Abstract
Trimethylguanosine Synthase catalyses transfer of two methyl groups to the m7G cap of RNA polymerase II transcribed snRNAs, snoRNAs, and telomerase RNA TLC1 to form a 2,2,7-trimethylguanosine cap. While in vitro studies indicate that Tgs1 functions as a monomer and the dimethylation of m7G caps is not a processive reaction, partially methylated sn(o)RNAs are typically not detected in living cells. Here we show that both yeast and human Tgs1p possess a conserved self-association property located at the N-terminus. A disruption of Tgs1 self-association led to a strong reduction of sn(o)RNA trimethylation as well as reduced nucleolar enrichment of Tgs1. Self-association of Tgs1p and its catalytic activity were also prerequisite to bypass the requirement for its accessory factor Swm2p for efficient pre-rRNA processing and snRNA trimethylation. The ability to self-associate might enable Tgs1 to efficiently dimethylate the caps of the targeted RNAs in vivo.
Highlights
The m7G caps of RNA polymerase II transcribed snRNAs, snoRNAs, telomerase RNA TLC1 and selenoprotein mRNAs are tri-methylated by the methyltransferase Tgs1p through two successive methyl-transfer reactions from AdoMet to the N2 position of 7-methylguanosine[1,2,3,4]
Tgs1p and Swm2p were fused to GAL4 DNA binding domain (GAL4-BD) to use as bait, or to GAL4 activation domain (GAL4-AD) to use as prey
Both bait and prey plasmids were co-transformed into the PJ69-4A reporting yeast strain[14], and the resulting yeast strains containing both plasmids were screened for Swm2p, or Tgs1p self-interaction
Summary
The m7G caps of RNA polymerase II transcribed snRNAs, snoRNAs, telomerase RNA TLC1 and selenoprotein mRNAs are tri-methylated by the methyltransferase Tgs1p through two successive methyl-transfer reactions from AdoMet to the N2 position of 7-methylguanosine[1,2,3,4]. The trimethylguanosine (TMG) cap modification is highly conserved throughout eukaryotes It is not essential for the viability of the budding yeast Saccharomyces cerevisiae, and deletion of TGS1 only causes a cold sensitive growth phenotype[1]. The steady state levels of snRNAs in tgs1Δ yeast are similar to that of wild type, deletion of TGS1 results in genome-wide pre-mRNA splicing defects[5], indicating a role for the TMG cap modification beyond simple snRNA/snoRNAs stabilisation. Upon deletion of SWM2, Tgs1p selectively fails to trimethylate snRNAs, suggesting that Swm2p provides substrate specificity to Tgs1p toward this class of RNAs12. We show that Tgs[1] contains a conserved self-association domain localised at its N-terminus, which is important for nucleolar accumulation and for efficient sn(o)RNA trimethylation in yeast. Only overexpression of a self-association competent and catalytic active Tgs1p eliminates the pre-rRNA processing, and snRNA trimethylation defects seen in swm2Δ strains
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