Gene expression in eukaryotes is a highly diverse process, involving regulation at both transcriptional and posttranscriptional levels (47
). 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
). In the yeast Saccharomyces cerevisiae
, the principal mRNA degradation pathway initiates with the removal of the poly(A) tail, followed by either decapping and 5′→3′ exonucleolytic decay or 3′→5′ exosome-mediated degradation (10
). 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 5′→3′degradation of the mRNA (4
). Messages undergoing leaky scanning (69
) or harboring upstream open reading frames (50
) represent some naturally occurring substrates that decay through this pathway.
The turnover of mRNAs is mediated by the interplay between a number of different cis
-acting sequences localized in the target substrate and the various trans-
acting factors that interact with them (70
). In both yeast and in mammalian systems, there are a large number of RNA-binding proteins (RBPs) that can act as either enhancers or inhibitors of stability and translation efficiency (70
). Many of these RBPs mediate their effects by selective binding to target motifs in the 3′ untranslated region (3′UTR). Examples include the UGU element for the yeast Puf proteins (32
) and the AU-rich elements (AREs) interacting with the mammalian ARE-binding proteins such as HuR, Hsp70, TTP, BRF1, AUF1, and KSRP (7
). In addition to the 3′UTR, cis
-acting elements may also be present in the 5′UTR (9
) or in the coding region of transcripts (51
). In addition to modulating stability, many of these 5′- and 3′UTR elements can also actively regulate translation by interacting with specific factors. Examples of this type of regulation include the translational repression of the tumor necrosis factor alpha (TNF-α) mRNA by the ARE-binding protein T-cell internal antigen 1 (TIA-1)/TIA-1-related protein (TIAR) (55
) or the control of ferritin mRNA translation through the iron response element-binding protein (20
The poly(U)-binding protein (Pub1p) is a yeast homologue of the mammalian ELAV-like proteins HuR and TIA-1/TIAR (1
). Pub1p has been recently implicated as a regulator of cellular mRNA decay (59
). This abundant 51-kDa RBP containing three RNA recognition motifs (43
) modulates the stability of targets that are degraded through at least two mechanistically distinct pathways. Similar to HuR, which stabilizes ARE-bearing transcripts degrading though the deadenylation-dependent pathway in mammalian cells (8
), Pub1p can specifically bind to a chimeric yeast mRNA bearing the TNF-α-ARE and stabilize this transcript (65
). Analogous to ARE-dependent regulation in mammalian systems, stabilization by Pub1p requires glucose, which activates the p38 MAP kinase pathway (65
). Additionally, Pub1p has also been shown to selectively bind to a stabilizer element (STE) located in the 5′UTR of the upstream open reading frame (upstream ORF)-containing transcripts YAP1 and GCN4 and to prevent their turnover through the NMD pathway (59
). These results demonstrate the Pub1p can bind to at least two classes of stability elements and modulate decay, based on cellular conditions.
Several lines of evidence also suggest that Pub1p may be involved in other aspects of mRNA metabolism. Both mammalian homologues of Pub1p, HuR and the TIA-1/TIAR, are involved in translational regulation. While HuR acts as a translational enhancer or repressor (42a
), the TIA-1 and TIAR proteins are involved in ARE-mediated translational repression (55
). Significantly, Pub1p has been shown to be associated with nonpolysomal mRNAs (1
); moreover, it binds to the 3′UTRs of endogenous ARE-containing transcripts such as MFA2 and TIF51A but does not modulate their stability under any conditions analyzed (65
; S. Vasudevan and S. W. Peltz, unpublished observations). Taken together, these results suggest that, in addition to regulating decay, Pub1p may also have a role in modulating translation efficiency. Furthermore, the TIA-1/TIAR proteins have also been implicated as activators of constitutive and alternative splicing (16
). As Pub1p is equally abundant in both the nucleus and cytoplasm (1
) and demonstrates interaction with Nab2p (31
), a factor involved in mRNA processing and export (26
), this additionally suggests that the protein might play nuclear roles, such as regulating the maturation and biogenesis of mRNAs.
The high cellular abundance of Pub1p and its ability to regulate the turnover of two known classes of transcripts makes it likely that Pub1p is a key trans
-acting factor mediating the stability of multiple mRNAs. In addition, the glucose dependence of Pub1p function suggests that this protein has the potential to mediate large changes in gene expression in response to cellular conditions. As expression and transcript stabilities of a large fraction of genes have recently been demonstrated to be regulated by RNA-binding proteins (22
) and can vary depending on cellular states such as changes in carbon source (14
), we hypothesize that Pub1p acts as a global regulator of gene expression engaged in coordinating the expression of a wide network of mRNAs. The goal of this work is to characterize the role of Pub1p in regulating mRNA stability and to identify classes of transcripts that associate with the protein.
To therefore distinguish classes of Pub1p-regulated transcripts, we have used cDNA arrays to query the yeast genome for potential Pub1p targets. Our results measuring global mRNA stability in wild-type and isogenic pub1Δ strains under glucose conditions show that nearly 10% of all yeast mRNAs decay in a Pub1p-dependent manner. The majority of these messages, representing 9% of all transcripts, exhibit destabilization in a pub1Δ strain. The remaining 1% of mRNAs, by contrast, are significantly stabilized. Furthermore, through an independent affinity purification approach, we demonstrate that Pub1p can directly associate with 368 cellular transcripts, which represent 6% of the genome. Classification and analysis of these mRNA populations reveal discrete subsets of transcripts whose decay is either directly or indirectly dependent on Pub1p. In addition, we also find groups of transcripts that demonstrate Pub1p binding without affecting mRNA half-lives, suggesting that Pub1p controls other aspects of their metabolism such as translation or mRNA export. Furthermore, each of these subsets of genes can be organized into distinct functional categories, suggesting a role of Pub1p in diverse biological processes. We identify putative regulatory motifs in the UTR regions of the Pub1p-associated mRNAs and demonstrate that transcripts exhibiting altered decay are specifically enriched in U-rich motifs, suggesting a role of these elements in modulating mRNA turnover. Finally, analysis of two candidate transcripts, RPS16B and SEC53, demonstrates that Pub1p-dependent regulation of stability requires sequences in their 3′UTR.
Taken together, these results implicate Pub1p as a major trans
-acting factor involved in cellular mRNA decay and suggest a novel role of Pub1p in regulating other posttranscriptional processes. Our work extends the observation of Grigull et al. measuring global decay using 1,10 phenanthroline (22
) by revealing that distinct groups of Pub1p targets exhibit coordinate regulation; moreover, a direct correlation between binding and stability can be established for a subset of these messages. Furthermore, we show the presence of conserved elements in the Pub1p target and set the stage for uncovering how Pub1p binding can lead to the modulation of mRNA decay rates or other posttranscriptional processes (33
). The results from this analysis join a growing body of studies measuring genome-wide stability and RNA-protein interactions (19
) and reiterate the extensive role RNA-binding proteins play in posttranscriptional control of gene expression.