Regulation of conditional gene expression by coupled transcription repression and RNA degradation.

Gene expression is determined by a combination of transcriptional and post-transcriptional regulatory events that were thought to occur independently. This report demonstrates that the genes associated with the Snf3p-Rgt2p glucose-sensing pathway are regulated by interconnected transcription repression and RNA degradation. Deletion of the dsRNA-specific ribonuclease III Rnt1p increased the expression of Snf3p-Rgt2p-associated transcription factors in vivo and the recombinant enzyme degraded their messenger RNA in vitro. Surprisingly, Rnt1ps effect on gene expression in vivo was both RNA and promoter dependent, thus linking RNA degradation to transcription. Strikingly, deletion of RNT1-induced promoter-specific transcription of the glucose sensing genes even in the absence of RNA cleavage signals. Together, the results presented here support a model in which co-transcriptional RNA degradation increases the efficiency of gene repression, thereby allowing an effective cellular response to the continuous changes in nutrient concentrations.

RNase III-mediated silencing of a glucose-dependent repressor in yeast.

Members of the RNase III family are found in all species examined with the exception of archaebacteria, where the functions of RNase III are carried out by the bulge-helix-bulge nuclease (BHB). In bacteria, RNase III contributes to the processing of many noncoding RNAs and directly cleaves several cellular and phage mRNAs. In eukaryotes, orthologs of RNase III participate in the biogenesis of many miRNAs and siRNAs, and this biogenesis initiates the degradation or translational repression of several mRNAs. However, the capacity of eukaryotic RNase IIIs to regulate gene expression by directly cleaving within the coding sequence of mRNAs remains speculative. Here we show that Rnt1p, a member of the RNase III family, selectively inhibits gene expression in baker's yeast by directly cleaving a stem-loop structure within the mRNA coding sequence. Analysis of mRNA expression upon the deletion of Rnt1p revealed an upregulation of the glucose-dependent repressor Mig2p. Mig2p mRNA became more stable upon the deletion of Rnt1p and resisted glucose-dependent degradation. In vitro, Rnt1p cleaved Mig2p mRNA and a silent mutation that disrupts Rnt1p signals blocked Mig2p mRNA degradation. These observations reveal a new RNase III-dependent mechanism of eukaryotic mRNA degradation.

Genome-wide prediction and analysis of yeast RNase III-dependent snoRNA processing signals.

In Saccharomyces cerevisiae, the maturation of both pre-rRNA and pre-small nucleolar RNAs (pre-snoRNAs) involves common factors, thereby providing a potential mechanism for the coregulation of snoRNA and rRNA synthesis. In this study, we examined the global impact of the double-stranded-RNA-specific RNase Rnt1p, which is required for pre-rRNA processing, on the maturation of all known snoRNAs. In silico searches for Rnt1p cleavage signals, and genome-wide analysis of the Rnt1p-dependent expression profile, identified seven new Rnt1p substrates. Interestingly, two of the newly identified Rnt1p-dependent snoRNAs, snR39 and snR59, are located in the introns of the ribosomal protein genes RPL7A and RPL7B. In vitro and in vivo experiments indicated that snR39 is normally processed from the lariat of RPL7A, suggesting that the expressions of RPL7A and snR39 are linked. In contrast, snR59 is produced by a direct cleavage of the RPL7B pre-mRNA, indicating that a single pre-mRNA transcript cannot be spliced to produce a mature RPL7B mRNA and processed by Rnt1p to produce a mature snR59 simultaneously. The results presented here reveal a new role of yeast RNase III in the processing of intron-encoded snoRNAs that permits independent regulation of the host mRNA and its associated snoRNA.