Abstract
The PUF family of RNA-binding proteins regulate gene expression post-transcriptionally. Saccharomyces cerevisiae Puf3p is characterised as binding nuclear-encoded mRNAs specifying mitochondrial proteins. Extensive studies of its regulation of COX17 demonstrate its role in mRNA decay. Using integrated genome-wide approaches we define an expanded set of Puf3p target mRNAs and quantitatively assessed the global impact of loss of PUF3 on gene expression using mRNA and polysome profiling and quantitative proteomics. In agreement with prior studies, our sequencing of affinity-purified Puf3-TAP associated mRNAs (RIP-seq) identified mRNAs encoding mitochondrially-targeted proteins. Additionally, we also found 720 new mRNA targets that predominantly encode proteins that enter the nucleus. Comparing transcript levels in wild-type and puf3∆ cells revealed that only a small fraction of mRNA levels alter, suggesting Puf3p determines mRNA stability for only a limited subset of its target mRNAs. Finally, proteomic and translatomic studies suggest that loss of Puf3p has widespread, but modest, impact on mRNA translation. Taken together our integrated multi-omics data point to multiple classes of Puf3p targets, which display coherent post-transcriptional regulatory properties and suggest Puf3p plays a broad, but nuanced, role in the fine-tuning of gene expression.
Highlights
Post-transcriptional regulation of mRNA is central to diverse cellular processes and plays an important role in the overall control of gene expression
COX17 has been a useful model for studies of mRNA decay mechanisms, but it is unclear how typical COX17 is of Puf3p target mRNAs
Puf3p binds sequence motifs frequently found within the 3′ untranslated regions (3′ UTR) of its target mRNAs
Summary
Post-transcriptional regulation of mRNA is central to diverse cellular processes and plays an important role in the overall control of gene expression. Post-transcriptional regulation can be achieved by several mechanisms including the recognition of mRNAs by multiple general and specific RNA binding proteins (RBPs) that modulate their fate[2]. RNAs bound to Puf3-TAP were captured via immunoprecipitation on IgG beads (RIP) and subsequently identified by microarrays (chip); 225 mRNAs predominantly encoding mitochondrial targeted proteins were bound significantly, implicating Puf3p in regulating the expression of multiple mitochondrial proteins. COX17 has been a useful model for studies of mRNA decay mechanisms, but it is unclear how typical COX17 is of Puf3p target mRNAs. Puf3p can act to localise specific mRNAs to mitochondria and to enhance mRNA degradation, recent studies suggest more complex roles for Puf3p. Puf3p was shown to interact with translating ribosomes[17] and in an RNA-dependent manner with multiple members of the ‘closed loop’ complex[21]. Most of the new mRNAs found were not mitochondrial, raising questions as to how comprehensive/selective each study was in identifying Puf3p target mRNAs
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