p68 RNA Helicase Is an Essential Human Splicing Factor That Acts at the U1 snRNA-5′ Splice Site Duplex

ABSTRACT Modulation of the interaction between U1 snRNP and the 5′ splice site (5′ss) is a key event that governs 5′ss recognition and spliceosome assembly. Using the methylene blue-mediated cross-linking method (Z. R. Liu, A. M. Wilkie, M. J. Clemens, and C. W. Smith, RNA 2:611-621, 1996), a 65-kDa protein (p65) was shown to interact with the U1-5′ss duplex during spliceosome assembly (Z. R. Liu, B. Sargueil, and C. W. Smith, Mol. Cell. Biol. 18:6910-6920, 1998). In this report, p65 was identified as p68 RNA helicase and shown to be essential for in vitro pre-mRNA splicing. Depletion of endogenous p68 RNA helicase does not affect the loading of the U1 snRNP to the 5′ss during early stage of splicing. However, dissociation of the U1 from the 5′ss is largely inhibited. The data suggest that p68 RNA helicase functions in destabilizing the U1-5′ss interactions. Furthermore, depletion of p68 RNA helicase arrested spliceosome assembly at the prespliceosome stage, suggesting that p68 may play a role in the transition from prespliceosome to spliceosome.

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Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site recognition

eLife ◽

10.7554/elife.04986 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 117

Author(s):

Yasushi Kondo ◽

Chris Oubridge ◽

Anne-Marie M van Roon ◽

Kiyoshi Nagai

Keyword(s):

Protein Interactions ◽

Splice Site ◽

Electrostatic Interactions ◽

Rna Binding ◽

Early Stage ◽

Splice Junction ◽

Small Nuclear Ribonucleoprotein ◽

U1 Snrna ◽

U1 Snrnp ◽

Rna Backbone

U1 snRNP binds to the 5′ exon-intron junction of pre-mRNA and thus plays a crucial role at an early stage of pre-mRNA splicing. We present two crystal structures of engineered U1 sub-structures, which together reveal at atomic resolution an almost complete network of protein–protein and RNA-protein interactions within U1 snRNP, and show how the 5′ splice site of pre-mRNA is recognised by U1 snRNP. The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5′-end of U1 snRNA. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA. The structure, together with RNA binding assays, shows that the selection of 5′-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5′-splice sites.

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ATPase/Helicase Activities of p68 RNA Helicase Are Required for Pre-mRNA Splicing but Not for Assembly of the Spliceosome

Molecular and Cellular Biology ◽

10.1128/mcb.25.17.7484-7493.2005 ◽

2005 ◽

Vol 25 (17) ◽

pp. 7484-7493 ◽

Cited By ~ 47

Author(s):

Chunru Lin ◽

Liuqing Yang ◽

Jenny J. Yang ◽

Youliang Huang ◽

Zhi-Ren Liu

Keyword(s):

Functional Role ◽

Rna Helicase ◽

Mrna Splicing ◽

Splicing Factor ◽

Present Evidence ◽

U1 Snrna ◽

P68 Rna Helicase ◽

Rna Unwinding

ABSTRACT We have previously demonstrated that p68 RNA helicase, as an essential human splicing factor, acts at the U1 snRNA and 5′ splice site (5′ss) duplex in the pre-mRNA splicing process. To further analyze the function of p68 in the spliceosome, we generated two p68 mutants (motif V, RGLD to LGLD, and motif VI, HRIGR to HLIGR). ATPase and RNA unwinding assays demonstrated that the mutations abolished the RNA-dependent ATPase activity and RNA unwinding activity. The function of p68 in the spliceosome was abolished by the mutations, and the mutations also inhibited the dissociation of U1 from the 5′ss, while the mutants still interacted with the U1-5′ss duplex. Interestingly, the nonactive p68 mutants did not prevent the transition from prespliceosome to the spliceosome. The data suggested that p68 RNA helicase might actively unwind the U1-5′ss duplex. The protein might also play a role in the U4.U6/U5 addition, which did not require the ATPase and RNA unwinding activities of p68. In addition, we present evidence here to demonstrate the functional role of p68 RNA helicase in the pre-mRNA splicing process in vivo. Our experiments also showed that p68 interacted with unspliced but not spliced mRNA in vivo.

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Yeast Pre-mRNA Splicing Requires a Pair of U1 snRNP-Associated Tetratricopeptide Repeat Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.18.1.353 ◽

1998 ◽

Vol 18 (1) ◽

pp. 353-360 ◽

Cited By ~ 24

Author(s):

Mitch R. McLean ◽

Brian C. Rymond

Keyword(s):

Transcriptional Repression ◽

Fusion Gene ◽

Mrna Splicing ◽

Tetratricopeptide Repeat ◽

Yeast Strains ◽

Wide Range ◽

U1 Snrna ◽

U1 Snrnp ◽

Snrnp Protein

ABSTRACT The U1 snRNP functions to nucleate spliceosome assembly on newly transcribed pre-mRNA. Saccharomyces cerevisiae is unusual among eukaryotes in the greatly extended length of its U1 snRNA and the apparent increased polypeptide complexity of the corresponding U1 snRNP. In this paper, we report the identification of a novel U1 snRNP protein, Prp42p, with unexpected properties. Prp42p was identified by its surprising structural similarity to the essential U1 snRNP protein, Prp39p. Both Prp39p and Prp42p possess multiple copies of a variant tetratricopeptide repeat, an element implicated in a wide range of protein assembly events. Yeast strains depleted of Prp42p by transcriptional repression of a GAL1::PRP42fusion gene arrest for splicing prior to pre-mRNA 5′ splice site cleavage. Prp42p was not observed in a recent biochemical analysis of purified U1 snRNPs from S. cerevisiae (28). Nevertheless, antibodies directed against an epitope-tagged version of Prp42p specifically precipitate U1 snRNA from yeast extracts. Furthermore, Prp42p is required for U1 snRNP biogenesis, because yeast strains depleted of Prp42p formed incomplete U1 snRNPs that failed to produce stable complexes with pre-mRNA in vitro. The evidence shows that Prp39p and Prp42p are both required to configure the atypical yeast U1 snRNP into a structure compatible with its evolutionarily conserved role in pre-mRNA splicing.

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Structural analysis of the spliceosomal RNA helicase Prp28 from the thermophilic eukaryoteChaetomium thermophilum

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16006038 ◽

2016 ◽

Vol 72 (5) ◽

pp. 409-416 ◽

Cited By ~ 4

Author(s):

Marcel J. Tauchert ◽

Ralf Ficner

Keyword(s):

Crystal Structure ◽

Structural Analysis ◽

Splice Site ◽

Base Pair ◽

Rna Helicase ◽

Mrna Splicing ◽

Independent Manner ◽

Dead Box ◽

Chaetomium Thermophilum ◽

U1 Snrnp

Prp28 (pre-mRNA-splicing ATP-dependent RNA helicase 28) is a spliceosomal DEAD-box helicase which is involved in two steps of spliceosome assembly. It is required for the formation of commitment complex 2 in an ATP-independent manner as well as for the formation of the pre-catalytic spliceosome, which in contrast is ATP-dependent. During the latter step, Prp28 is crucial for the integration of the U4/U6·U5 tri-snRNP since it displaces the U1 snRNP and allows the U6 snRNP to base-pair with the 5′-splice site. Here, the crystal structure of Prp28 from the thermophilic fungusChaetomium thermophilumis reported at 3.2 Å resolution and is compared with the available structures of homologues.

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Proximity of the U12 snRNA with both the 5′ Splice Site and the Branch Point during Early Stages of Spliceosome Assembly

Molecular and Cellular Biology ◽

10.1128/mcb.25.12.4813-4825.2005 ◽

2005 ◽

Vol 25 (12) ◽

pp. 4813-4825 ◽

Cited By ~ 8

Author(s):

Mikko J. Frilander ◽

Xiaojuan Meng

Keyword(s):

Branch Point ◽

Splice Site ◽

Cross Linking ◽

Catalytic Core ◽

Spliceosome Assembly ◽

Site Specific ◽

Early Stages ◽

Branch Point Sequence

ABSTRACT U12 snRNA is required for branch point recognition in the U12-dependent spliceosome. Using site-specific cross-linking, we have captured an unexpected interaction between the 5′ end of the U12 snRNA and the −2 position upstream of the 5′ splice site of P120 and SCN4a splicing substrates. The U12 snRNA nucleotides that contact the 5′ exon are the same ones that form the catalytically important helix Ib with U6atac snRNA in the spliceosome catalytic core. However, the U12/5′ exon interaction is transient, occurring prior to the entry of the U4atac/U6atac.U5 tri-snRNP to the spliceosome. This suggests that the helix Ib region of U12 snRNA is positioned near the 5′ splice site early during spliceosome assembly and only later interacts with U6atac to form helix Ib. We also provide evidence that U12 snRNA can simultaneously interact with 5′ exon sequences near 5′ splice site and the branch point sequence, suggesting that the 5′ splice site and branch point sequences are separated by <40 to 50 Å in the complex A of the U12-dependent spliceosome. Thus, no major rearrangements are subsequently needed to position these sites for the first step of catalysis.

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Pyrimidine tracts between the 5' splice site and branch point facilitate splicing and recognition of a small Drosophila intron.

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2774 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2774-2780 ◽

Cited By ~ 20

Author(s):

C F Kennedy ◽

S M Berget

Keyword(s):

Drosophila Melanogaster ◽

Branch Point ◽

Splice Site ◽

Minimum Size ◽

Cross Linking ◽

In Vitro Splicing ◽

Precursor Rna ◽

Minimal Region

The minimum size for splicing of a vertebrate intron is approximately 70 nucleotides. In Drosophila melanogaster, more than half of the introns are significantly below this minimum yet function well. Such short introns often lack the pyrimidine tract located between the branch point and 3' splice site common to metazoan introns. To investigate if small introns contain special sequences that facilitate their recognition, the sequences and factors required for the splicing of a 59-nucleotide intron from the D. melanogaster mle gene have been examined. This intron contains only a minimal region of interrupted pyrimidines downstream of the branch point. Instead, two longer, uninterrupted C-rich tracts are located between the 5' splice site and branch point. Both of these sequences are required for maximal in vivo and in vitro splicing. The upstream sequences are also required for maximal binding of factors to the 5' splice site, cross-linking of U2AF to precursor RNA, and assembly of the active spliceosome, suggesting that sequences upstream of the branch point influence events at both ends of the small mle intron. Thus, a very short intron lacking a classical pyrimidine tract between the branch point and 3' splice site requires accessory pyrimidine sequences in the short region between the 5' splice site and branch point.

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Competing Upstream 5′ Splice Sites Enhance the Rate of Proximal Splicing

Molecular and Cellular Biology ◽

10.1128/mcb.01071-09 ◽

2010 ◽

Vol 30 (8) ◽

pp. 1878-1886 ◽

Cited By ~ 24

Author(s):

Martin J. Hicks ◽

William F. Mueller ◽

Peter J. Shepard ◽

Klemens J. Hertel

Keyword(s):

Splice Site ◽

Site Selection ◽

Regulatory Element ◽

Regulatory Elements ◽

Splice Sites ◽

Base Pairing ◽

Splice Site Selection ◽

U1 Snrna ◽

Pairing Potential

ABSTRACT Alternative 5′ splice site selection is one of the major pathways resulting in mRNA diversification. Regulation of this type of alternative splicing depends on the presence of regulatory elements that activate or repress the use of competing splice sites, usually leading to the preferential use of the proximal splice site. However, the mechanisms involved in proximal splice site selection and the thermodynamic advantage realized by proximal splice sites are not well understood. Here, we have carried out a systematic analysis of alternative 5′ splice site usage using in vitro splicing assays. We show that observed rates of splicing correlate well with their U1 snRNA base pairing potential. Weak U1 snRNA interactions with the 5′ splice site were significantly rescued by the proximity of the downstream exon, demonstrating that the intron definition mode of splice site recognition is highly efficient. In the context of competing splice sites, the proximity to the downstream 3′ splice site was more influential in dictating splice site selection than the actual 5′ splice site/U1 snRNA base pairing potential. Surprisingly, the kinetic analysis also demonstrated that an upstream competing 5′ splice site enhances the rate of proximal splicing. These results reveal the discovery of a new splicing regulatory element, an upstream 5′ splice site functioning as a splicing enhancer.

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Splicing Functions and Global Dependency on Fission Yeast Slu7 Reveal Diversity in Spliceosome Assembly

Molecular and Cellular Biology ◽

10.1128/mcb.00007-13 ◽

2013 ◽

Vol 33 (16) ◽

pp. 3125-3136 ◽

Cited By ~ 5

Author(s):

Shataparna Banerjee ◽

Piyush Khandelia ◽

Geetha Melangath ◽

Samirul Bashir ◽

Vijaykrishna Nagampalli ◽

...

Keyword(s):

Splice Site ◽

Genetic Interactions ◽

Splicing Factors ◽

Spliceosome Assembly ◽

Content Type ◽

Missense Mutant ◽

Stable Association ◽

Content Factor ◽

The Impact

The multiple short introns inSchizosaccharomyces pombegenes with degeneratecissequences and atypically positioned polypyrimidine tracts make an interesting model to investigate canonical and alternative roles for conserved splicing factors. Here we report functions and interactions of theS. pombe slu7+(spslu7+) gene product, known fromSaccharomyces cerevisiaeand humanin vitroreactions to assemble into spliceosomes after the first catalytic reaction and to dictate 3′ splice site choice during the second reaction. By using a missense mutant of this essentialS. pombefactor, we detected a range of global splicing derangements that were validated in assays for the splicing status of diverse candidate introns. We ascribe widespread, intron-specific SpSlu7 functions and have deduced several features, including the branch nucleotide-to-3′ splice site distance, intron length, and the impact of its A/U content at the 5′ end on the intron's dependence on SpSlu7. The data imply dynamic substrate-splicing factor relationships in multiintron transcripts. Interestingly, the unexpected early splicing arrest inspslu7-2revealed a role before catalysis. We detected a salt-stable association with U5 snRNP and observed genetic interactions withspprp1+, a homolog of human U5-102k factor. These observations together point to an altered recruitment and dependence on SpSlu7, suggesting its role in facilitating transitions that promote catalysis, and highlight the diversity in spliceosome assembly.

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