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

Schultz, Annemarie; Nottrott, Stephanie; Hartmuth, Klaus; Lührmann, Reinhard

doi:10.1074/jbc.m603350200

Cited by 35 publications

(40 citation statements)

References 25 publications

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“…4B,F; apparent K d z25 mM). This result is in agreement with the previous observation that truncations of stem I, which were deleterious to the stability of the hPrp31-15.5K-U4 59SL complex, had little effect in the analogous U4atac-based complex (Schultz et al 2006).…”

Section: Reconstitution Of Hprp31supporting

confidence: 83%

Structural basis for the dual U4 and U4atac snRNA-binding specificity of spliceosomal protein hPrp31

Liu¹,

Ghalei²,

Lührmann³

et al. 2011

RNA

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Human proteins 15.5K and hPrp31 are components of the major spliceosomal U4 snRNP and of the minor spliceosomal U4atac snRNP. The two proteins bind to related 59-stem loops (59SLs) of the U4 and U4atac snRNAs in a strictly sequential fashion. The primary binding 15.5K protein binds at K-turns that exhibit identical sequences in the two snRNAs. However, RNA sequences contacted by the secondary binding hPrp31 differ in U4 and U4atac snRNAs, and the mechanism by which hPrp31 achieves its dual specificity is presently unknown. We show by crystal structure analysis that the capping pentaloops of the U4 and U4atac 59SLs adopt different structures in the ternary hPrp31-15.5K-snRNA complexes. In U4atac snRNA, a noncanonical base pair forms across the pentaloop, based on which the RNA establishes more intimate interactions with hPrp31 compared with U4 snRNA. Stacking of hPrp31-His270 on the noncanonical base pair at the base of the U4atac pentaloop recapitulates intramolecular stabilizing principles known from the UUCG and GNRA families of RNA tetraloops. Rational mutagenesis corroborated the importance of the noncanonical base pair and the U4atac-specific hPrp31-RNA interactions for complex stability. The more extensive hPrp31-U4atac snRNA interactions are in line with a higher stability of the U4atac compared with the U4-based ternary complex seen in gel-shift assays, which may explain how U4atac snRNA can compete with the more abundant U4 snRNA for the same protein partners in vivo.Keywords: major and minor spliceosome; pre-mRNA splicing; RNA-protein complex; U4 snRNP and U4atac snRNP; X-ray crystallography

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Section: Reconstitution Of Hprp31supporting

confidence: 83%

Structural basis for the dual U4 and U4atac snRNA-binding specificity of spliceosomal protein hPrp31

Liu¹,

Ghalei²,

Lührmann³

et al. 2011

RNA

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“…As shown in Figure 1, the 59 region of U4atac snRNA is involved in the stem I and stem II interactions with U6atac snRNA with a stem-loop structure between them. This stem-loop structure has been shown to bind the same 15.5 kd protein and to adopt the same structure as U4 snRNA (Nottrott et al 1999;Schultz et al 2006). Thus, these elements did not appear to be likely targeting sequences.…”

Section: A Region Of U4atac Snrna Is Also Required For Splicing In Vitromentioning

confidence: 99%

The conserved 3′ end domain of U6atac snRNA can direct U6 snRNA to the minor spliceosome

Dietrich¹,

Padgett²,

Shukla³

2009

RNA

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U6 and U6atac snRNAs play analogous critical roles in the major U2-dependent and minor U12-dependent spliceosomes, respectively. Previous results have shown that most of the functional cores of these two snRNAs are either highly similar in sequence or functionally interchangeable. Thus, a mechanism must exist to restrict each snRNA to its own spliceosome. Here we show that a chimeric U6 snRNA containing the unique and highly conserved 39 end domain of U6atac snRNA is able to function in vivo in U12-dependent spliceosomal splicing. Function of this chimera required the coexpression of a modified U4atac snRNA; U4 snRNA could not substitute. Partial deletions of this element in vivo, as well as in vitro antisense experiments, showed that the 39 end domain of U6atac snRNA is necessary to direct the U4atac/U6atac.U5 tri-snRNP to the forming U12-dependent spliceosome. In vitro experiments also uncovered a role for U4atac snRNA in this targeting.

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“…We would expect that, since binding of the PRPF31 protein requires prior 15.5K protein binding to U4atac snRNA Schultz et al 2006a;Liu et al 2007Liu et al , 2011, defects in binding to the 15.5K protein would also lead to defects in binding of the PRPF31 protein. In order to confirm this hypothesis, we tested the binding of recombinant human PRPF31 protein to RNA-15.5K complexes using an electrophoretic super shift assay (Schultz et al 2006a).…”

Section: Protein Binding By Mutant U4atac Snrnasmentioning

confidence: 99%

“…The K-turn motif of U4atac snRNA is comprised of a canonical stem (C-stem) that has two Watson-Crick G-C base pairs, an internal purine-rich loop containing two tandem-sheared G-A base pairs, and a noncanonical stem (NC-stem) that has one G-U and one Watson-Crick G-C base pair attached to an external pyrimidine-rich pentaloop (Cojocaru et al 2005;Woźniak et al 2005;Schultz et al 2006a). The 50G>A, 50G>C and 51G>A MOPD I mutations FIGURE 1.…”

Section: Distribution Of Mopd I Mutations In U4atac Snrnamentioning

confidence: 99%

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Biochemical defects in minor spliceosome function in the developmental disorder MOPD I

Jafarifar

Dietrich

Hiznay

et al. 2014

RNA

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Biallelic mutations of the human RNU4ATAC gene, which codes for the minor spliceosomal U4atac snRNA, cause the developmental disorder, MOPD I/TALS. To date, nine separate mutations in RNU4ATAC have been identified in MOPD I patients. Evidence suggests that all of these mutations lead to abrogation of U4atac snRNA function and impaired minor intron splicing. However, the molecular basis of these effects is unknown. Here, we use a variety of in vitro and in vivo assays to address this question. We find that only one mutation, 124G>A, leads to significantly reduced expression of U4atac snRNA, whereas four mutations, 30G>A, 50G>A, 50G>C and 51G>A, show impaired binding of essential protein components of the U4atac/U6atac di-snRNP in vitro and in vivo. Analysis of MOPD I patient fibroblasts and iPS cells homozygous for the most common mutation, 51G>A, shows reduced levels of the U4atac/U6atac.U5 tri-snRNP complex as determined by glycerol gradient sedimentation and immunoprecipitation. In this report, we establish a mechanistic basis for MOPD I disease and show that the inefficient splicing of genes containing U12-dependent introns in patient cells is due to defects in minor tri-snRNP formation, and the MOPD I-associated RNU4ATAC mutations can affect multiple facets of minor snRNA function.

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

Cited by 35 publications

References 25 publications

Structural basis for the dual U4 and U4atac snRNA-binding specificity of spliceosomal protein hPrp31

Structural basis for the dual U4 and U4atac snRNA-binding specificity of spliceosomal protein hPrp31

The conserved 3′ end domain of U6atac snRNA can direct U6 snRNA to the minor spliceosome

Biochemical defects in minor spliceosome function in the developmental disorder MOPD I

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