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PubMed2025

Proper 5'-3' cotranslational mRNA decay in yeast requires import of Xrn1 to the nucleus.

The budding yeast Xrn1 protein shuttles between the nucleus, where it stimulates transcription, and the cytoplasm, where it executes the major cytoplasmic mRNA decay. In the cytoplasm, apart from catalyzing 5'→3' decay onto non translated mRNAs, Xrn1 can follow the last translating ribosome to degrade the decapped mRNA template, a process known as "cotranslational mRNA decay". We have previously observed that the import of Xrn1 to the nucleus is required for efficient cytoplasmic mRNA decay. Here by using an Xrn1 mutant that cannot enter the nucleus, but is otherwise functional in ribonuclease activity, we show that nuclear import is necessary for proper global cotranslational decay of mRNAs along coding regions and also affects degradation in the of 5' region of a large group of mRNAs, which comprise about 20% of the transcriptome. Furthermore, a principal component analysis of the genomic datasets of this mutant and other Xrn1 mutants also shows that lack of a cytoplasmic 5'→3' exoribonuclease is the primary cause of the physiological defects seen in a xrn1Δ mutant, but also suggests that Xrn1 import into the nucleus is necessary for its full in vivo functions.

Exoribonucleases↗

PubMed1990

Translation and a 42-nucleotide segment within the coding region of the mRNA encoded by the MAT alpha 1 gene are involved in promoting rapid mRNA decay in yeast.

In yeast, the mRNA encoded by the MAT alpha 1 gene is unstable (t1/2 = 5 min) and the mRNAs encoded by the ACT1 gene (t1/2 = 30 min) and the PGK1 gene (t1/2 = 45 min) are stable. To understand the RNA structural features that dictate mRNA decay rates in yeast, we have constructed PGK1/MAT alpha 1 and ACT1/MAT alpha 1 gene fusions and analyzed the decay rates of the resultant chimeric transcripts. Fusion of a MAT alpha 1 segment containing 73% of the coding region and the 3' untranslated region to either of the stable genes is sufficient to cause rapid decay of the chimeric mRNAs (t1/2 = 6-7.5 min). Sequences required for this rapid decay are not found in the MAT alpha 1 3' untranslated region but are located within a 42-nucleotide segment of the coding region that has a high content (8 out of 14) of rare codons. Introduction of a translational stop codon upstream of this region stabilizes the hybrid mRNAs, indicating that the rapid decay promoted by these sequences is dependent on ribosomal translocation.

Base Sequence↗

PubMed2005

Gene set coregulated by the Saccharomyces cerevisiae nonsense-mediated mRNA decay pathway.

The nonsense-mediated mRNA decay (NMD) pathway has historically been thought of as an RNA surveillance system that degrades mRNAs with premature translation termination codons, but the NMD pathway of Saccharomyces cerevisiae has a second role regulating the decay of some wild-type mRNAs. In S. cerevisiae, a significant number of wild-type mRNAs are affected when NMD is inactivated. These mRNAs are either wild-type NMD substrates or mRNAs whose abundance increases as an indirect consequence of NMD. A current challenge is to sort the mRNAs that accumulate when NMD is inactivated into direct and indirect targets. We have developed a bioinformatics-based approach to address this challenge. Our approach involves using existing genomic and function databases to identify transcription factors whose mRNAs are elevated in NMD-deficient cells and the genes that they regulate. Using this strategy, we have investigated a coregulated set of genes. We have shown that NMD regulates accumulation of ADR1 and GAL4 mRNAs, which encode transcription activators, and that Adr1 is probably a transcription activator of ATS1. This regulation is physiologically significant because overexpression of ADR1 causes a respiratory defect that mimics the defect seen in strains with an inactive NMD pathway. This strategy is significant because it allows us to classify the genes regulated by NMD into functionally related sets, an important step toward understanding the role NMD plays in the normal functioning of yeast cells.

Binding Sites↗

PubMed1995

Identification and characterization of a sequence motif involved in nonsense-mediated mRNA decay.

In both prokaryotes and eukaryotes, nonsense mutations in a gene can enhance the decay rate or reduce the abundance of the mRNA transcribed from that gene, and we call this process nonsense-mediated mRNA decay. We have been investigating the cis-acting sequences involved in this decay pathway. Previous experiments have demonstrated that, in addition to a nonsense codon, specific sequences 3' of a nonsense mutation, which have been defined as downstream elements, are required for mRNA destabilization. The results presented here identify a sequence motif (TGYYGATGYYYYY, where Y stands for either T or C) that can predict regions in genes that, when positioned 3' of a nonsense codon, promote rapid decay of its mRNA. Sequences harboring two copies of the motif from five regions in the PGK1, ADE3, and HIS4 genes were able to function as downstream elements. In addition, four copies of this motif can function as an independent downstream element. The sequences flanking the motif played a more significant role in modulating its activity when fewer copies of the sequence motif were present. Our results indicate the sequences 5' of the motif can modulate its activity by maintaining a certain distance between the sequence motif and the termination codon. We also suggest that the sequences 3' of the motif modulate the activity of the downstream element by forming RNA secondary structures. Consistent with this view, a stem-loop structure positioned 3' of the sequence motif can enhance the activity of the downstream element. This sequence motif is one of the few elements that have been identified that can predict regions in genes that can be involved in mRNA turnover. The role of these sequences in mRNA decay is discussed.

Alcohol Oxidoreductases↗

PubMed2005

Identification of in vivo P-glycoprotein mRNA decay intermediates in normal liver but not in liver tumors.

Post-transcriptional regulation at the level of mRNA stability is one important mechanism for over-expression of P-glycoprotein (Pgp) genes observed in cultured cells and in animals. A previous study has shown that mRNA half-lives for Pgp genes in normal liver were less than 2 h, in contrast to greater than 12 h measured in a transplantable liver tumor line. This lower turnover rate of Pgp mRNA may, in large part, contribute to the abundance of Pgp mRNA in liver tumors. The current study sought to investigate the underlying mechanism for the lower turnover rate of Pgp2 mRNA previously determined in liver tumors. As a first approach, we set out to understand the Pgp2 mRNA decay in both normal liver and liver tumors by first identifying and characterizing Pgp2 mRNA degradation intermediates. In this study, we showed that the sensitive ligation-mediated polymerase chain reaction (LM-PCR) method can be used to detect a homogenous pool of in vitro transcribed RNA down to 0.4 ng. By employing gene-specific primers in the LM-PCR method, we successfully identified four Pgp2 mRNA decay intermediates in normal liver. All four decay intermediates detected correspond to the 5' coding region of Pgp2 mRNA, and surprisingly no decay intermediates which correspond to 3' untranslated region, 3' coding region or middle coding region were found using LM-PCR. The identified decay intermediates are unique to the normal liver as they were absent or present at very low level in all three liver tumor samples analyzed. This observation supports our previous findings that the Pgp mRNA turnover rate is lower in liver tumors than in normal liver. These findings have implications for our understanding of the regulation of Pgp mRNA turnover in normal and malignant tissues.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2006

Tethering KSRP, a decay-promoting AU-rich element-binding protein, to mRNAs elicits mRNA decay.

Inherently unstable mRNAs contain AU-rich elements (AREs) in their 3' untranslated regions that act as mRNA stability determinants by interacting with ARE-binding proteins (ARE-BPs). We have destabilized two mRNAs by fusing sequence-specific RNA-binding proteins to KSRP, a decay-promoting ARE-BP, in a tethering assay. These results support a model that KSRP recruits mRNA decay machinery/factors to elicit decay. The ability of tethered KSRP to elicit mRNA decay depends on functions of known mRNA decay enzymes. By targeting the Rev response element of human immunodeficiency virus type 1 by using Rev-KSRP fusion protein, we degraded viral mRNA, resulting in a dramatic reduction of viral replication. These results provide a foundation for the development of novel therapeutic strategies to inhibit specific gene expression in patients with acquired or hereditary diseases.

3' Untranslated Regions↗

PubMed2002

Precision and functional specificity in mRNA decay.

Posttranscriptional processing of mRNA is an integral component of the gene expression program. By using DNA microarrays, we precisely measured the decay of each yeast mRNA, after thermal inactivation of a temperature-sensitive RNA polymerase II. The half-lives varied widely, ranging from approximately 3 min to more than 90 min. We found no simple correlation between mRNA half-lives and ORF size, codon bias, ribosome density, or abundance. However, the decay rates of mRNAs encoding groups of proteins that act together in stoichiometric complexes were generally closely matched, and other evidence pointed to a more general relationship between physiological function and mRNA turnover rates. The results provide strong evidence that precise control of the decay of each mRNA is a fundamental feature of the gene expression program in yeast.

Glycolysis↗

PubMed1988

Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.

DNA↗

PubMed1997

Upf1p, Nmd2p, and Upf3p are interacting components of the yeast nonsense-mediated mRNA decay pathway.

Rapid turnover of nonsense-containing mRNAs in Saccharomyces cerevisiae is dependent on Upf1p, Nmd2p, and Upf3p, the products of the UPF1, NMD2/UPF2, and UPF3 genes, respectively. We showed previously that Upf1p and Nmd2p interact and that this interaction is required for nonsense-mediated mRNA decay (F. He and A. Jacobson, Genes Dev. 9:437-454, 1995; F. He, A. H. Brown, and A. Jacobson, RNA 2:153-170, 1996). In this study we have used the yeast two-hybrid system to define other protein-protein interactions among the essential components of this decay pathway. Nmd2p-Upf3p and Upf1p-Upf3p interactions were identified, and the respective domains involved in these interactions were delineated by deletion analysis. The domains of Upf1p and Upf3p putatively involved in their mutual interaction were found to correspond to the domains on the two proteins which interact with Nmd2p, suggesting that Nmd2p bridges Upf1p and Upf3p. This conclusion was reinforced by experiments showing that: (i) deletion of NMD2 completely abolishes interactions between Upf1p and Upf3p and (ii) overexpression of full-length Nmd2p or Nmd2p fragments that retain Upf1p- and Upf3p-interacting domains promotes 10- to 200-fold enhancement of Upf1p-Nmd2p-Upf3p complex formation. These results; the observation that cells harboring either single or multiple deletions of UPF1, NMD2, and UPF3 inhibit nonsense-mediated mRNA decay to the same extent; and an analysis of the possible targets of a dominant-negative NMD2 allele indicate that Upf1p, Nmd2p, Upf3p, and at least one other factor are functionally dependent, interacting components of the yeast nonsense-mediated mRNA decay pathway.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Utilizing the GCN4 leader region to investigate the role of the sequence determinants in nonsense-mediated mRNA decay.

In the yeast Saccharomyces cerevisiae, premature translation termination promotes rapid degradation of mRNAs. Accelerated decay requires the presence of specific cis-acting sequences which have been defined as downstream elements. It has been proposed that the role of the downstream element may be to promote translational reinitiation or ribosomal pausing. The GCN4 gene produces an mRNA that contains four short upstream open reading frames (uORFs) preceding the GCN4 protein-coding region in which translational initiation and reinitiation events occur. It was anticipated that these uORFs would function in a manner analogous to nonsense codons, promoting rapid degradation of the mRNA. However, the GCN4 transcript was not degraded by the nonsense-mediated mRNA decay pathway. We have investigated the role of the leader region of the GCN4 transcript in an effort to identify possible sequence elements that inactivate this decay pathway. We show that the GCN4 leader region does not harbor a downstream element needed to promote mRNA decay. In addition, using hybrid GCN4-PGK1 transcripts, we demonstrate that if a translational reinitiation signal precedes a downstream element, the mRNA will no longer be sensitive to nonsense-mediated decay. Furthermore, we demonstrate that the downstream element is functional only after a translational initiation and termination cycle has been completed but is unable to promote nonsense-mediated mRNA decay if it is situated 5' of a translational initiation site. Based on these results, the role of the downstream element will be discussed.

Base Sequence↗

PubMed2006

Upf1/Upf2 regulation of 3' untranslated region splice variants of AUF1 links nonsense-mediated and A+U-rich element-mediated mRNA decay.

AUF1 is an RNA-binding protein that targets mRNAs containing A+U-rich elements (AREs) for rapid cytoplasmic turnover. Alternative pre-mRNA splicing produces five variants of AUF1 mRNA that differ in the composition of their 3'-untranslated regions (3'-UTRs). Previous work suggested that this heterogeneity in 3'-UTR sequence could regulate AUF1 expression by two potential mechanisms. First, AUF1 may regulate its own expression by binding to AREs in 3'-UTR splice variants that retain intron 9. The second potential mechanism, and the focus of this report, is regulation of a subset of 3'-UTR splice variants by the nonsense-mediated mRNA decay (NMD) pathway. Two of the five AUF1 mRNA 3'-UTR variants position the translational termination codon more than 50 nucleotides upstream of an exon-exon junction, creating a potential triggering signal for NMD in mammalian cells. Disruption of cellular NMD pathways by RNA interference-mediated knockdown of Upf1/Rent1 or Upf2/Rent2 or transfection of a dominant-negative Upf1 mutant specifically enhanced expression of these two candidate NMD substrate mRNAs in cells, involving stabilization of each transcript. Ribonucleoprotein immunoprecipitation experiments revealed that both Upf1 and Upf2 can associate with an NMD-sensitive AUF1 mRNA 3'-UTR variant in cells. Finally, quantitation of AUF1 mRNA 3'-UTR splice variants during murine embryonic development showed that the expression of NMD-sensitive AUF1 mRNAs is specifically enhanced as development proceeds, contributing to dynamic changes in AUF1 3'-UTR structures during embryogenesis. Together, these studies provide the first evidence of linkage between the nonsense- and ARE-mediated mRNA decay pathways, which may constitute a new mechanism regulating the expression of ARE-containing mRNAs.

3' Untranslated Regions↗

PubMed2005

SMG7 is a 14-3-3-like adaptor in the nonsense-mediated mRNA decay pathway.

In metazoa, regulation of the phosphorylation state of UPF1 is crucial for nonsense-mediated mRNA decay (NMD), a process by which aberrant mRNAs containing nonsense mutations are degraded. UPF1 is targeted for dephosphorylation by three related proteins, SMG5, SMG6, and SMG7. We report here the crystal structure of the N-terminal domain of SMG7. The structure reveals that SMG7 contains a 14-3-3-like domain. Residues that bind phosphoserine-containing peptides in 14-3-3 are conserved at the equivalent positions in SMG7. Mutation of these residues impairs UPF1 binding to SMG7 in vitro and UPF1 recruitment to cytoplasmic mRNA decay foci in vivo, suggesting that SMG7 acts as an adaptor in targeting mRNAs associated with phosphorylated UPF1 for degradation. The 14-3-3 site of SMG7 is conserved in SMG5 and SMG6. These data also imply that the homologous human Est1 might have a 14-3-3 function at telomeres, and that phosphorylation events may be important for telomerase regulation.

14-3-3 Proteins↗

PubMed1988

mRNA decay rates in late-developing Dictyostelium discoideum cells are heterogeneous, and cyclic AMP does not act directly to stabilize cell-type-specific mRNAs.

We reevaluated the use of 32PO4 pulse-chases for analyzing mRNA decay rates in late-developing Dictyostelium cells. We found that completely effective PO4 chases could not be obtained in developing cells and that, as a consequence, the decay rates exhibited by some mRNAs were influenced by the rates at which they were transcribed. In developing cells disaggregated in the presence of cyclic AMP, the poly(A)+ mRNA population turned over with an apparent half-life of 4 h, individual mRNA decay rates were heterogeneous, and some prestalk and prespore mRNAs appeared to decay with biphasic kinetics. In cells disaggregated in the absence of cyclic AMP, all prestalk and prespore mRNAs decayed with biphasic kinetics. During the first 1 to 1.5 h after disaggregation in the absence of cyclic AMP, the cell-type-specific mRNAs were selectively degraded, decaying with half-lives of 20 to 30 min; thereafter, the residual prestalk and prespore mRNA molecules decayed at rates that were similar to those measured in the presence of cyclic AMP. This short-term labilization of cell-type-specific mRNAs was observed even for those species not requiring cyclic AMP for their accumulation in developing cells. The observation that cell-type specific mRNAs can decay at similar rates in disaggregated cells with or without cyclic AMP indicates that this compound does not act directly to stabilize prestalk and prespore mRNAs during development and that its primary role in the maintenance of cyclic-AMP-dependent mRNAs is likely to be transcriptional.

Cell Aggregation↗

PubMed1996

Interaction between Nmd2p and Upf1p is required for activity but not for dominant-negative inhibition of the nonsense-mediated mRNA decay pathway in yeast.

Rapid turnover of nonsense-containing mRNAs in the yeast Saccharomyces cerevisiae is dependent on the products of the UPF1 (Upf1p), NMD2/UPF2 (Nmd2p) and UPF3 (Upf3p) genes. Mutations in each of these genes lead to the selective stabilization of mRNAs containing early nonsense mutations without affecting the decay rates of most other mRNAs. NMD2 was recently identified in a two-hybrid screen as a gene that encodes a Upf1p-interacting protein. To identify the amino acids essential to this interaction, we used two-hybrid analysis as well as missense, nonsense, and deletion mutants of NMD2, and mapped the Upf1p-interacting domain of Nmd2p to a 157-amino acid segment at its C-terminus. Mutations in this domain that disrupt interaction with Upf1p also disrupt nonsense-mediated mRNA decay. A dominant-negative deletion allele of NMD2 identified previously includes the Upf1p-interacting domain. However, mutations in the Upf1p-interacting domain do not affect dominant-negative inhibition of mRNA decay caused by this allele, suggesting interaction with yet another factor. These results, and the observation that deletion of a putative nuclear localization signal and a putative transmembrane domain also inactivate nonsense-mediated mRNA decay, suggest that Nmd2p may contain as many as four important functional domains.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Inhibition of nonsense-mediated mRNA decay rescues the phenotype in Ullrich's disease.

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance system that eliminates aberrant mRNAs containing premature translation termination codons (PTCs). We evaluated the role of NMD in of Ullrich's disease. The patient has a frameshift mutation with a PTC in the collagen VI alpha2 gene causing the loss of collagen VI and functional defects in extracellular matrix (ECM). The pharmacological block of NMD caused upregulation of the mutant collagen VI alpha2 subunit, resulting in collagen VI assembly and partially functional ECM formation. Our results suggest that NMD inhibitors can be used as a therapeutic tool to rescue some human genetic diseases exacerbated by NMD.

Androstadienes↗

PubMed2005

Nonsense-mediated mRNA decay: molecular insights and mechanistic variations across species.

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance pathway that ensures the rapid degradation of mRNAs containing premature translation termination codons (PTCs), thereby preventing the synthesis of truncated and potentially harmful proteins. In addition, this pathway regulates the expression of approximately 10% of the transcriptome and is essential in mice. Although NMD is conserved in eukaryotes, recent studies in several organisms have revealed that different mechanisms have evolved to discriminate natural from premature stop codons and to degrade the targeted mRNAs. With the elucidation of the first crystal structures of components of the NMD machinery, the way is paved towards a molecular understanding of the protein interaction network underlying this process.

Animals↗

PubMed2003

Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation.

Messenger RNA decay, which is a regulated process intimately linked to translation, begins with the deadenylation of the poly(A) tail at the 3' end. However, the precise mechanism triggering the first step of mRNA decay and its relationship to translation have not been elucidated. Here, we show that the translation termination factor eRF3 mediates mRNA deadenylation and decay in the yeast Saccharomyces cerevisiae. The N-domain of eRF3, which is not necessarily required for translation termination, interacts with the poly(A)-binding protein PABP. When this interaction is blocked by means of deletion or overexpression of the N-domain of eRF3, half-lives of all mRNAs are prolonged. The eRF3 mutant lacking the N-domain is deficient in the poly(A) shortening. Furthermore, the eRF3-mediated mRNA decay requires translation to proceed, especially ribosomal transition through the termination codon. These results indicate that the N-domain of eRF3 mediates mRNA decay by regulating deadenylation in a manner coupled to translation.

Codon↗

PubMed1999

Analysis of mRNA decay pathways in Saccharomyces cerevisiae.

The analysis of mRNA turnover often requires a knowledge of the pathway by which a particular mRNA is being degraded. In this article we describe experimental procedures that can be used to determine the mechanism of degradation for yeast transcripts. These approaches include the insertion of strong secondary structures to block exonuclease cleavage and thereby trap decay intermediates. In addition, mRNA decay pathways can be analyzed by using regulatable promoters to perform transcriptional pulse-chase experiments, thereby allowing the determination of precursor-product relationships during the mRNA decay process. Finally, the mechanism of mRNA degradation can also now be determined by using trans-acting mutations specific for distinct mRNA turnover pathways. Most importantly, the combination of these three approaches can often clearly define the mechanism(s) by which a given transcript is degraded.

Genes, Fungal↗