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.
    Full-text · Article · Apr 2015 · G3-Genes Genomes Genetics
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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.
    Full-text · Article · Feb 2014 · Nucleic Acids Research
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    • "We used our collection of viable Msl5 mutants to illuminate synthetic genetic interactions between Msl5 and Mud2, Nam8, and Tgs1. The results suggested 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 59 splice site (Chang et al. 2012). Here, we queried the genetic interactions of Cbc2-Y24A with our collection of Msl5 mutants that grow as well as wild-type MSL5 cells. "
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    ABSTRACT: Nuclear cap binding protein complex (CBC) is a heterodimer of a small subunit (Cbc2 in yeast) that binds the m7G cap and a large subunit (Sto1 in yeast) that interacts with karyopherins. In order to probe the role of cap recognition in yeast CBC function, we introduced alanine mutations (Y24A, F91A, D120A, D122A, R129A, and R133A) and N-terminal deletions (NΔ21 and NΔ42) in the cap-binding pocket of Cbc2. These lesions had no effect on vegetative growth, but they ameliorated the cold-sensitivity of tgs1Δ cells that lack trimethylguanosine caps (a phenotype attributed to ectopic association of CBC with the m7G cap of the normally TMG-capped U1 snRNA), thereby attesting to their impact on cap binding in vivo. Further studies of the Cbc2-Y24A variant revealed synthetic lethality or sickness with null mutations of proteins involved in early steps of spliceosome assembly (Nam8, Mud1, Swt21, Mud2, Ist3, and Brr1) and with otherwise benign mutations of Msl5, the essential branchpoint binding protein. Whereas the effects of weakening CBC-cap interactions are buffered by other actors in the splicing pathway during mitotic growth, the NΔ42 allele causes a severe impediment to yeast sporulation and meiosis. RNA analysis revealed a selective defect in the splicing of MER3 and SAE3 transcripts in cbc2-NΔ42 diploids during attempted sporulation. An intronless MER3 cDNA fully restored sporulation and spore viability in the cbc2-NΔ42 strain, signifying that MER3 splicing is a limiting transaction. These studies reveal a new level of splicing control during meiosis that is governed by nuclear CBC.
    Preview · Article · Oct 2012 · RNA
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