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RNA. 2010 May;16(5):1018-31. doi: 10.1261/rna.2082610. Epub 2010 Apr 1.

Mutational analyses of trimethylguanosine synthase (Tgs1) and Mud2: proteins implicated in pre-mRNA splicing.

Author information

  • 1Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.

Abstract

Yeast and human Tgs1 are orthologous RNA cap (guanine-N2) methyltransferases that convert m(7)G 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 tgs1Delta and mud2Delta 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 pi-cation interaction between Trp766 and the m(7)G 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 tgs1Delta mud2Delta and mud1Delta mud2Delta strains. Other changes in Mud2 elicited distinct phenotypes in tgs1Delta versus mud1Delta backgrounds. mud2Delta also caused a severe growth defect in cells lacking the Tgs1-binding protein encoded by the nonessential gene YNR004w (now renamed SWM2, synthetic with mud2Delta). Mud2 mutational effects in the swm2Delta background paralleled those for mud1Delta. The requirements for Mud2 function are apparently more stringent when yeast cells lack TMG caps than when they lack Mud1 or Swm2.

PMID:
20360394
[PubMed - indexed for MEDLINE]
PMCID:
PMC2856874
Free PMC Article

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