Global Analysis of Pub1p Targets Reveals a Coordinate Control of Gene Expression through Modulation of Binding and Stability

Abstract

Regulation of mRNA turnover is an important cellular strategy for posttranscriptional control of gene expression, mediated by the interplay of cis-acting sequences and associated trans-acting factors. Pub1p, an ELAV-like yeast RNA-binding protein with homology to T-cell internal antigen 1 (TIA-1)/TIA-1-related protein (TIAR), is an important modulator of the decay of two known classes of mRNA. Our goal in this study was to determine the range of mRNAs whose stability is dependent on Pub1p, as well as to identify specific transcripts that directly bind to this protein. We have examined global mRNA turnover in isogenic PUB1 and pub1⌬ strains through gene expression analysis and demonstrate that 573 genes exhibit a significant reduction in half-life in a pub1⌬ strain. We also examine the binding specificity of Pub1p using affinity purification followed by microarray analysis to comprehensively distinguish between direct and indirect targets and find that Pub1p significantly binds to 368 cellular transcripts. Among the Pub1p-associated mRNAs, 53 transcripts encoding proteins involved in ribosomal biogenesis and cellular metabolism are selectively destabilized in the pub1⌬ strain. In contrast, genes involved in transporter activity demonstrate association with Pub1p but display no measurable changes in transcript stability. Characterization of two candidate genes, SEC53 and RPS16B, demonstrate that both Pub1p-dependent regulation of stability and Pub1p binding require 3 untranslated regions, which harbor distinct sequence motifs. These results suggest that Pub1p binds to discrete subsets of cellular transcripts and posttranscriptionally regulates their expression at multiple levels.Gene expression in eukaryotes is a highly diverse process, involving regulation at both transcriptional and posttranscriptional levels (47, 70). The process of mRNA turnover is an important posttranscriptional control point that helps to modulate the cellular abundance of a transcript. A large number of clinically relevant transcripts exhibit regulated decay in response to cellular signals, and the deregulation of their decay rates directly correlates with disease states (58,66,70). In the yeast Saccharomyces cerevisiae, the principal mRNA degradation pathway initiates with the removal of the poly(A) tail, followed by either decapping and 5Ј33Ј exonucleolytic decay or 3Ј35Ј exosome-mediated degradation (10, 64). In addition to this pathway, aberrant mRNAs harboring premature termination codons are degraded by an alternate nonsense-mediated decay (NMD) pathway that functions to ensure quality control of gene expression. NMD is initiated when a premature termination codon is recognized during translation termination and stimulates rapid deadenylation-independent decapping, followed by 5Ј33Јdegradation of the mRNA (4). Messages undergoing leaky scanning (69) or harboring upstream open reading frames (50, 59) represent some naturally occurring substrates that decay through this pathway.The turnover of mRNAs is mediated by the interplay between...

Mating Pheromone in Cryptococcus neoformans Is Regulated by a Transcriptional/Degradative “Futile” Cycle

“…Perturbations of MF␣ transcriptional levels after VAD1 gene dosing manipulation also correlated with MF␣ protein levels and filamentation, suggesting the central role of MF␣ in sexual development (15). This is similar to the role of Pub1p, an ELAV-like yeast RNAbinding protein with homology to TIA-1 (T-cell internal antigen 1)/ TIAR (TIA-1-related protein), which binds to discrete subsets of cellular transcripts, regulating their expression at multiple levels (45). Indeed, Vad1 of C. neoformans shows homology to the Dhh1 RNA-binding protein of S. cerevisiae, a component of the cytoplasmic mRNA degradation complex, having 74% identity at the protein level (20,40,48,49).…”

“…Finding a role for MF␣1 3Ј-UTR would suggest a function in RNA stability affecting processes such as deadenylation, which is dependent on the 3Ј-UTR (45). To test these possibilities, we expressed MF␣1-GFP, replacing the MF␣1 3Ј-UTR with an unrelated EF1-␣ 3Ј-UTR, which has been successfully used to express proteins in C. neoformans (25).…”

Yeast translational response to high salinity: Global analysis reveals regulation at multiple levels

“…These motifs contain A-rich, U-rich, or A:U-rich (ARE) sequences and are highly similar to elements that were found as binding sites for the ARE-binding protein Pub1 ( Fig. 9B; Duttagupta et al 2005). Application of the Tomtom algorithm (Gupta et al 2007) to measure the statistical similarity between a Pub1 motif and its corresponding Hog1 motif revealed for all pairs a significant similarity (E À4 for all pairs and the lowest was for the U-rich motifs in the 39 UTRs [E = 10 À8 ]).…”

Cis-regulatory elements explain most of the mRNA stability variation across genes in yeast

“…4A, Materials and Methods). These include three described motifs: the Puf3 binding motif TGTAAATA (FDR = 3.2 × 10 −5 , median fold-change 1.29) (Gerber et al 2004;Gupta et al 2014), the Whi3 binding motif TGCAT (FDR = 7 × 10 −4 , median fold-change 1.24) (Colomina et al 2008;Cai and Futcher 2013), and a poly(U) motif TTTTTTA (FDR = 0.09, median fold-change 1.20), which can be bound by Pub1 (Duttagupta et al 2005), or is part of the long poly(U) stretch that forms a looping structure with a poly(A) tail (Geisberg et al 2014). Moreover, an uncharacterized motif, ATATTC, was associated with lower mRNA half-life (FDR = 2 × 10 −5 , median fold-change 1.24).…”

“…Here we use the formal definition of CRE, i.e., a regulatory element affecting expression of the gene it belongs to in an allele-specific manner (Rockman and Kruglyak 2006;Skelly et al 2009). CREs affecting mRNA stability include but are not limited to secondary structure (Rabani et al 2008;Geisberg et al 2014), sequence motifs present in the 3 ′ UTR including binding sites of RNA-binding proteins (Olivas and Parker 2000;Duttagupta et al 2005;Shalgi et al 2005;Hogan et al 2008;Hasan et al 2014), and, in higher eukaryotes, microRNAs (Lee et al 1993). Moreover, translation-related features are frequently associated with mRNA stability.…”