A compensatory base change in human U2 snRNA can suppress a branch site mutation.

Zhuang, Yuan; Weiner, Alan M.

doi:10.1101/gad.3.10.1545

Cited by 289 publications

(220 citation statements)

References 43 publications

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“…The mutation which increased NS2 interaction with Crm1 was K96E/L103P, as described by Lopez-Bueno et al (14). The U2 snRNA mutations were made in the wild-type human U2 snRNA gene, obtained from Alan Weiner (29), by using standard techniques. The branch point-binding 5Ј ends of the human and mouse U2 snRNA are identical.…”

Section: Cells and Virusmentioning

confidence: 99%

Replication of Minute Virus of Mice DNA Is Critically Dependent on Accumulated Levels of NS2

Choi

Newman

Burger

et al. 2005

J Virol

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Following transfection of murine fibroblasts, the lymphotropic strain of minute virus of mice (MVMi) does not efficiently produce progeny single-strand DNA (ssDNA). However, changing a single nucleotide in the MVMi 3 splice site to that found in the fibrotropic strain MVMp enabled full DNA replication and production of ssDNA. This change enhanced excision of the large intron and the production of NS2, likely by improving interaction, in fibroblasts with the branch point-binding U2 snRNA. One function of NS2 involves interaction with the nuclear export protein Crm1. The defect in production of MVMi ssDNA in fibroblasts can also be overcome by introducing a mutation in MVMi NS2 that enhances its interaction with Crm1. Although MVMi contains a 3 splice site that performs poorly in fibroblasts, MVMi generated at least as much R2 and NS2 in murine lymphocytes as did MVMp in fibroblasts. Therefore, it appears that MVMp has acquired a mutation that improves the excision of the large intron, as it adapted to fibroblasts to accommodate the need for NS2 for replication in these cells, and that the ratio of NS1 to NS2 may play a larger role in the host range of MVM than previously appreciated.Two strains of minute virus of mice, fibrotropic strain MVMp and lymphotropic strain MVMi, are reciprocally restricted for growth in murine cells (2,10,15,23,25,26). MVMp productively infects murine fibroblasts but not murine lymphocytes, while MVMi does the opposite. The major viral determinant of this host range resides within the capsid. The primary block to restrictive infections is intracellular, following binding to cells but before deposition of viral DNA in the nucleus (1,3,4,11,12,20).Characterization of MVMp and MVMi has also led to the identification of another determinant that affects viral replication in a host cell-dependent manner. Analysis of MVMi virus selected for growth on fibroblasts and analysis of the replication of engineered MVMi and MVMp chimeras on murine lymphocytes have demonstrated that a region encompassing the large-intron 3Ј splice site and P38 TATA box also plays a role in the efficiency of viral DNA replication in these two cell types (7, 12). The role that this region plays in replication has not been well characterized, but in both cases, differences in the ratio of mRNA R1 to R2, which encode NS1 and NS2, respectively, have been observed (4, 7, 11). The importance of the NS region in cell type-specific replication has also been noted for cells of neuronal origin (22).Previous studies in our lab have shown that even minor alterations of the MVM large-intron 3Ј splice site can have significant effects on the steady-state ratio of R1 to R2 and, hence, on the ratio of the viral nonstructural proteins NS1 and NS2 (19,28). Differences between MVMi and MVMp in this area have been previously noted, and ways in which these differences might affect RNA processing in the two strains have been discussed previously (19). Recently, two reports have In this study we have shown that, following transfection of mur...

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Section: Cells and Virusmentioning

confidence: 99%

Replication of Minute Virus of Mice DNA Is Critically Dependent on Accumulated Levels of NS2

Choi

Newman

Burger

et al. 2005

J Virol

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“…This recognition involves a base-pairing interaction between the 5' end region of U1 and the 5' splice site of the pre-mRNA. The U2 snRNP then recognizes the branch site through a base-pairing interaction involving the branch site recognition region of U2, thereby bulging out the branch point adenosine of the pre-mRNA (Parker et al, 1987;Zhuang and Weiner, 1989). The joining of U1 and U2 results in the formation of a pre-splicing complex (complex A).…”

Section: Introductionmentioning

confidence: 99%

Pseudouridines in spliceosomal snRNAs

2011

Protein Cell

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Spliceosomal RNAs are a family of small nuclear RNAs (snRNAs) that are essential for pre-mRNA splicing. All vertebrate spliceosomal snRNAs are extensively pseudouridylated after transcription. Pseudouridines in spliceosomal snRNAs are generally clustered in regions that are functionally important during splicing. Many of these modified nucleotides are conserved across species lines. Recent studies have demonstrated that spliceosomal snRNA pseudouridylation is catalyzed by two different mechanisms: an RNA-dependent mechanism and an RNA-independent mechanism. The functions of the pseudouridines in spliceosomal snRNAs (U2 snRNA in particular) have also been extensively studied. Experimental data indicate that virtually all pseudouridines in U2 snRNA are functionally important. Besides the currently known pseudouridines (constitutive modifications), recent work has also indicated that pseudouridylation can be induced at novel positions under stress conditions, thus strongly suggesting that pseudouridylation is also a regulatory modification.

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“…The spliceosome assembles de novo on each premRNA molecule, and distinct intermediates in the assembly pathway can be observed in vitro+ The first binding events form the commitment or early complex (CC or E) and are ATP independent+ In this complex, the 59 splice site is recognized by U1 snRNP and mammalian proteins SF1 and U2AF 65 , or yeast proteins BBP and MUD2, bind the branch region and pyrimidine tract, respectively+ In the mammalian system, recent evidence suggests that U2 snRNP is loosely associated at this time, but not yet stably engaged (Das et al+, 2000), although such an association has not been detected in yeast (Liao et al+, 1992)+ In both mammals and yeast, the first ATP-dependent step is the stable binding of U2 snRNP to the pre-mRNA branch site (Cheng & Abelson, 1987;Konarska & Sharp, 1987;Krämer, 1988;Pruzan et al+, 1990;Michaud & Reed, 1991;Liao et al+, 1992), in part, through U2 snRNA•branch region base pairing (Parker et al+, 1987;Wu & Manley, 1989;Zhuang & Weiner, 1989)+ This forms complex A in the mammalian system+ Subsequently, a larger complex, B, is formed by association of U4/U5/U6 tri-snRNP+ Complex C follows B after significant rearrangements and is the active spliceosome, containing U2/5/6 snRNPs and splicing intermediates (reviewed in Moore et al+, 1993)+ In the mammalian system, both the mechanistic basis for the ATP requirement during U2 snRNP addition and the factor(s) utilizing the ATP remain unclear+ U2 snRNP is a 17S complex composed of U2 snRNA and approximately 20 proteins that form ordered subdomains (Krämer et al+, 1999;reviewed in Will & Lührmann, 1997)+ A major U2 protein component, SF3, composed of two heteromeric complexes SF3a and SF3b, has been implicated in stabilization of the U2 particle via contact to the RNA 59 to the branch region (also called the anchoring region; Gozani et al+, 1996)+ One SF3b protein, SF3b-155, additionally contacts the RNA on the 39 side of the branch region (Gozani et al+, 1998), and another SF3b-associated protein, U2-p14, contacts RNA within the branch region )+ In both yeast and mammals, SF3 can dissociate from U2 snRNP in vitro, indicating that a remodeling of the particle may be important for its function (Krämer, 1996;Pauling et al+, 2000)+ Other factors, including U2AF 35 and SR proteins, also contribute to stable U2 snRNP binding+ Yeast U2 addition also requires the presence of two DExH/D ATPases (Prp5p and Sub2p), but their targets are not yet known (Ruby et al+, 1993;Kistler & Guthrie, 2001;Libri et al+, 2001;Z...…”

Section: Introductionmentioning

confidence: 99%

The ATP requirement for U2 snRNP addition is linked to the pre-mRNA region 5′ to the branch site

Newnham

Query

2001

RNA

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Association of U2 snRNP with the pre-mRNA branch region is the first ATP-dependent step in spliceosome assembly. The basis of this energy dependence is not known. Previously, we identified minimal intron-derived substrates that form complexes with U2 independent of ATP. Here, we identify the intron region linked to the ATP dependence of this step by comparing these substrates to longer RNAs that recapitulate the ATP requirement. This region needed to impose ATP dependence lies immediately 59 to the branch site. Sequences ranging from 6 to 14 nt yield a near linear inhibitory effect on efficiency of complex formation with U2 snRNP, with 18 nt yielding near maximal ATP dependence. This region is not protected prior to U2 addition, and RNase H targeting of the region within nuclear extract converts an ATP-dependent substrate into an ATP-independent one. Within this region, there is no sequence specificity linked with the ATP requirement, as neither a specific sequence is needed, nor even nucleobases. These data and the results of other modifications suggest models in which the 18-nt region is a target for interactions with U2 snRNP in an ATP-bound or -activated conformation.

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A compensatory base change in human U2 snRNA can suppress a branch site mutation.

Cited by 289 publications

References 43 publications

Replication of Minute Virus of Mice DNA Is Critically Dependent on Accumulated Levels of NS2

Replication of Minute Virus of Mice DNA Is Critically Dependent on Accumulated Levels of NS2

Pseudouridines in spliceosomal snRNAs

The ATP requirement for U2 snRNP addition is linked to the pre-mRNA region 5′ to the branch site

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