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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. Yak1p deletion alters the half‐life of Puf3p target mRNAs. Our findings outline a metabolism‐driven regulatory switch, whereby, in mitochondria‐independent conditions, Puf3p recruits Pop2p and the decay machinery to bound mRNAs for rapid decay. Conversely, in mitochondria‐reliant conditions, the association of Puf3p with Yak1p increases, placing Yak1p proximal to neighboring Pop2p. Subsequent Pop2p phosphorylation reduces the Puf3p–Pop2p interaction and stabilizes Puf3p target mRNAs. [ABSTRACT FROM AUTHOR]

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. Pumilio proteins belong to the highly conserved PUF family of eukaryotic RNA-binding proteins that post-transcriptionally regulate gene expression through binding conserved motifs in the 3' untranslated region (UTR) of mRNA targets known as PUF Recognition Elements (PREs). The 3'UTRs of LRRK2, SNCA and SAT1 each contain multiple putative PREs. Knockdown (KD) of the two human Pumilio homologs (Pumilio 1 and Pumilio 2) in a neurodegenerative model cell line, SH-SY5Y, resulted in increased SNCA and LRRK2 mRNA, as well as alpha-synuclein levels, suggesting these genes are normally repressed by the Pumilio proteins. Some studies have indicated a relationship between Pumilio and microRNA activities on the same target, especially when their binding sites are close together. LRRK2, SNCA, and SAT1 each contain several putative microRNA-binding sites within the 3'UTR, some of which reside near PREs. Small RNA-seq and microRNA qPCR assays were performed in both wild type and Pumilio KD SH-SY5Y cells to analyze global and differential microRNA expression. One thousand four hundred and four microRNAs were detected across wild type and Pumilio KD cells. Twenty-one microRNAs were differentially expressed between treatments, six of which were previously established to be altered in Parkinson's disease patient samples or research models. Expression of ten miRs predicted to target LRRK2 and SNCA was verified by RT-qPCR. Collectively, our results demonstrate that Pumilios and microRNAs play a multi-faceted role in regulating Parkinson's disease-associated genes. [ABSTRACT FROM AUTHOR]

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. 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

Reports the results of electrophoretic gel mobility shift titrations, which analyzed the formation and stability of a purine motif triple helix in the presence of monovalent cations{M(+)}, and monitored oligonucleotide aggregation under these conditions. Details of materials and methods; Results and discussion.

Suggests that the design of high-affinity oligonucleotide probes for alternate strand recognition of natural DNA sequences may be more complex and difficult to achieve than for artificial sequences that were optimized for triple helix formation. Electrophoretic mobility shift titrations; Dimethyl sulfate footprinting; Model studies.

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. Here we show that YHB1 mRNA is destabilized by Puf1p, Puf4p, and Puf5p through two overlapping Puf recognition elements (PREs) in the YHB1 3' UTR. Overexpression of any of the three Pufs is sufficient to fully rescue wild-type decay in the absence of other Pufs, and overexpression of Puf4p or Puf5p can enhance the rate of wild-type decay. YHB1 mRNA decay stimulation by Puf proteins is also responsive to cellular stress. YHB1 mRNA is stabilized in galactose and high culture density, indicating inactivation of the Puf proteins. This condition-specific inactivation of Pufs is overcome by Puf overexpression, and Puf4p/Puf5p overexpression during nitric oxide exposure reduces the steady-state level of endogenous YHB1 mRNA, resulting in slow growth. Puf inactivation is not a result of altered expression or localization. Puf1p and Puf4p can bind target mRNA in inactivating conditions; however, Puf5p binding is reduced. This work demonstrates how multiple Puf proteins coordinately regulate YHB1 mRNA to protect cells from nitric oxide stress., (© 2015 Russo and Olivas. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

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. In a novel finding, multiple Puf proteins, including Puf1p, regulate both of these mRNAs in combination. TIF1 mRNA decay can be stimulated individually by Puf1p and Puf5p, but the combination of both proteins is required for full regulation. This Puf-mediated decay requires the presence of two UGUA binding sites within the TIF1 3' UTR, with one site regulated by Puf5p and the other by both Puf1p and Puf5p. Alteration of the UGUA site in the tif1 3' UTR to more closely resemble the Puf3p binding site broadens the specificity to include regulation by Puf3p. The stability of the endogenously transcribed HXK1 mRNA, cellular levels of Hxk1 protein activity, and HXK1 3' UTR-directed decay are affected by Puf1p and Puf5p as well as Puf4p. Together these results identify the first mRNA targets of Puf1p-mediated decay, describe similar yet distinct combinatorial control of two new target mRNAs by the yeast Puf proteins, and suggest the importance of direct testing to evaluate RNA-regulatory mechanisms.

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. In addition, two reliable methods to quantitate mRNA levels, northern blot analysis and real-time PCR, are outlined and compared.

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. Mutation of the predicted RNA-binding positions of Puf3p to those found in Puf5p demonstrated that a single amino acid change in Puf3p abolished detectable binding to COX17. Since this amino acid position in both Puf3p and Puf5p is predicted to contact an adenine in the respective target RNAs, the amino acid in Puf3p must play a more critical role in promoting COX17 interaction. In contrast, an amino acid change in the third repeat of Puf3p, which interacts with the only divergent nucleotide between the Puf3p and Puf5p targets, had no effect on binding COX17. These results argue that a simple set of rules cannot reliably link specific amino acid positions with target specificity. Each of these amino acid changes in Puf3p enhanced binding to the Puf5p target HO RNA, suggesting a different mode of binding to this target. Finally, we identified an outer surface loop that was dispensable for binding but was required to promote both rapid deadenylation and subsequent decapping of the COX17 mRNA, most likely as a point of protein-protein interactions.

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., (Copyright 2004 RNA Society)