Mutational analyses of trimethylguanosine synthase (Tgs1) and Mud2: Proteins implicated in pre-mRNA splicing Journal Article

   

Authors:	Chang, J.; Schwer, B.; Shuman, S.
Article Title:	Mutational analyses of trimethylguanosine synthase (Tgs1) and Mud2: Proteins implicated in pre-mRNA splicing
Abstract:	Yeast and human Tgs1 are orthologous RNA cap (guanine-N2) methyltransferases that convert m7G caps into the 2,2,7-trimethylguanosine (TMG) caps characteristic of spliceosomal snRNAs. TMG caps are dispensable for vegetative yeast growth, but are essential in the absence of Mud2, the putative yeast homolog of human splicing factor U2AF. Here we exploited the synthetic lethal interactions of tgs1D and mud2D mutations to identify essential structural features of the Tgs1 and Mud2 proteins. Thirty-two new mutations were introduced into human Tgs1 and surveyed for their effects on function in vivo in yeast and on the two sequential guanine-N2 methylation reactions in vitro. The structure-function data highlight a strictly essential p-cation interaction between Trp766 and the m7G base and a network of important enzymic contacts to the cap triphosphate via Lys646, Tyr771, Arg807, and Lys836. Mud2 is a 527-amino acid polypeptide composed of a hydrophilic N-terminal domain and a C-terminal RRM domain. We found that the RRM domain is necessary but not sufficient for Mud2 function in complementing growth of tgs1D mud2D and mud1D mud2D strains. Other changes in Mud2 elicited distinct phenotypes in tgs1D versus mud1D backgrounds. mud2D also caused a severe growth defect in cells lacking the Tgs1-binding protein encoded by the nonessential gene YNR004w (now renamed SWM2, synthetic with mud2D). Mud2 mutational effects in the swm2D background paralleled those for mud1D. The requirements for Mud2 function are apparently more stringent when yeast cells lack TMG caps than when they lack Mud1 or Swm2. Copyright © 2010 RNA Society.
Keywords:	unclassified drug; methylation; mutation; nonhuman; phenotype; amino acid substitution; protein binding; in vivo study; protein interaction; in vitro study; tyrosine; mutational analysis; methyltransferase; methyltransferases; amino acid sequence; molecular sequence data; sequence homology, amino acid; messenger rna; guanosine derivative; saccharomyces cerevisiae; rna caps; recombinant proteins; ribonucleoproteins; models, molecular; mutagenesis, site-directed; protein structure, tertiary; binding sites; yeast; saccharomyces cerevisiae proteins; structure analysis; protein derivative; yeast cell; lysine; rna splicing; rna precursors; tryptophan; rna, fungal; polypeptide; trimethylguanosine synthase; rrm domain; snrnps; tmg caps; mud2 protein; genetic complementation test
Journal Title:	RNA
Volume:	16
Issue:	5
ISSN:	1355-8382
Publisher:	Cold Spring Harbor Laboratory Press
Date Published:	2010-05-01
Start Page:	1018
End Page:	1031
Language:	English
DOI:	10.1261/rna.2082610
PUBMED:	20360394
PROVIDER:	scopus
PMCID:	PMC2856874
DOI/URL:	http://www.scopus.com/inward/record.url?eid=2-s2.0-77951154264&partnerID=40&md5=f0ef1064b5ed39ec18672e55b32e5de5
Notes:	--- - "Export Date: 20 April 2011" - "CODEN: RNARF" - "Source: Scopus"

https://synapse.mskcc.org/synapse/works/has_related_data_chk/3847