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PubMed2004

Nonsense-mediated mRNA decay: terminating erroneous gene expression.

Nonsense-mediated mRNA decay is a surveillance pathway that reduces errors in gene expression by eliminating aberrant mRNAs that encode incomplete polypeptides. Recent experiments suggest a working model whereby premature and normal translation termination events are distinct as a consequence of the spatial relationship between the termination codon and mRNA binding proteins, a relationship partially established by nuclear pre-mRNA processing. Aberrant termination then leads to both translational repression and an increased susceptibility of the mRNA to multiple ribonucleases.

3' Untranslated Regions↗

PubMed2004

Leaky termination at premature stop codons antagonizes nonsense-mediated mRNA decay in S. cerevisiae.

The Nonsense-Mediated mRNA Decay (NMD) pathway mediates the rapid degradation of mRNAs that contain premature stop mutations in eukaryotic organisms. It was recently shown that mutations in three yeast genes that encode proteins involved in the NMD process, UPF1, UPF2, and UPF3, also reduce the efficiency of translation termination. In the current study, we compared the efficiency of translation termination in a upf1Delta strain and a [PSI(+)] strain using a collection of translation termination reporter constructs. The [PSI(+)] state is caused by a prion form of the polypeptide chain release factor eRF3 that limits its availability to participate in translation termination. In contrast, the mechanism by which Upf1p influences translation termination is poorly understood. The efficiency of translation termination is primarily determined by a tetranucleotide termination signal consisting of the stop codon and the first nucleotide immediately 3' of the stop codon. We found that the upf1Delta mutation, like the [PSI(+)] state, decreases the efficiency of translation termination over a broad range of tetranucleotide termination signals in a unique, context-dependent manner. These results suggest that Upf1p may associate with the termination complex prior to polypeptide chain release. We also found that the increase in readthrough observed in a [PSI(+)]/upf1Delta strain was larger than the readthrough observed in strains carrying either defect alone, indicating that the upf1Delta mutation and the [PSI(+)] state influence the termination process in distinct ways. Finally, our analysis revealed that the mRNA destabilization associated with NMD could be separated into two distinct forms that correlated with the extent the premature stop codon was suppressed. The minor component of NMD was a 25% decrease in mRNA levels observed when readthrough was >/=0.5%, while the major component was represented by a larger decrease in mRNA abundance that was observed only when readthrough was </=0.5%. This low threshold for the onset of the major component of NMD indicates that mRNA surveillance is an ongoing process that occurs throughout the lifetime of an mRNA.

Codon, Nonsense↗

PubMed1987

Autogenous regulation of histone mRNA decay by histone proteins in a cell-free system.

We tested the hypothesis that histone mRNA turnover is accelerated in the presence of free histone proteins. In an in vitro mRNA decay system, histone mRNA was degraded four- to sixfold faster in reaction mixtures containing core histones and a cytoplasmic S130 fraction than in reaction mixtures lacking these components. The decay rate did not change significantly when histones or S130 was added separately, suggesting either that the histones were modified and thereby activated by S130 or that additional factors besides histones were required. RecA, SSB (single-stranded binding), and histone proteins all formed complexes with histone mRNA, but only histones induced accelerated histone mRNA turnover. Therefore, the effect was not the result of random RNA-protein interactions. Moreover, histone proteins did not induce increased degradation of gamma globin mRNA, c-myc mRNA, or total poly(A)- or poly(A)+ polysomal mRNAs. This autoregulatory mechanism is consistent with the observed accumulation of cytoplasmic histone proteins in cells after DNA synthesis stops, and it can account, in part, for the rapid disappearance of histone mRNA at the end of S phase.

Cell-Free System↗

PubMed2004

The structural basis for the interaction between nonsense-mediated mRNA decay factors UPF2 and UPF3.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism by which eukaryotic cells detect and degrade transcripts containing premature termination codons. Three 'up-frameshift' proteins, UPF1, UPF2 and UPF3, are essential for this process in organisms ranging from yeast to human. We present a crystal structure at a resolution of 1.95 A of the complex between the interacting domains of human UPF2 and UPF3b, which are, respectively, a MIF4G (middle portion of eIF4G) domain and an RNP domain (ribonucleoprotein-type RNA-binding domain). The protein-protein interface is mediated by highly conserved charged residues in UPF2 and UPF3b and involves the beta-sheet surface of the UPF3b RNP domain, which is generally used by these domains to bind nucleic acids. We show that the UPF3b RNP does not bind RNA, whereas the UPF2 construct and the complex do. Our results advance understanding of the molecular mechanisms underlying the NMD quality control process.

Amino Acid Sequence↗

PubMed2004

Genetics of the DST-mediated mRNA decay pathway using a transgene-based selection.

mRNA sequences that control abundance, localization and translation initiation have been identified, yet the factors that recognize these sequences are largely unknown. In this report, a transgene-based strategy designed to isolate mutants of Arabidopsis thaliana that fail to recognize these sequences is described. In this strategy, a selectable gene and a screenable marker gene are put under the control of the sequence element being analysed and mutants are selected with altered abundance of the corresponding marker RNAs. The selection of mutants deficient in recognition of the DST (downstream) mRNA degradation signal is used as a test-case to illustrate some of the technical aspects that have facilitated success. Using this strategy, we report the isolation of a new mutant, dst3, deficient in the DST-mediated mRNA decay pathway. The targeted genetic strategy described circumvents certain technical limitations of biochemical approaches. Hence, it provides a means to investigate a variety of other mechanisms responsible for post-transcriptional regulation.

Hydrolysis↗

PubMed2006

Functions of the nonsense-mediated mRNA decay pathway in Drosophila development.

Nonsense-mediated mRNA decay (NMD) is a cellular surveillance mechanism that degrades transcripts containing premature translation termination codons, and it also influences expression of certain wild-type transcripts. Although the biochemical mechanisms of NMD have been studied intensively, its developmental functions and importance are less clear. Here, we describe the isolation and characterization of Drosophila "photoshop" mutations, which increase expression of green fluorescent protein and other transgenes. Mapping and molecular analyses show that photoshop mutations are loss-of-function mutations in the Drosophila homologs of NMD genes Upf1, Upf2, and Smg1. We find that Upf1 and Upf2 are broadly active during development, and they are required for NMD as well as for proper expression of dozens of wild-type genes during development and for larval viability. Genetic mosaic analysis shows that Upf1 and Upf2 are required for growth and/or survival of imaginal cell clones, but this defect can be overcome if surrounding wild-type cells are eliminated. By contrast, we find that the PI3K-related kinase Smg1 potentiates but is not required for NMD or for viability, implying that the Upf1 phosphorylation cycle that is required for mammalian and Caenorhabditis elegans NMD has a more limited role during Drosophila development. Finally, we show that the SV40 3' UTR, present in many Drosophila transgenes, targets the transgenes for regulation by the NMD pathway. The results establish that the Drosophila NMD pathway is broadly active and essential for development, and one critical function of the pathway is to endow proliferating imaginal cells with a competitive growth advantage that prevents them from being overtaken by other proliferating cells.

Animals↗

PubMed1978

Translation and mRNA decay.

Degradation of messenger RNA from the lactose operon (lac mRNA) was measured during the inhibition of protein synthesis by chloramphenicol (CM) or of translation-initiation by kasugamycin (KAS). With increasing CM concentration mRNA decay becomes slower, but there is no direct proportionality between rates of chemical decay and polypeptide synthesis. During exponential growth lac mRNA is cleaved endonucleolytically (Blundell and Kennell, 1974). At a CM concentration which completely inhibits all polypeptide synthesis this cleavage is blocked. In contrast, if only the initiation of translation is blocked by addition of KAS, the cleavage rate as well as the rate of chemical decay are increased significantly without delay. These faster rates do not result from immediate degradation of the lengthening stretch of ribosome-free proximal message, since the full-length size is present and the same discrete message sizes are generated during inhibition. These results suggest that neither ribosomes nor translation play an active role in the degradative process. Rather, targets can be protected by the proximity of a ribosome, and without nearly ribosomes the probability of cleavage becomes very high. During normal growth there is a certain probability that any message is in such a vulnerable state, and the fraction of vulnerable molecules determines the inactivation rate of that species.

Aminoglycosides↗

PubMed1991

Genetic studies of cleavage-initiated mRNA decay and processing of ribosomal 9S RNA show that the Escherichia coli ams and rne loci are the same.

We show in the present paper that the cleavages initiating decay of the ompA mRNA are suppressed both in the Escherichia coli ams(ts) strain (originally defined by a prolonged bulk mRNA half-life) and in the me(ts) strain (originally defined by aberrant 9S RNA processing). The temperature-sensitive defects of both these strains are complemented by a recombinant lambda phage containing a genomic segment that carries the putative ams locus. A 5.8 kb fragment from this genomic DNA segment was cloned into a low-copy plasmid and used to transform the ams(ts) and rne(ts) strains. This resulted in growth at the non-permissive temperature and a reoccurrence of the cleavages initiating decay of the ompA mRNA. Deletion analyses of this 5.8 kb fragment indicated that the putative ams open reading frame could complement both the Ams(ts) and the Rne(ts) phenotype with regard to the ompA cleavages. In addition we showed that the ams(ts) strain suppresses 9S RNA processing to 5S RNA to the same extent as the rne(ts) strain, and that the rne(ts0 strain has a prolonged bulk mRNA half-life, as was reported for the ams(ts) strain. Therefore we suggest that ams and rne reflect the same gene locus; one which is involved both in mRNA decay and RNA processing. We discuss how this gene locus may related to the previously characterized endoribonucleolytic activities of RNase E and RNase K.

Bacteriophage lambda↗

PubMed1997

Polysome-associated mRNAs are substrates for the nonsense-mediated mRNA decay pathway in Saccharomyces cerevisiae.

In eukaryotic cells, premature termination of translation at nonsense codons has been implicated as the cause of a variety of posttranscriptional events, including rapid mRNA decay in the cytoplasm or the nucleus, altered splice site selection, and exon skipping. In the yeast Saccharomyces cerevisiae, nonsense codons promote accelerated mRNA decay, and we sought to determine the cellular location in which this degradation occurs. In this report, we demonstrate that six different mRNAs, including nonsense-containing transcripts of the LEU2, HIS4, PGK1, and CYH2 genes, and two wild-type mRNAs (the MAT(alpha)1 and CYH2 mRNAs), were stabilized when the translation elongation inhibitor cycloheximide was added to cellular growth media. Subsequent removal of cycloheximide resulted in resumption of translation and degradation of wild-type and nonsense-containing mRNAs. A significant fraction of the CYH2 pre-mRNA that accumulated in the presence of cycloheximide was associated with polysomes, but disappeared from that fraction when decay resumed in the absence of the drug. Moreover, the abundance of the spliced and unspliced forms of the untranslated U3 snRNA was shown to be unaffected in strains harboring mutations that stabilize nonsense-containing mRNAs. Taken together, these observations indicate that nonsense-containing mRNAs in yeast are degraded within the polysome compartment of the cell.

Codon, Nonsense↗

PubMed2001

Polysomal ribonuclease 1 exists in a latent form on polysomes prior to estrogen activation of mRNA decay.

Estrogen induces a global change in the translation profile of Xenopus hepatocytes, replacing serum protein synthesis with production of the yolk protein precursor vitellogenin. This is accomplished by the coordinate destabilization of serum protein mRNAs and the transcriptional induction and subsequent stabilization of vitellogenin mRNA. Previous work identified an endonuclease activity whose appearance on polysomes correlated with the disappearance of serum protein mRNAs. This enzyme, polysomal ribonuclease 1 (PMR1), is a novel member of the peroxidase gene family. The current study examined the association of PMR1 with its mRNA targets on polysomes and mRNPs. The highest amount of polysome-bound PMR1 was observed prior to estrogen induction of mRNA decay. Its distribution on sucrose density gradients matched the absorbance profile of polysome-bound mRNA, suggesting that PMR1 forms a latent complex with mRNA. Following dissociation with EDTA the 62 kDa PMR1 sedimented with a larger complex of >670 kDa. Estrogen induces a 22-fold increase in unit enzymatic activity of polysome-bound PMR1, and a time-dependent loss of PMR1 from polysomes in a manner that mirrors the disappearance of albumin mRNA. These data suggest that the key step in the extensive estrogen-induced change in mRNA decay in Xenopus liver is activation of a latent mRNA endonuclease associated with its target mRNA.

Animals↗

PubMed2000

Arabidopsis thaliana poly (A) binding protein 2 (PAB2) functions in yeast translational and mRNA decay processes.

The single yeast gene (PAB1) encoding poly (A) binding protein (PABP) has several roles in post-transcriptional processes, including translation initiation and mRNA decay. PABP is encoded by a large gene family in plants. Within Arabidopsis thaliana, the several characterized PABP genes exhibit an extreme degree of sequence divergence and are differentially expressed. Arabidopsis PAB2 is expressed in distinct tissues or during defined developmental windows in most plant organs. In this study we demonstrate that PAB2 restores viability to a yeast pab1 mutant strain. Yeast strains containing wild-type, null (PAB2s) and temperature sensitive (PAB2ts) alleles of PAB2 were used to explore the molecular functions of the plant protein. PAB2 can participate in poly (A) tail shortening, thus demonstrating that it interacts with the yeast poly(A) nuclease complex. PAB2 is required for translation, helping to maintain intact polysome structures. Consistent with its role in translation initiation, poly (A) was found to enhance PAB2 binding to Arabidopsis eIF-iso4G in vitro. In addition, PAB2 can partially restore the linkage between deadenylation, decapping and mRNA decay in yeast. Taken together, our results suggest that Arabidopsis PAB2 participates in many of the same complex post-transcriptional processes identified for yeast PAB1, and is functionally distinct from other characterized Arabidopsis PABPs.

Alleles↗

PubMed2000

Hfq (HF1) stimulates ompA mRNA decay by interfering with ribosome binding.

The adaptation of mRNA stability to environmental changes is a means of cells to adjust the level of gene expression. The Escherichia coli ompA mRNA has served as one of the paradigms for regulated mRNA decay in prokaryotes. The stability of the transcript is known to be correlated inversely with the bacterial growth rate. Thus, the regulation of ompA mRNA stability meets the physiological needs to adjust the level of ompA expression to the rate of cell division. Recently, host factor I (Hfq/HF1) was shown to be involved in the regulation of ompA mRNA stability under slow growth conditions. Here, we present the first direct demonstration that 30S ribosomes bound to the ompA 5'-UTR protect the transcript from RNase E cleavage in vitro. However, the 30S protection was found to be abrogated in the presence of Hfq. Toeprinting and in vitro translation assays revealed that translation of ompA is repressed in the presence of Hfq. These in vitro studies are corroborated by in vivo expression studies demonstrating that the reduced synthesis rate of OmpA effected by Hfq results in functional inactivation of the ompA mRNA. The data are discussed in terms of a model wherein Hfq regulates the stability of ompA mRNA by competing with 30S ribosomes for binding to the ompA 5'-UTR.

5' Untranslated Regions↗

PubMed2005

Multiple transcripts regulate glucose-triggered mRNA decay of the lactate transporter JEN1 from Saccharomyces cerevisiae.

The Saccharomyces cerevisiae JEN1 gene encoding the lactate transporter undergoes strong catabolic repression at both transcriptional and post-transcriptional levels. JEN1 mRNA decay is greatly accelerated upon the addition of a pulse of glucose, fructose or mannose to induced cell cultures. Mapping of the 5'UTRs and 3'UTRs of JEN1 transcripts revealed multiple transcription start-sites located at position -51, +391 or +972, depending on the cell culture conditions. The presence of the JEN1(+391) transcript correlated with rapid glucose-triggered mRNA degradation of the JEN1(-51) transcript, whereas when the small transcript started at position +972, the JEN1(-51) mRNA turnover rate was unaffected. Overexpressed JEN1(+391) transcript accelerated JEN1(-51) mRNA decay in all conditions tested but was not translated. We propose that the JEN1(+391) transcript may have a "sensor-like" function, regulating glucose-triggered degradation of JEN1(-51) protein-coding mRNA.

Dose-Response Relationship, Drug↗

PubMed2004

Translation repression by GLD-1 protects its mRNA targets from nonsense-mediated mRNA decay in C. elegans.

Previously, we identified multiple in vivo mRNA targets of the maxi-KH/STAR domain protein GLD-1 by their ability to interact with GLD-1 in cytoplasmic extracts and, for all targets tested thus far, GLD-1 functions as a translational repressor. However, here we show that GLD-1 stabilizes the mRNAs of two targets, gna-2 (T23G11.2) and Y75B12B.1. gna-2 mRNA has two upstream open reading frames (uORF), resulting in two premature stop codons. We found that gna-2 mRNA is a naturally occurring mRNA target of nonsense-mediated mRNA decay (NMD) and that the binding of GLD-1 protects gna-2 mRNA from NMD, likely by repressing translation of the uORFs. Therefore, gna-2 mRNA comes under two posttranscriptional controls: (1) translation regulation by a specific translational repressor, GLD-1; and (2) uORF elicited regulation, mainly through NMD. As a result, these two posttranscriptional controls together provide precise temporal and spatial control of gene expression. Consistent with this novel mode of regulation, when GLD-1 mRNA targets acquire premature stop codon mutations, GLD-1 protects them from NMD. Analysis of several mRNA targets containing premature stop codons suggests that in translation repression, GLD-1 either represses ribosome assembly on the target mRNA, or subsequent ribosome elongation to the premature stop codon.

3' Untranslated Regions↗

PubMed2006

Specific inhibition of nonsense-mediated mRNA decay components, SMG-1 or Upf1, rescues the phenotype of Ullrich disease fibroblasts.

Nonsense-mediated mRNA decay (NMD) is an mRNA quality-control mechanism that degrades aberrant mRNAs containing premature translation termination codons (PTCs). The essential proteins for NMD include SMG-1, a protein kinase, and Upf1, a substrate of SMG-1 with RNA helicase activity. In this study, we evaluated the effects of NMD inhibition by siRNA-mediated knockdown of SMG-1 or Upf1 on the phenotype of Ullrich disease, an autosomal recessive congenital muscular dystrophy. The patient studied showed a homozygous frameshift mutation with a PTC in the collagen VI alpha2 gene, which encodes a truncated but partially functional protein. The patient's fibroblasts showed a nearly complete loss of the triple-helical collagen VI protein and functional defects in the extracellular matrix (ECM) due to the crucial deficiency of the collagen VI alpha2 protein. We have shown that siRNA-mediated knockdown of SMG-1 or Upf1 causes the up-regulation of the mutant triple-helical collagen VI, resulting in the formation of partially functional ECM. We suggest that the inhibition of NMD may be useful as a therapeutic approach to treat some human genetic diseases exacerbated by NMD.

Adult↗

PubMed2006

Quantitative analysis of CAPN3 transcripts in LGMD2A patients: involvement of nonsense-mediated mRNA decay.

Limb girdle muscular dystrophy type 2A (LGMD2A) is caused by single or small nucleotide changes widespread along the CAPN3 gene, which encodes the muscle-specific proteolytic enzyme calpain-3. About 356 unique allelic variants of CAPN3 have been identified to date. We performed analysis of the CAPN3 gene in LGMD2A patients at both the mRNA level using reverse transcription-PCR, and at the DNA level using PCR and denaturing high performance liquid chromatography. In four patients, we detected homozygous occurrence of a missense mutation or an in-frame deletion at the mRNA level although the DNA was heterozygous for this mutation in conjunction with a frame-shift mutation. The relationship observed in 12 patients between the quantity of CAPN3 mRNA, determined using real-time PCR, and the genotype leads us to propose that CAPN3 mRNAs which contain frame-shift mutations are degraded by nonsense-mediated mRNA decay. Our results illustrate the importance of DNA analysis for reliable establishment of mutation status, and provide a new insight into the process of mRNA decay in cells of LGMD2A patients.

Adolescent↗

PubMed2003

Widespread predicted nonsense-mediated mRNA decay of alternatively-spliced transcripts of human normal and disease genes.

We have recently shown that a third of reliably-inferred alternative mRNA isoforms are candidates for nonsense-mediated mRNA decay (NMD), an mRNA surveillance system (Lewis et al., 2003; PROC: Natl Acad. Sci. USA, 100, 189-192). Rather than being translated to yield protein, these transcripts are expected to be degraded and may be subject to regulated unproductive splicing and translation (RUST). Our initial experimental studies are consistent with these predictions and suggest an unappreciated role for NMD in several human diseases.

Alternative Splicing↗

PubMed1997

Premature termination codon mutations in the type VII collagen gene in recessive dystrophic epidermolysis bullosa result in nonsense-mediated mRNA decay and absence of functional protein.

The severe mutilating Hallopeau-Siemens type of recessive dystrophic epidermolysis bullosa (HS-RDEB) is characterized by the absence of anchoring fibrils that consist of type VII collagen. We have previously identified premature termination codon (PTC) mutations in both alleles of the type VII collagen gene (COL7A1) in HS-RDEB patients. In this study we have defined the mechanism by which these mutations elicit their phenotypic consequences in a family. The extent of nonsense-mediated mRNA decay induced by these mutations was assessed by quantitation of the level of expression of the corresponding mRNA from each of the mutant alleles by RT-PCR of parental RNA. The level of expression of the paternal mutant allele with a PTC in exon 2 was approximately 30% of that of the wild-type allele whereas that of the maternal mutant allele with a PTC in exon 104 was reduced to about 80% of the normal allele. Immunoprecipitation of newly synthesized type VII collagen with a monoclonal antibody revealed reduced quantities of alpha1(VII) polypeptides in both parents' cells, whereas their synthesis was entirely absent in the proband's keratinocytes. Thus, a consequence of these premature termination codon mutations in COL7A1 is nonsense-mediated mRNA decay, with a dramatic reduction in type VII collagen synthesis, and the absence of anchoring fibrils in the proband. These results establish a mechanistic link between the presence of premature termination codon mutations in both alleles of COL7A1 and the clinical phenotype of HS-RDEB.

Alleles↗