Structure–function analysis and genetic interactions of the yeast branchpoint binding protein Msl5

Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
Nucleic Acids Research (Impact Factor: 9.11). 01/2012; 40(10):4539-52. DOI: 10.1093/nar/gks049
Source: PubMed


Saccharomyces cerevisiae Msl5 (branchpoint binding protein) orchestrates spliceosome assembly by binding the branchpoint sequence 5'-UACUAAC and establishing cross intron-bridging interactions with other components of the splicing machinery. Reciprocal tandem affinity purifications verify that Msl5 exists in vivo as a heterodimer with Mud2 and that the Msl5-Mud2 complex is associated with the U1 snRNP. By gauging the ability of mutants of Msl5 to complement msl5Δ, we find that the Mud2-binding (amino acids 35-54) and putative Prp40-binding (PPxY(100)) elements of the Msl5 N-terminal domain are inessential, as are the C-terminal proline-rich domain (amino acids 382-476) and two zinc-binding CxxCxxxxHxxxxC motifs (amino acids 273-286 and 299-312). A subset of conserved branchpoint RNA-binding amino acids in the central KH-QUA2 domain (amino acids 146-269) are essential pairwise (Ile198-Arg190; Leu256-Leu259) or in trios (Leu169-Arg172-Leu176), whereas other pairs of RNA-binding residues are dispensable. We used our collection of viable Msl5 mutants to interrogate synthetic genetic interactions, in cis between the inessential structural elements of the Msl5 polypeptide and in trans between Msl5 and yeast splicing factors (Mud2, Nam8 and Tgs1) that are optional for vegetative growth. The results suggest a network of important but functionally buffered protein-protein and protein-RNA interactions between the Mud2-Msl5 complex at the branchpoint and the U1 snRNP at the 5' splice site.

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    • "tgs1Δ yeast cells display apparently normal steady-state snRNA levels (Mouaikel et al. 2002; Hausmann et al. 2007) and no overt aberrations in the RNA or protein contents of their spliceosomal snRNPs, except for the acquisition of the nuclear capbinding complex (CBC) as a stoichiometric component of the U1 snRNP (Schwer et al. 2011). S. cerevisiae can grow in the absence of Tgs1 because the effects of ablating the TMG cap of the spliceosomal U snRNAs are genetically buffered, either by spliceosome assembly factors that are themselves inessential for vegetative growth (Hausmann et al. 2008; Wilmes et al. 2008; Chang et al. 2010) or by otherwise dispensable domains of the essential branchpoint binding protein Msl5 (Chang et al. 2012). Copyright © 2015 Qiu et al. doi: 10.1534/g3.115.016675 "
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    ABSTRACT: The trimethylguanosine (TMG) caps of small nuclear (sn) RNAs are synthesized by the enzyme Tgs1, via sequential methyl additions to the N2 atom of the m(7)G cap. Whereas TMG caps are inessential for Saccharomyces cerevisiae vegetative growth at 25˚ to 37˚, tgs1∆ cells that lack TMG caps fail to thrive at 18˚. The cold-sensitive defect correlates with ectopic stoichiometric association of nuclear cap-binding complex (CBC) with the residual m(7)G cap of the U1 snRNA and is suppressed fully by Cbc2 mutations that weaken cap binding. Here we show that normal growth of tgs1∆ cells at 18˚ is also restored by a C-terminal deletion of 77 amino acids from the Snp1 subunit of yeast U1 snRNP. These results underscore the U1 snRNP as a focal point for TMG cap function in vivo. Casting a broader net, we conducted a dosage suppressor screen for genes that allowed survival of tgs1∆ cells at 18˚. We thereby recovered RPO26 (encoding a shared subunit of all three nuclear RNA polymerases) and RPO31 (encoding the largest subunit of RNA polymerase III) as moderate and weak suppressors of tgs1∆ cold sensitivity, respectively. A structure-guided mutagenesis of Rpo26, using rpo26∆ complementation and tgs1∆ suppression as activity readouts, defined Rpo26-(78-155) as a minimized functional domain. Alanine scanning identified Glu89, Glu124, Arg135, and Arg136 as essential for rpo26∆ complementation. The E124A and R135A alleles retained tgs1∆ suppressor activity, thereby establishing a separation-of-function. These results illuminate the structure-activity profile of an essential RNA polymerase component. Copyright © 2015 Author et al.
    G3-Genes Genomes Genetics 04/2015; 5(7). DOI:10.1534/g3.115.016675 · 3.20 Impact Factor
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    • "However, when structures are available, they can be exploited to program mutations with specific functional defects and then systematically test an allelic series for synthetic genetic interactions with other spliceosome components or splicing factors. This has been applied to the m7G-cap binding pocket of yeast Cbc2 (34), guided by the crystal structure of the homologous human CBC•m7G-cap complex (35,36), and to the branchpoint RNA-binding site of yeast Msl5 (25,26), directed by the NMR structure of the human homolog SF1 bound to an RNA containing the yeast branchpoint consensus sequence (37). "
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    ABSTRACT: Yhc1 and U1C are homologous essential subunits of the yeast and human U1 snRNP, respectively, that are implicated in the establishment and stability of the complex of U1 bound to the pre-mRNA 5' splice site (5'SS). Here, we conducted a mutational analysis of Yhc1, guided by the U1C NMR structure and low-resolution crystal structure of human U1 snRNP. The N-terminal 170-amino acid segment of the 231-amino acid Yhc1 polypeptide sufficed for vegetative growth. Although changing the zinc-binding residue Cys6 to alanine was lethal, alanines at zinc-binding residues Cys9, His24 and His30 were not. Benign alanine substitutions at conserved surface residues elicited mutational synergies with other splicing components. YHC1-R21A was synthetically lethal in the absence of Mud2 and synthetically sick in the absence of Nam8, Mud1 and Tgs1 or in the presence of variant U1 snRNAs. YHC1 alleles K28A, Y12A, T14A, K22A and H15A displayed a progressively narrower range of synergies. R21A and K28A bypassed the essentiality of DEAD-box protein Prp28, suggesting that they affected U1•5'SS complex stability. Yhc1 Arg21 fortifies the U1•5'SS complex via contacts with SmD3 residues Glu37/Asp38, mutations of which synergized with mud2Δ and bypassed prp28Δ. YHC1-(1-170) was synthetically lethal with mutations of all components interrogated, with the exception of Nam8.
    Nucleic Acids Research 02/2014; 42(7). DOI:10.1093/nar/gku097 · 9.11 Impact Factor
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    ABSTRACT: Most eukaryotic primary transcripts include segments, or introns, that will be accurately removed during RNA biogenesis. This process, known as pre-messenger RNA splicing, is catalyzed by the spliceosome, accurately selecting a set of intronic marks from others apparently equivalent. This identification is critical, as incorrectly spliced RNAs can be toxic for the organism. One of these marks, the dinucleotide AG, signals the intronic 3' end, or 3' splice site (ss). In this review we will focus on those intronic features that have an impact on 3' ss selection. These include the location and type of neighboring sequences, and their distance to the 3' end. We will see that their interplay is needed to select the right intronic end, and that this can be modulated by additional intronic elements that contribute to alternative splicing, whereby diverse RNAs can be generated from identical precursors. This complexity, still poorly understood, is fundamental for the accuracy of gene expression. In addition, a clear knowledge of 3' ss selection is needed to fully decipher the coding potential of genomes.
    WIREs RNA 09/2012; 3(5):707-17. DOI:10.1002/wrna.1131 · 6.02 Impact Factor
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