A Panoramic View of Yeast Noncoding RNA Processing

Abstract

Predictive analysis using publicly available yeast functional genomics and proteomics data suggests that many more proteins may be involved in biogenesis of ribonucleoproteins than are currently known. Using a microarray that monitors abundance and processing of noncoding RNAs, we analyzed 468 yeast strains carrying mutations in protein-coding genes, most of which have not previously been associated with RNA or RNP synthesis. Many strains mutated in uncharacterized genes displayed aberrant noncoding RNA profiles. Ten factors involved in noncoding RNA biogenesis were verified by further experimentation, including a protein required for 20S pre-rRNA processing (Tsr2p), a protein associated with the nuclear exosome (Lrp1p), and a factor required for box C/D snoRNA accumulation (Bcd1p). These data present a global view of yeast noncoding RNA processing and confirm that many currently uncharacterized yeast proteins are involved in biogenesis of noncoding RNA.

The Specificities of Four Yeast Dihydrouridine Synthases for Cytoplasmic tRNAs

“…By contrast, these primer extension stops are both missing in alkali-treated RNA from a dus1-⌬ strain (lane g). This result confirms that Dus1p modifies U 16 tRNA Tyr has dihydrouridine residues at five positions in the D loop as well as at D 47 . We proposed above that Dus3p might be responsible for formation of D 47 , based on our quantitation of the dihydrouridine content of tRNA Tyr from a dus3-⌬ strain (Fig.…”

“…This suggests that Dus4p activity accounts for formation of two of the six dihydrouridine residues in tRNA Tyr . These sites could be D 20 Microarray Analysis Largely Confirms the Preliminary Model of Dus Protein Specificity-It was previously observed that the absence of dihydrouridine in tRNAs from a dus1 mutant strain could be detected by microarray, using a protocol in which the fluors are coupled directly to the cellular RNA and hybridized to an array of oligonucleotides (16). It was noted that only oligonucleotides covering tRNA positions 16 and 17 reported significant preferential hybridization to RNA from a dus1 mutant relative to RNA from a wild type strain (positive ratios), and it was concluded that the loss of dihydrouridine modification in the dus1 mutant results in higher binding affinity.…”

“…As shown ( Fig. 2A and Table III), the D 16 and D 17 modifications of tRNA Phe are due to Dus1p, based on analysis of the nucleoside content of purified tRNA Phe from a dus1 mutant strain. Primer extension analysis of alkali-treated RNA with a tRNA Phe primer confirms this result (Fig.…”

“…Furthermore, for reasons that are unclear, a number of bands appeared even in the absence of alkali treatment of wild type tRNA Tyr (lane e), and the density of dihydrouridine residues precludes visualization of all five dihydrouridine modifications in treated wild type tRNA (lane f). Because it cannot be compared, there is no score for tRNA Tyr We note that the signal for D 16 is too weak to ascribe to Dus1p or any other Dus protein.…”

“…(15) were grown in 50 ml of SC medium to ϳ1 ϫ 10 7 cells/ml in parallel with a wild type (BY4743) culture. Total RNA was extracted using hot phenol as previously described (16), ethanol-precipitated and quantified by measuring the A 260 . Contaminating DNA was removed by treatment with DNase I (Fermentas) according to the manufacturer's instructions followed by phenol extraction and precipitation.…”