70K, U1A, and U1C (38). To further probe the interactions between RBFOX2 and U1 snRNP, RBFOX2 and U1-specific proteins were immunoprecipitated from MELC nuclear extracts expressing Fox2-FLAG or U1C-FLAG with anti-FLAG, anti-U1C, anti-Fox2, and anti-U1 70K antibodies and analyzed for the presence of the associated proteins.…”

Section: Rbfox2mentioning

confidence: 99%

RBFOX2 Promotes Protein 4.1R Exon 16 Selection via U1 snRNP Recruitment

Huang

Park

et al. 2012

Molecular and Cellular Biology

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The erythroid differentiation-specific splicing switch of protein 4.1R exon 16, which encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability, is modulated by the differentiation-induced splicing factor RBFOX2. We have now characterized the mechanism by which RBFOX2 regulates exon 16 splicing through the downstream intronic element UGCAUG. Exon 16 possesses a weak 5= splice site (GAG/GTTTGT), which when strengthened to a consensus sequence (GAG/GTAAGT) leads to near-total exon 16 inclusion. Impaired RBFOX2 binding reduces exon 16 inclusion in the context of the native weak 5= splice site, but not the engineered strong 5= splice site, implying that RBFOX2 achieves its effect by promoting utilization of the weak 5= splice site. We further demonstrate that RBFOX2 increases U1 snRNP recruitment to the weak 5= splice site through direct interaction between its C-terminal domain (CTD) and the zinc finger region of U1C and that the CTD is required for the effect of RBFOX2 on exon 16 splicing. Our data suggest a novel mechanism for exon 16 5= splice site activation in which the binding of RBFOX2 to downstream intronic splicing enhancers stabilizes the pre-mRNA-U1 snRNP complex through interactions with U1C.A lternative splicing is a eukaryotic regulatory mechanism that allows for the generation of numerous protein isoforms with often diverse biological functions from a single gene (4,26,41). It begins with the spliceosome, which is assembled stepwise by the addition of discrete small nuclear ribonucleoprotein particles (snRNPs) and numerous accessory non-snRNP splicing factors (23, 33). The excision of introns followed by the joining of exons depends on the recognition and usage of 5= and 3= splice sites (5= ss and 3= ss, respectively) by the splicing machinery (19, 34). The initial splicing step is comprised of 5= ss recognition by U1 snRNP and binding of U2 auxiliary factor (U2AF) to the 3= ss. These factors and additional proteins form the E or commitment complex, which bridges the intron and brings the splice sites close together. U2AF then recruits U2 snRNP to form the A complex. Subsequent binding of the U4-U6-U5 tri-snRNP and many other factors result in a fully assembled spliceosome that supports a series of rearrangements via RNA-RNA and RNA-protein interactions and activates the catalytic steps of cleavage, exon joining, and intron release (4, 26).The splice site signals that define the 5= ss and 3= ss of an alternatively spliced exon are often weak. How and when they are used is believed to be modulated by a complex interplay of positive (splicing enhancers) and negative (splicing silencers) cis elements and trans-acting factors (4, 26). These form the basis for alternative splicing. Target prediction for specific splicing factors is difficult, largely due to the small size and degeneracy of splicing factorbinding motifs. An exception to this degeneracy is the hexanucleotide UGCAUG, which has been shown to be an important element for the splicing of several exons (3,5,14,16,20,24,...

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The 5′-terminal sequence of U1 RNA complementary to the consensus 5′ splice site of hnRNA is single-stranded in intact U1 snRNP particles

Cited by 37 publications

References 31 publications

The genomic binding sites of a noncoding RNA

The genomic binding sites of a noncoding RNA

Novel Splicing Factor RBM25 Modulates Bcl-x Pre-mRNA 5′ Splice Site Selection

RBFOX2 Promotes Protein 4.1R Exon 16 Selection via U1 snRNP Recruitment

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