Eukaryotic post-transcriptional regulation is often specified by control elements within mRNA 3'- untranslated regions (3'-UTRs). In order to identify proteins that regulate specific mRNA decay rates in Saccharomyces cerevisae, we analyzed the role of five members of the Puf family present in the yeast genome (referred to as JSN1/PUF1, PUF2, PUF3, PUF4 and MPT5/PUF5). Yeast strains lacking all five Puf proteins showed differential expression of numerous yeast mRNAs. Examination of COX17 mRNA indicates that Puf3p specifically promotes decay of this mRNA by enhancing the rate of deadenylation and subsequent turnover. Puf3p also binds to the COX17 mRNA 3'-UTR in vitro. This indicates that the function of Puf proteins as specific regulators of mRNA deadenylation has been conserved throughout eukaryotes. In contrast to the case in Caenorhabditis elegans and Drosophila, yeast Puf3p does not affect translation of COX17 mRNA. These observations indicate that Puf proteins are likely to play a role in the control of transcript-specific rates of degradation in yeast by interacting directly with the mRNA turnover machinery.

The steady-state abundance of an mRNA is determined by the balance between transcription and decay. Although regulation of transcription has been well studied both experimentally and computationally, regulation of transcript stability has received little attention. We developed an algorithm, MatrixREDUCE, that discovers the position-specific affinity matrices for unknown RNAbinding factors and infers their condition-specific activities, using only genomic sequence data and steady-state mRNA expression data as input. We identified and computationally characterized the binding sites for six mRNA stability regulators in Saccharomyces cerevisiae, which include two members of the Pumilio-homology domain (Puf) family of RNA-binding proteins, Puf3p and Puf4p. We provide computational and experimental evidence that regulation of mRNA stability by these factors is modulated in response to a variety of environmental stimuli.cis-regulatory element ͉ gene expression ͉ microarray ͉ mRNA decay ͉ Puf protein

Puf proteins are regulators of diverse eukaryotic processes including stem cell maintenance, organelle biogenesis, oogenesis, neuron function, and memory formation. At the molecular level, Puf proteins promote translational repression and/or degradation of target mRNAs by first interacting with conserved cis-elements in the 3' untranslated region (UTR). Once bound to an mRNA, Puf proteins elicit RNA repression by complex interactions with protein cofactors and regulatory machinery involved in translation and degradation. Recent work has dramatically increased our understanding of the targets of Puf protein regulation, as well as the mechanisms by which Puf proteins recognize and regulate those mRNA targets. Crystal structure analysis of several Puf-RNA complexes has demonstrated that while Puf proteins are extremely conserved in their RNA-binding domains, Pufs attain target specificity by utilizing different structural conformations to recognize 8-10 nt sequences. Puf proteins have also evolved modes of protein interactions that are organism and transcript-specific, yet two common mechanisms of repression have emerged: inhibition of cap-binding events to block translation initiation, and recruitment of the CCR4-POP2-NOT deadenylase complex for poly(A) tail removal. Finally, multiple schemes to regulate Puf protein activity have been identified, including post-translational mechanisms that allow rapid changes in the repression of mRNA targets.

The Puf family of RNA-binding proteins regulates mRNA translation and decay via interactions with 3 untranslated regions (3 UTRs) of target mRNAs. In yeast, Puf3p binds the 3 UTR of COX17 mRNA and promotes rapid deadenylation and decay. We have investigated the sequences required for Puf3p recruitment to this 3 UTR and have identified two separate binding sites. These sites are specific for Puf3p, as they cannot bind another Puf protein, Puf5p. Both sites use a conserved UGUANAUA sequence, whereas one site contains additional sequences that enhance binding affinity. In vivo, presence of either site partially stimulates COX17 mRNA decay, but full decay regulation requires the presence of both sites. No other sequences outside the 3 UTR are required to mediate this decay regulation. The Puf repeat domain of Puf3p is sufficient not only for in vitro binding to the 3 UTR, but also in vivo stimulation of COX17 mRNA decay. These experiments indicate that the essential residues involved in mRNA decay regulation are wholly contained within this RNA-binding domain.

The genome sequences from increasing numbers of organisms allow for rapid and organized examination of gene expression. Yet current computational-based paradigms for gene recognition are limited and likely to miss genes expressing non-coding RNAs or mRNAs with small open reading frames (ORFs). We have utilized two strategies to determine if there are additional transcripts in the yeast Saccharomyces cerevisiae that were not identified in previous analyses of the genome. In one approach, we identified strong consensus polymerase III promoters based on sequence, and determined experimentally if these promoters drive the expression of an RNA polymerase III transcript. This approach led to the identification of a new, non-essential 170 nt non-coding RNA. An alternative strategy analyzed RNA expression from large sequence gaps>2 kb between predicted ORFs. Fifteen unique RNA transcripts ranging in size from 161 to 1200 nt were identified from a total of 59 sequence gaps. Several of these RNAs contain unusually small potential ORFs, while one is clearly non-coding and appears to be a small nucleolar RNA. These results suggest that there are likely to be additional previously unidentified non-coding RNAs in yeast, and that new paradigms for gene recognition will be required to identify all expressed genes from an organism.