Direct probing of RNA structure and RNA-protein interactions in purified HeLa cell’s and yeast spliceosomal U4/U6.U5 tri-snRNP particles 1 1Edited by J. Doudna

Mougin, Annie; Gottschalk, Alexander; Fabrizio, Patrizia; Lührmann, Reinhard; Branlant, Christiane

doi:10.1006/jmbi.2002.5451

Cited by 43 publications

(46 citation statements)

References 66 publications

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“…Interestingly, our data are fully compatible with extensive comparison by RNA structure probing of the U4/U6.U5 trisnRNP (containing hPrp31) and 10 S U4/U6 di-snRNP (lacking it) (26). For example, sites of strong cleavage by the doublestrand-specific nuclease V1 were observed on stem I of U4/U6 snRNAs, irrespective of the presence or absence of hPrp31 (U4-G48 and, to a lesser degree, U4-C49), indicating that hPrp31 makes contact with stem I from one side only.…”

Section: Discussionsupporting

confidence: 87%

“…For example, sites of strong cleavage by the doublestrand-specific nuclease V1 were observed on stem I of U4/U6 snRNAs, irrespective of the presence or absence of hPrp31 (U4-G48 and, to a lesser degree, U4-C49), indicating that hPrp31 makes contact with stem I from one side only. This is further corroborated by discrete V1 cleavages at the 5Ј end of stem I (U4-U21 and U4-C22), which were only observed in the absence of hPrp31 (26). In sum, these observations suggest that what we observe in the U4/U6 complex is directly applicable to the situation in the tri-snRNP.…”

Section: Discussionsupporting

confidence: 79%

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RNA Structural Requirements for the Association of the Spliceosomal hPrp31 Protein with the U4 and U4atac Small Nuclear Ribonucleoproteins

Schultz¹,

Nottrott²,

Hartmuth

et al. 2006

Journal of Biological Chemistry

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The kink-turn, a stem I-internal loop-stem II structure of the 5 stem-loop of U4 and U4atac small nuclear (sn) RNAs bound by 15.5K protein is required for binding of human Prp31 protein (hPrp31) during U4 and U4atac snRNP assembly. In box C/D snoRNPs a similar kink-turn with bound 15.5K protein is required for selective binding of proteins NOP56 and NOP58. Here we analyzed RNA structural requirements for association of hPrp31 with U4 snRNP in vitro by hydroxyl radical footprinting. hPrp31 induced protection of the terminal penta-loop, as well as of stems I and II flanking the kink-turn. Similar protection was found with U4/U6 snRNA duplex prebound with 15.5K protein. A detailed mutational analysis of the U4 snRNA elements by electrophoretic mobility shift analysis revealed that stem I could not be shortened, although it tolerated sequence alterations. However, introduction of a third Watson-Crick base pair into stem II significantly reduced hPrp31 binding. While stem I of U4atac snRNA showed relaxed binding requirements, its stem II requirements were likewise restricted to two base pairs. In contrast, as shown previously, stem II of the kink-turn motif in box C/D snoRNAs is comprised of three base pairs, and NOP56 and NOP58 require a G-C pair at the central position. This indicates that hPrp31 binding specificity is achieved by the recognition of the two base pair long stem II of the U4 and U4atac snRNAs and suggests how discrimination is achieved by RNA structural elements during assembly of U4/U6 and U4atac/ U6atac snRNPs and box C/D snoRNPs.The processing of pre-mRNA in the nucleus is catalyzed by a large RNA-protein complex, the spliceosome (1-3). The spliceosome comprises, apart from the pre-mRNA substrate, the U1, U2, U4, U5, and U6 snRNPs, 4 as well as numerous splicing factors, that is, proteins that do not make up an integral part of the snRNPs. Each snRNP is a stable complex, consisting of a single RNA molecule and various proteins, of which some (the so-called Sm proteins) are common to all snRNPs, while others are specific to a particular snRNP (2). The individual snRNPs interact with one another in a dynamic manner during the splicing cycle. For example, at the beginning of this cycle the U4 snRNP and the U6 snRNP are associated firmly with one another by two extended stretches of RNA-RNA base pairs. Two intermolecular helices of the U4/U6 snRNA are separated by an intramolecular 5Ј-terminal hairpin loop (5Ј stem-loop) of the U4 snRNA (4 -7). The 5Ј-terminal stem-loop of the U4 snRNA (Fig. 1B) consists of a stem I, an intramolecular loop and then a stem II. The latter ends in a pentanucleotide loop ("penta-loop"). Thus at this stage the U4 and U6 snRNP form a single particle, the U4/U6 di-snRNP. This complex, in turn, associates with the U5 snRNP and thus the U4/U6⅐U5 tri-snRNP is formed, which is integrated as such into the precatalytic spliceosome. During the rearrangement of the fully assembled spliceosome to its catalytically active state, the intermolecular base pairing between the U4 and U6 sn...

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Section: Discussionsupporting

confidence: 87%

Section: Discussionsupporting

confidence: 79%

RNA Structural Requirements for the Association of the Spliceosomal hPrp31 Protein with the U4 and U4atac Small Nuclear Ribonucleoproteins

Schultz¹,

Nottrott²,

Hartmuth

et al. 2006

Journal of Biological Chemistry

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“…Here we demonstrate that in a binary system, the RH domain of Prp8 contacts the single-stranded region of U4 adjacent to U4/U6 stem I. Structure probing of purified human and yeast tri-snRNP complexes indicated that this U4 singlestranded region is either inaccessible (in the case of humans) or somewhat less accessible (in the case of yeast), which is at least consistent with Prp8 interacting with this region of the U4 snRNA in the U4/U6.U5 trisnRNP (Mougin et al 2002). As mentioned above, the cold-sensitive U4 mutation (U4-cs1), which stabilizes the U4/U6 interaction that must be disrupted during activation of the spliceosome, is suppressed by several Prp8 mutations, including those mapping to its RH domain.…”

Section: Discussionmentioning

confidence: 71%

The Prp8 RNase H-like domain inhibits Brr2-mediated U4/U6 snRNA unwinding by blocking Brr2 loading onto the U4 snRNA

et al. 2012

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The spliceosomal RNA helicase Brr2 catalyzes unwinding of the U4/U6 snRNA duplex, an essential step for spliceosome catalytic activation. Brr2 is regulated in part by the spliceosomal Prp8 protein by an unknown mechanism. We demonstrate that the RNase H (RH) domain of yeast Prp8 binds U4/U6 small nuclear RNA (snRNA) with the single-stranded regions of U4 and U6 preceding U4/U6 stem I, contributing to its binding. Via cross-linking coupled with mass spectrometry, we identify RH domain residues that contact the U4/U6 snRNA. We further demonstrate that the same single-stranded region of U4 preceding U4/U6 stem I is recognized by Brr2, indicating that it translocates along U4 and first unwinds stem I of the U4/U6 duplex. Finally, we show that the RH domain of Prp8 interferes with U4/U6 unwinding by blocking Brr2's interaction with the U4 snRNA. Our data reveal a novel mechanism whereby Prp8 negatively regulates Brr2 and potentially prevents premature U4/U6 unwinding during splicing. They also support the idea that the RH domain acts as a platform for the exchange of U6 snRNA for U1 at the 59 splice site. Our results provide insights into the mechanism whereby Brr2 unwinds U4/U6 and show how this activity is potentially regulated prior to spliceosome activation.[Keywords: pre-mRNA splicing; RNA helicase; RNA-protein complex; RNA-protein cross-linking; spliceosome catalytic activation] Supplemental material is available for this article. Received July 11, 2012; revised version accepted September 18, 2012. Pre-mRNA splicing is catalyzed by a multisubunit RNAprotein enzyme, the spliceosome, which carries out two successive trans-esterification reactions that lead to removal of an intron and the ligation of its flanking exons. Spliceosomes are formed via the stepwise recruitment of small nuclear ribonucleoprotein particles (snRNPs) and numerous non-snRNP proteins to the pre-mRNA substrate (for review, see Wahl et al. 2009). Initially, U1 and U2 snRNPs bind the 59 splice site (59 SS) and the branch site (BS) of the pre-mRNA's intron, respectively. This is followed by the recruitment of the U4/U6.U5 tri-snRNP to the spliceosome, yielding the B complex, which does not yet have an active center. The U4 and U6 snRNAs are extensively base-paired in the tri-snRNP, thereby keeping U6 small nuclear RNA (snRNA) catalytically inert (Staley and Guthrie 1998). For catalytic activation of the spliceosome, U4 snRNA must be displaced from U6 snRNA, which allows the formation of new U2/U6 base-pairing interactions and a catalytically important U6 internal stem-loop (ISL) ). Concomitant with or prior to this, the base-pairing interaction between the U1 snRNA and the 59 SS must be disrupted to allow the highly conserved ACAGAGA sequence of U6 snRNA to base-pair with the 59 end of the intron. This newly formed U2-U6-pre-mRNA RNA interaction network is thought to comprise the heart of the catalytic center of the spliceosome (Madhani and Guthrie 1992;Villa et al. 2002).Brr2 catalyzes the dissociation of the U4/U6 duplex and thus plays a ke...

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“…S1). This insertion falls in the central domain of U4, in the middle of the hypothetical U4/U6 stem III, which is evolutionarily conserved, but for which there is no experimental support (10,11). In addition, the 5′ stem-loop of U6 is ∼10 bp longer than that in other species (Fig.…”

Section: Resultsmentioning

confidence: 99%

Dramatically reduced spliceosome in Cyanidioschyzon merolae

Stark

Dunn²,

Dunn

et al. 2015

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

143

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The human spliceosome is a large ribonucleoprotein complex that catalyzes pre-mRNA splicing. It consists of five snRNAs and more than 200 proteins. Because of this complexity, much work has focused on the Saccharomyces cerevisiae spliceosome, viewed as a highly simplified system with fewer than half as many splicing factors as humans. Nevertheless, it has been difficult to ascribe a mechanistic function to individual splicing factors or even to discern which are critical for catalyzing the splicing reaction. We have identified and characterized the splicing machinery from the red alga Cyanidioschyzon merolae, which has been reported to harbor only 26 intron-containing genes. The U2, U4, U5, and U6 snRNAs contain expected conserved sequences and have the ability to adopt secondary structures and form intermolecular base-pairing interactions, as in other organisms. C. merolae has a highly reduced set of 43 identifiable core splicing proteins, compared with ∼90 in budding yeast and ∼140 in humans. Strikingly, we have been unable to find a U1 snRNA candidate or any predicted U1-associated proteins, suggesting that splicing in C. merolae may occur without the U1 small nuclear ribonucleoprotein particle. In addition, based on mapping the identified proteins onto the known splicing cycle, we propose that there is far less compositional variability during splicing in C. merolae than in other organisms. The observed reduction in splicing factors is consistent with the elimination of spliceosomal components that play a peripheral or modulatory role in splicing, presumably retaining those with a more central role in organization and catalysis.pre-mRNA splicing | spliceosome core | U1 snRNP | genome reduction | splicing mechanism P re-mRNA splicing occurs by two transesterification reactions that are catalyzed by the spliceosome, a large macromolecular assembly of five snRNAs and more than 200 proteins in humans (1). These components are thought to assemble onto each new pre-mRNA transcript in an ordered fashion through the recognition and binding of three highly conserved sequences in the transcript: the 5′ splice site, the branch site, and the 3′ splice site (2, 3). Some of these interactions occur via direct RNA/RNA base pairing between the transcript and snRNAs; for example, both U1 and U6 snRNAs base pair to the 5′ splice site of the pre-mRNA transcript, and, similarly, U2 snRNA base pairs to the branch site (3).Given the complexity of the human spliceosome, it is of considerable interest to find a more tractable splicing system with fewer components to study the core processes of splicing (assembly, catalysis, and fidelity). The Saccharomyces cerevisiae (yeast) spliceosome has been proposed as a simplified model system, because it contains only about 100 proteins (4). Indeed, substantial progress in understanding the spliceosome has been made by studying yeast splicing (3,5). Nevertheless, the yeast spliceosome is still a highly complex system in which to investigate the role of individual proteins, let alone attempt ...

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Direct probing of RNA structure and RNA-protein interactions in purified HeLa cell’s and yeast spliceosomal U4/U6.U5 tri-snRNP particles 1 1Edited by J. Doudna

Cited by 43 publications

References 66 publications

RNA Structural Requirements for the Association of the Spliceosomal hPrp31 Protein with the U4 and U4atac Small Nuclear Ribonucleoproteins

RNA Structural Requirements for the Association of the Spliceosomal hPrp31 Protein with the U4 and U4atac Small Nuclear Ribonucleoproteins

The Prp8 RNase H-like domain inhibits Brr2-mediated U4/U6 snRNA unwinding by blocking Brr2 loading onto the U4 snRNA

Dramatically reduced spliceosome in Cyanidioschyzon merolae

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