Yeast Puf3p-mediated mRNA decay is regulated by carbon source-specific differential interaction of Puf3p with Pop2p and Yak1p.

Puf3p regulates the stability of nuclear-encoded mRNAs acting in mitochondrial biogenesis and function in Saccharomyces cerevisiae. This work identifies the phosphorylation of Pop2p, a component of the deadenylase complex, as being critical for adapting Puf3p-mediated mRNA decay upon carbon source alterations. We demonstrate that the Puf3p-Pop2p association diminishes in mitochondria-reliant conditions and establish Yak1p, a kinase that phosphorylates Pop2p at threonine 97, as a new player in Puf3p-mediated regulation of mRNA decay.

Regulation of Parkinson's disease-associated genes by Pumilio proteins and microRNAs in SH-SY5Y neuronal cells.

Parkinson's disease is the second most common age-related, neurodegenerative disease. A small collection of genes has been linked to Parkinson's disease including LRRK2, SAT1, and SNCA, the latter of which encodes the protein alpha-synuclein that aggregates in Lewy bodies as a hallmark of the disease. Overexpression of even wild-type versions of these genes can lead to pathogenesis, yet the regulatory mechanisms that control protein production of the genes are not fully understood.

Multiple Puf proteins regulate the stability of ribosome biogenesis transcripts.

Cells must make careful use of the resources available to them. A key area of cellular regulation involves the biogenesis of ribosomes. Transcriptional regulation of ribosome biogenesis factor genes through alterations in histone acetylation has been well studied.

Conditional regulation of Puf1p, Puf4p, and Puf5p activity alters YHB1 mRNA stability for a rapid response to toxic nitric oxide stress in yeast.

Puf proteins regulate mRNA degradation and translation through interactions with 3' untranslated regions (UTRs). Such regulation provides an efficient method to rapidly alter protein production during cellular stress. YHB1 encodes the only protein to detoxify nitric oxide in yeast.

Carbon source-dependent alteration of Puf3p activity mediates rapid changes in the stabilities of mRNAs involved in mitochondrial function.

The Puf family of RNA-binding proteins regulates gene expression primarily by interacting with the 3' untranslated region (3' UTR) of targeted mRNAs and inhibiting translation and/or stimulating decay. Physical association and computational analyses of yeast Puf3p identified >150 potential mRNA targets involved in mitochondrial function. However, only COX17 has been established as a target of Puf3p-mediated deadenylation and decapping.

Roles of Puf proteins in mRNA degradation and translation.

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.

Identification of changes in gene expression by quantitation of mRNA levels.

Sequence elements within mRNA-untranslated regions and their binding partners are key controllers of mRNA stability. Changes in mRNA stability can often be detected by changes in steady-state mRNA abundance, or a more careful analysis of mRNA half-lives can be performed following transcriptional repression. This chapter presents methods to isolate RNA from both yeast and mammalian cells for either steady-state or half-life analyses.

Puf1p acts in combination with other yeast Puf proteins to control mRNA stability.

The eukaryotic Puf proteins bind 3' untranslated region (UTR) sequence elements to regulate the stability and translation of their target transcripts, and such regulatory events are critical for cell growth and development. Several global genome analyses have identified hundreds of potential mRNA targets of the Saccharomyces cerevisiae Puf proteins; however, only three mRNA targets for these proteins have been characterized thus far. After direct testing of nearly 40 candidate mRNAs, we established two of these as true mRNA targets of Puf-mediated decay in yeast, HXK1 and TIF1.

Profiling condition-specific, genome-wide regulation of mRNA stability in yeast.

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 RNA-binding factors and infers their condition-specific activities, using only genomic sequence data and steady-state mRNA expression data as input.

Yeast Puf3 mutants reveal the complexity of Puf-RNA binding and identify a loop required for regulation of mRNA decay.

The eukaryotic Puf proteins regulate mRNA translation and degradation by binding the 3' untranslated regions of target mRNAs. Crystal structure analysis of a human Puf bound to RNA suggested a modular mode of binding, with specific amino acids within each of eight repeat domains contacting a single nucleotide of the target RNA. Here we study the mechanism by which the yeast Puf3p binds and stimulates the degradation of COX17 mRNA.

Recruitment of the Puf3 protein to its mRNA target for regulation of mRNA decay in yeast.

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.