Comparative parallel analysis of RNA ends identifies mRNA substrates of a tRNA splicing endonuclease-initiated mRNA decay pathway.

Eukaryotes share a conserved messenger RNA (mRNA) decay pathway in which bulk mRNA is degraded by exoribonucleases. In addition, it has become clear that more specialized mRNA decay pathways are initiated by endonucleolytic cleavage at particular sites. The transfer RNA (tRNA) splicing endonuclease (TSEN) has been studied for its ability to remove introns from pre-tRNAs.

Origin, conservation, and loss of alternative splicing events that diversify the proteome in Saccharomycotina budding yeasts.

Many eukaryotes use RNA processing, including alternative splicing, to express multiple gene products from the same gene. The budding yeast has been successfully used to study the mechanism of splicing and the splicing machinery, but alternative splicing in yeast is relatively rare and has not been extensively studied. Alternative splicing of is widely conserved, but yeast and a few other eukaryotes have replaced this one alternatively spliced gene with a pair of duplicated, unspliced genes as part of a whole genome doubling (WGD).

Analysis of 2'-phosphotransferase (Tpt1p) from Saccharomyces cerevisiae: evidence for a conserved two-step reaction mechanism.

Tpt1p is an essential protein responsible for the 2'-phosphotransferase step of tRNA splicing in Saccharomyces cerevisiae, in which the splice junction 2'-phosphate of ligated tRNA is transferred to NAD to form mature tRNA and ADP-ribose 1''-2'' cyclic phosphate. We showed previously that Tpt1p is a member of a family of functional 2'-phosphotransferases found in eukaryotes, eubacteria, and archaea, that the Escherichia coli protein (KptA) is highly specific for 2'-phosphorylated RNAs despite the lack of obvious natural substrates, and that KptA acts on a trinucleotide substrate through an intermediate in which RNA is ADP-ribosylated at the 2'-phosphate. This mechanism is similar to a proposed mechanism of NAD-dependent histone deacetylases.

Analysis of recombinant yeast decapping enzyme.

A critical step in the turnover of yeast mRNAs is decapping. Two yeast proteins, Dcp1p and Dcp2p, are absolutely required for decapping, although their precise roles in the decapping reaction have not been established. To determine the function of both Dcp1p and Dcp2p in decapping, we purified recombinant versions of these proteins from Escherichia coli and examined their properties.