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PubMed2006

BRF1 protein turnover and mRNA decay activity are regulated by protein kinase B at the same phosphorylation sites.

BRF1 posttranscriptionally regulates mRNA levels by targeting ARE-bearing transcripts to the decay machinery. We previously showed that protein kinase B (PKB) phosphorylates BRF1 at Ser92, resulting in binding to 14-3-3 and impairment of mRNA decay activity. Here we identify an additional regulatory site at Ser203 that cooperates in vivo with Ser92. In vitro kinase labeling and wortmannin sensitivity indicate that Ser203 phosphorylation is also performed by PKB. Mutation of both serines to alanine uncouples BRF1 from PKB regulation, leading to constitutive mRNA decay even in the presence of stabilizing signals. BRF1 protein is labile because of proteasomal degradation (half-life, <3 h) but becomes stabilized upon phosphorylation and is less stable in PKBalpha(-/-) cells. Surprisingly, phosphorylation-dependent protein stability is also regulated by Ser92 and Ser203, with parallel phosphorylation required at these sites. Phosphorylation-dependent binding to 14-3-3 is abolished only when both sites are mutated. Cell compartment fractionation experiments support a model in which binding to 14-3-3 sequesters BRF1 through relocalization and prevents it from executing its mRNA decay activity, as well as from proteasomal degradation, thereby maintaining high BRF1 protein levels that are required to reinstate decay upon dissipation of the stabilizing signal.

Alanine↗

PubMed2003

Nonsense-mediated mRNA decay in Drosophila: at the intersection of the yeast and mammalian pathways.

The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature stop codons (PTCs). In Caenorhabditis elegans, seven genes (smg1-7) playing an essential role in NMD have been identified. Only SMG2-4 (known as UPF1-3) have orthologs in Saccharomyces cerevisiae. Here we show that the Drosophila orthologs of UPF1-3, SMG1, SMG5 and SMG6 are required for the degradation of PTC-containing mRNAs, but that there is no SMG7 ortholog in this organism. In contrast, orthologs of SMG5-7 are encoded by the human genome and all three are required for NMD. In human cells, exon boundaries have been shown to play a critical role in defining PTCs. This role is mediated by components of the exon junction complex (EJC). Contrary to expectation, however, we show that the components of the EJC are dispensable for NMD in Drosophila cells. Consistently, PTC definition occurs independently of exon boundaries in Drosophila. Our findings reveal that despite conservation of the NMD machinery, different mechanisms have evolved to discriminate premature from natural stop codons in metazoa.

Amino Acid Sequence↗

PubMed2006

Crystal structure of the UPF2-interacting domain of nonsense-mediated mRNA decay factor UPF1.

UPF1 is an essential eukaryotic RNA helicase that plays a key role in various mRNA degradation pathways, notably nonsense-mediated mRNA decay (NMD). In combination with UPF2 and UPF3, it forms part of the surveillance complex that detects mRNAs containing premature stop codons and triggers their degradation in all organisms studied from yeast to human. We describe the 3 A resolution crystal structure of the highly conserved cysteine-histidine-rich domain of human UPF1 and show that it is a unique combination of three zinc-binding motifs arranged into two tandem modules related to the RING-box and U-box domains of ubiquitin ligases. This UPF1 domain interacts with UPF2, and we identified by mutational analysis residues in two distinct conserved surface regions of UPF1 that mediate this interaction. UPF1 residues we identify as important for the interaction with UPF2 are not conserved in UPF1 homologs from certain unicellular parasites that also appear to lack UPF2 in their genomes.

Amino Acid Sequence↗

PubMed2004

An activated 5' cryptic splice site in the human ALG3 gene generates a premature termination codon insensitive to nonsense-mediated mRNA decay in a new case of congenital disorder of glycosylation type Id (CDG-Id).

A defect of the dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl mannosyltransferase encoded by the ALG3 gene (alias NOT56L) causes congenital disorder of glycosylation type Id (CDG-Id). In this work, a new mutation in the ALG3 gene causing atypical splicing is described with characterization of expression levels and transcript stabilities of the different splice products. A silent mutation in exon 1 of the ALG3 gene (c.165C<T) resulted in a deletion in the corresponding transcripts (c.160_196del) due to the activation of a cryptic donor splice site. Expression studies revealed that negligible amounts of normal transcripts were present in the patient. The deletion in the ALG3 gene generated a premature termination codon (PTC) coding for an ALG3 protein truncated after the first N-terminal transmembranous domain (p.Val54fsX66). Nonsense mediated decay (NMD) of mRNA is a general mechanism for clearing of RNA molecules containing suitable PTCs. However, suppression of NMD using cycloheximide had no influence on ALG3 transcript levels, although the PTCs of the transcript fulfill the criteria for the initiation of NMD. The results presented in this work demonstrate that factors abrogating NMD of the ALG3 gene exists and that the ALG3 gene can serve as a valuable tool for further investigations of the regulation of NMD.

Amino Acid Sequence↗

PubMed2004

Splicing of U12-type introns deposits an exon junction complex competent to induce nonsense-mediated mRNA decay.

Metazoan cells have two pathways for intron removal involving the U2- and U12-type spliceosomes, which contain mostly nonoverlapping sets of small nuclear ribonucleoproteins. We show that in vitro splicing of a U12-type intron assembles an exon junction complex (EJC) that is comparably positioned and contains many of the same components as that deposited by the U2-type spliceosome. The presence of a U12-type intron downstream of a premature termination codon within an open reading frame (ORF) induces nonsense-mediated decay of the mRNA in vivo. These findings suggest a common pathway for EJC assembly by the two spliceosomes and highlight the evolutionary age of the EJC and its downstream functions in gene expression.

Cell Line↗

PubMed2006

The canonical UPF1-dependent nonsense-mediated mRNA decay is inhibited in transcripts carrying a short open reading frame independent of sequence context.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs carrying premature translation termination codons. Generally, NMD is elicited if translation terminates >50-54 nucleotides (nt) upstream of an exon-exon junction. We have previously reported that human beta-globin mRNAs carrying 5'-proximal nonsense mutations (e.g., beta15) accumulate to normal levels, suggesting an exception to the "50-54-nt boundary rule." In the present report, we demonstrate that the strength of the UPF1-dependent NMD of mutant beta-globin mRNAs is specifically determined by the proximity of the nonsense codon to the initiation AUG. This conclusion is supported by a parallel effect of the short ORF size on NMD of nonsense-containing alpha-globin mRNAs. To determine whether the short-ORF effect on NMD response is conserved in heterologous transcripts, we assessed its effects on a set of beta-globin/triosephosphate isomerase (TPI) hybrid mRNAs and on the TPI mRNA. Our data support the conclusion that nonsense mutations resulting in a short ORF are able to circumvent the full activity of the canonical UPF1-dependent NMD pathway.

5' Untranslated Regions↗

PubMed2001

The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay.

We recently reported that spliceosomes alter messenger ribonucleoprotein particle (mRNP) composition by depositing several proteins 20-24 nucleotides upstream of mRNA exon-exon junctions. When assembled in vitro, this so-called 'exon-exon junction complex' (EJC) contains at least five proteins: SRm160, DEK, RNPS1, Y14 and REF. To better investigate its functional attributes, we now describe a method for generating spliced mRNAs both in vitro and in vivo that either do or do not carry the EJC. Analysis of these mRNAs in Xenopus laevis oocytes revealed that this complex is the species responsible for enhancing nucleocytoplasmic export of spliced mRNAs. It does so by providing a strong binding site for the mRNA export factors REF and TAP/p15. Moreover, by serving as an anchoring point for the factors Upf2 and Upf3, the EJC provides a direct link between splicing and nonsense-mediated mRNA decay. Finally, we show that the composition of the EJC is dynamic in vivo and is subject to significant evolution upon mRNA export to the cytoplasm.

Animals↗

PubMed2002

Regulated alpha-globin mRNA decay is a cytoplasmic event proceeding through 3'-to-5' exosome-dependent decapping.

The alpha-globin mRNA contains a C-rich stability element (CRE) in its 3' untranslated region (3' UTR) which is critical for the stability of this long-lived mRNA. A protein complex, termed the alpha-complex, forms on the CRE and has been shown to contribute to stabilization of the mRNA by at least two mechanisms, first by interacting with the poly(A)-binding protein (PABP) to prevent deadenylation, and second by protecting the mRNA from attack by an erythroid endoribonuclease. In this report, we demonstrate that the alpha-globin 3' UTR can confer stability on a heterologous mRNA in cells, and this stability is dependent on the alpha-complex. Moreover, the stability was exclusively detected with cytoplasmic mRNA, suggesting that the regulation of alpha-globin mRNA stability is a cytoplasmic event. An additional mechanism by which the alpha-complex can confer stability on an RNA in vitro was also identified and shown to involve inhibition of 3' to 5' exonucleolytic degradation. Furthermore, using an in vitro mRNA decay system, we were able to follow the demise of the alpha-globin RNA and demonstrate that the decay was initiated by deadenylation followed by 3'-to-5' decay carried out by the exosome and ultimately hydrolysis of the residual cap structure.

3' Untranslated Regions↗

PubMed2005

A new function for nonsense-mediated mRNA-decay factors.

mRNAs often contain premature-termination (nonsense) codons as a result of mutations and RNA splicing errors. These nonsense codons cause rapid decay of the mRNAs that contain them, a phenomenon called nonsense-mediated mRNA decay (NMD). This response is thought to be a quality-control mechanism that protects cells from truncated dominant-negative proteins. Surprisingly, recent evidence strongly suggests that the NMD factors UPF1, UPF2, UPF3B, RNPS1, Y14 and MAGOH also promote translation of normal mRNAs in mammalian cells. This, along with an earlier discovery that NMD factors appear to dictate efficient translation termination, suggests that NMD factors do not merely function in RNA surveillance. These findings lead to the interesting question of why NMD factors evolved; are they for RNA-quality control or to promote efficient translation initiation and termination?

Animals↗

PubMed2005

The role of SMG-1 in nonsense-mediated mRNA decay.

SMG-1, a member of the PIKK (phosphoinositide 3-kinase related kinases) family, plays a critical role in the mRNA quality control system termed nonsense-mediated mRNA decay (NMD). NMD protects the cells from the accumulation of aberrant mRNAs with premature termination codons (PTCs) that encode nonfunctional or potentially harmful truncated proteins. SMG-1 directly phosphorylates Upf1, another key component of NMD, and this phosphorylation occurs upon recognition of PTC on post-spliced mRNA during the initial round of translation. At present, a variety of tools are available that can specifically suppress NMD, and it is possible to examine the contribution of NMD in a variety of physiological and pathological conditions.

Animals↗

PubMed2005

Alternative splicing and nonsense-mediated mRNA decay regulate mammalian ribosomal gene expression.

Messenger RNAs containing premature stop codons are generally targeted for degradation through nonsense-mediated mRNA decay (NMD). This mechanism degrades aberrant transcripts derived from mutant genes containing nonsense or frameshift mutations. Wild-type genes also give rise to transcripts targeted by NMD. For example, some wild-type genes give rise to alternatively spliced transcripts that are targeted for decay by NMD. In Caenorhabditis elegans, the ribosomal protein (rp) L12 gene generates a nonsense codon-bearing alternatively spliced transcript that is induced in an autoregulatory manner by the rpL12 protein. By pharmacologically blocking the NMD pathway, we identified alternatively spliced mRNA transcripts derived from the human rpL3 and rpL12 genes that are natural targets of NMD. The deduced protein sequence of these alternatively spliced transcripts suggests that they are unlikely to encode functional ribosomal proteins. Overexpression of rpL3 increased the level of the alternatively spliced rpL3 mRNA and decreased the normally expressed rpL3. This indicates that rpL3 regulates its own production by a negative feedback loop and suggests the possibility that NMD participates in this regulatory loop by degrading the non-functional alternatively spliced transcript.

Alternative Splicing↗

PubMed2005

Exon-junction complex components specify distinct routes of nonsense-mediated mRNA decay with differential cofactor requirements.

Messenger RNAs (mRNAs) bearing premature translation termination codons (PTCs) are degraded by nonsense-mediated mRNA decay (NMD). For mammalian NMD, current models propose a linear pathway that involves the splicing-dependent deposition of exon-junction complexes (EJCs) and the sequential action of the NMD factors UPF3, UPF2, and UPF1. We show here that different EJC proteins serve as entry points for the formation of distinguishable NMD-activating mRNPs. Specifically, Y14, MAGOH, and eIF4A3 can activate NMD in an UPF2-independent manner, whereas RNPS1-induced NMD requires UPF2. We identify the relevant regions of RNPS1, eIF4A3, Y14, and MAGOH, which are essential for NMD and provide insights into the formation of complexes, that classify alternative NMD pathways. These results are integrated into a nonlinear model for mammalian NMD involving alternative routes of entry that converge at a common requirement of UPF1.

Animals↗

PubMed1994

Functional hierarchy of AUUUA motifs in mediating rapid interleukin-3 mRNA decay.

In mast cells, expression of interleukin-3 (IL-3) is induced following IgE-receptor activation via calcium-dependent mRNA stabilization. We performed a mutational analysis of the 8 AUUUA motifs located in the 3'-untranslated region of IL-3 mRNA, and analyzed the effect on mRNA stability in PB-3c mast cells. Similar to endogenous IL-3 mRNA, exogenous IL-3 transcripts had a short half-life and were stabilized following stimulation of calcium influx by ionomycin. Specific mutation of 3 AUUUA motifs had almost the same stabilizing effect as deletion of the entire AU-rich region. Mutating even a single critical motif led to a weak, but significant mRNA stabilization. Ionomycin treatment did not further enhance the expression of mutationally stabilized transcripts. Our data fit a model, where within a cluster of 6 AUUUA motifs, 3 adjacent motifs are required for binding of a factor which mediates rapid IL-3 mRNA decay. Activity of such a factor might be under control of a calcium-dependent pathway.

Animals↗

PubMed2001

A strategy for disease gene identification through nonsense-mediated mRNA decay inhibition.

Premature termination codons (PTCs) have been shown to initiate degradation of mutant transcripts through the nonsense-mediated messenger RNA (mRNA) decay (NMD) pathway. We report a strategy, termed gene identification by NMD inhibition (GINI), to identify genes harboring nonsense codons that underlie human diseases. In this strategy, the NMD pathway is pharmacologically inhibited in cultured patient cells, resulting in stabilization of nonsense transcripts. To distinguish stabilized nonsense transcripts from background transcripts upregulated by drug treatment, drug-induced expression changes are measured in control and disease cell lines with complementary DNA (cDNA) microarrays. Transcripts are ranked by a nonsense enrichment index (NEI), which relates expression changes for a given transcript in NMD-inhibited control and patient cell lines. The most promising candidates can be selected using information such as map location or biological function; however, an important advantage of the GINI strategy is that a priori information is not essential for disease gene identification. GINI was tested on colon cancer and Sandhoff disease cell lines, which contained previously characterized nonsense mutations in the MutL homolog 1 (MLH1) and hexosaminidase B (HEXB) genes, respectively. A list of genes was produced in which the MLH1 and HEXB genes were among the top 1% of candidates, thus validating the strategy.

Adaptor Proteins, Signal Transducing↗

PubMed2004

SMG-1 is a phosphatidylinositol kinase-related protein kinase required for nonsense-mediated mRNA Decay in Caenorhabditis elegans.

Eukaryotic messenger RNAs containing premature stop codons are selectively and rapidly degraded, a phenomenon termed nonsense-mediated mRNA decay (NMD). Previous studies with both Caenohabditis elegans and mammalian cells indicate that SMG-2/human UPF1, a central regulator of NMD, is phosphorylated in an SMG-1-dependent manner. We report here that smg-1, which is required for NMD in C. elegans, encodes a protein kinase of the phosphatidylinositol kinase superfamily of protein kinases. We identify null alleles of smg-1 and demonstrate that SMG-1 kinase activity is required in vivo for NMD and in vitro for SMG-2 phosphorylation. SMG-1 and SMG-2 coimmunoprecipitate from crude extracts, and this interaction is maintained in smg-3 and smg-4 mutants, both of which are required for SMG-2 phosphorylation in vivo and in vitro. SMG-2 is located diffusely through the cytoplasm, and its location is unaltered in mutants that disrupt the cycle of SMG-2 phosphorylation. We discuss the role of SMG-2 phosphorylation in NMD.

Animals↗

PubMed2006

Functions of hUpf3a and hUpf3b in nonsense-mediated mRNA decay and translation.

The exon-junction complex (EJC) components hUpf3a and hUpf3b serve a dual function: They promote nonsense-mediated mRNA decay (NMD), and they also regulate translation efficiency. Whether these two functions are interdependent or independent of each other is unknown. We characterized the function of the hUpf3 proteins in a lambdaN/boxB-based tethering system. Despite the high degree of sequence similarity between hUpf3b and hUpf3a, hUpf3a is much less active than hUpf3b to induce NMD and to stimulate translation. We show that induction of NMD by hUpf3 proteins requires interaction with Y14, Magoh, BTZ, and eIF4AIII. The protein region that mediates this interaction and discriminates between hUpf3a and hUpf3b in NMD function is located in the C-terminal domain and fully contained within a small sequence that is highly conserved in Upf3b but not Upf3a proteins. Stimulation of translation is independent of this interaction and is determined by other regions of the hUpf3 protein, indicating the presence of different downstream pathways of hUpf3 proteins either in NMD or in translation.

Amino Acid Sequence↗

PubMed1996

Development of an in vitro mRNA decay system for Escherichia coli: poly(A) polymerase I is necessary to trigger degradation.

Using a novel Escherichia coli in vitro decay system in which polysomes are the source of both enzymes and mRNA, we demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA turnover. The in vitro decay of two different mRNAs (trxA and lpp) is triggered by the addition of ATP only when polysomes are prepared from s strain carrying the wild-type gene for PAP I (pcnB+). The relative decay rates of these two messages are similar in vitro and in vivo. Poly(A) tails are formed on both mRNAs, but no poly(A) are detected on the 3' end of mature 23S rRNA. The size distribution of poly(A) tails generated in vitro, averaging 50 nt in length, is comparable to that previously reported in vivo. PAP I activity is associated exclusively with the polysomes. Exogenously added PAP I does not restore mRNA decay to PAP I-polysomes, suggesting that, in vivo, PAP I may be part of a multiprotein complex. The potential of this in vitro system for analyzing mRNA decay in E. coli is discussed.

Adenosine Monophosphate↗

PubMed1999

Mutations in the MOF2/SUI1 gene affect both translation and nonsense-mediated mRNA decay.

Recent studies have demonstrated that cells have evolved elaborate mechanisms to rid themselves of aberrant proteins and transcripts. The nonsense-mediated mRNA decay pathway (NMD) is an example of a pathway that eliminates aberrant mRNAs. In yeast, a transcript is recognized as aberrant and is rapidly degraded if a specific sequence, called the DSE, is present 3' of a premature termination codon. Results presented here show that strains harboring the mof2-1, mof4-1, mof5-1, and mof8-1 alleles, previously demonstrated to increase the efficiency of programmed -1 ribosomal frameshifting, decrease the activity of the NMD pathway. The effect of the mof2-1 allele on NMD was characterized in more detail. Previous results demonstrated that the wild-type MOF2 gene is identical to the SUI1 gene. Studies on the mof2-1 allele of the SUI1 gene indicate that in addition to its role in recognition of the AUG codon during translation initiation and maintenance of the appropriate reading frame during translation elongation, the Mof2 protein plays a role in the NMD pathway. The Mof2p/Sui1 p is conserved throughout nature and the human homolog of the Mof2p/Sui1p functions in yeast cells to activate NMD. These results suggest that factors involved in NMD are general modulators that act in several aspects of translation and mRNA turnover.

Alleles↗