Many or all of the sites of pseudouridine (⌿) formation in eukaryotic rRNA are selected by site-specific base-pairing with members of the box H + ACA class of small nucleolar RNAs (snoRNAs). Database searches previously identified strong homology between the rat nucleolar protein Nap57p, its yeast homolog Cbf5p, and the Escherichia coli ⌿ synthase truB/P35. We therefore tested whether Cbf5p is required for synthesis of ⌿ in the yeast rRNA. After genetic depletion of Cbf5p, formation of ⌿ in the pre-rRNA is dramatically inhibited, resulting in accumulation of the unmodified rRNA. Protein A-tagged Cbf5p coprecipitates all tested members of the box H + ACA snoRNAs but not box C + D snoRNAs or other RNA species. Genetic depletion of Cbf5p leads to depletion of all box H + ACA snoRNAs. These include snR30, which is required for pre-rRNA processing. Depletion of Cbf5p also results in a pre-rRNA processing defect similar to that seen on depletion of snR30. We conclude that Cbf5p is likely to be the rRNA ⌿ synthase and is an integral component of the box H + ACA class of snoRNPs, which function to target the enzyme to its site of action.

We have identified a nuclear pathway that rapidly degrades unspliced pre-mRNAs in yeast. This involves 3'-->5' degradation by the exosome complex and 5'-->3' degradation by the exonuclease Rat1p. 3'-->5' degradation is normally the major pathway and is regulated in response to carbon source. Inhibition of pre-mRNA degradation resulted in increased levels of pre-mRNAs and spliced mRNAs. When splicing was inhibited by mutation of a splicing factor, inhibition of turnover resulted in 20- to 50-fold accumulation of pre-mRNAs, accompanied by increased mRNA production. Splicing of a reporter construct with a 3' splice site mutation was also increased on inhibition of turnover, showing competition between degradation and splicing. We propose that nuclear pre-mRNA turnover represents a novel step in the regulation of gene expression.

Methylations at position N of internal adenosines (mAs) are the most abundant and widespread mRNA modifications. These modifications play crucial roles in reproduction, growth, and development by controlling gene expression patterns at the posttranscriptional level. Their function is decoded by readers that share the YTH domain, which forms a hydrophobic pocket that directly accommodates the mA residues. While the physiological and molecular functions of YTH readers have been extensively studied in animals, little is known about plant readers, even though mAs are crucial for plant survival and development. Viridiplantae contains high numbers of YTH domain proteins. Here, we performed comprehensive evolutionary analysis of YTH domain proteins and demonstrated that they are highly likely to be actual readers with redundant as well as specific functions. We also show that the ECT2 protein from binds to mA-containing RNAs in vivo and that this property relies on the mA binding pocket carried by its YTH domain. ECT2 is cytoplasmic and relocates to stress granules upon heat exposure, suggesting that it controls mRNA fate in the cytosol. Finally, we demonstrate that ECT2 acts to decode the mA signal in the trichome and is required for their normal branching through controlling their ploidy levels.