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At least 1,333 records · Page 74Linked to original sources
PubMed2006

NMDA induces post-transcriptional regulation of alpha2-guanylyl-cyclase-subunit expression in cerebellar granule cells.

Activation of N-methyl-D-aspartate (NMDA) glutamate receptors commonly affects gene expression in different neurons. We reported previously that chronic treatment of rat cerebellar granule cells with NMDA (24 hours) upregulates the expression of mRNA encoding the alpha2 subunit of the nitric-oxide-sensitive guanylyl cyclase. However, the molecular mechanisms involved in this process remained to be elucidated. Here, we have performed mRNA-decay experiments using the transcriptional inhibitor actinomycin D, providing evidence that the half-life of alpha2 mRNA is significantly prolonged in cells exposed to NMDA. The role of the 3' untranslated region of the alpha2 transcripts in NMDA-induced mRNA stabilisation was examined and an association between the RNA-binding proteins AUF1 and ELAV-like protein 1 (HuR/HuA), and endogenous alpha2 mRNA was demonstrated in vivo, as revealed by coimmunoprecipitation experiments with specific antibodies against AUF1 and HuR. Further studies indicated that stimulation of the NMDA receptor induces a downregulation in AUF1 levels stabilising the alpha2 mRNA transcripts. These events are triggered through a mechanism that depends on formation of nitric oxide, and on the subsequent activation of guanylyl cyclase and cGMP dependent protein kinases.

3' Untranslated Regions↗
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↗
PubMed2005

The poly(A)-limiting element enhances mRNA accumulation by increasing the efficiency of pre-mRNA 3' processing.

The poly(A)-limiting element (PLE) is a conserved sequence originally found in the 3' UTR of Xenopus albumin mRNA whose presence restricts the length of the poly(A) tail on both pre-mRNA and fully processed mRNA to <20 nt. Results presented in this study show that the PLE also increases the cytoplasmic level of reporter beta-globin mRNA. Transcription run-on shows this increase was not due to increased reporter gene transcription, and experiments with tetracycline repressor-controlled reporter mRNA showed the PLE does not alter the rate of mRNA decay. Both RT-PCR and RNase protection assay showed the PLE caused a 50% increase in the 3' processing of reporter beta-globin mRNA in vivo. This was confirmed in vitro, where PLE-containing RNA was cleaved in HeLa nuclear extract at a rate 80% faster than a control RNA bearing an inactive element. These results indicate that the PLE regulates the length of the poly(A) tail and the efficiency of 3' processing. In addition, they show that PLE-containing mRNA with a <20-nt poly(A) tail is as stable as mRNA with a 100- to 200-nt poly(A) tail.

3' Untranslated Regions↗
PubMed2003

RNA editing and regulation of Drosophila 4f-rnp expression by sas-10 antisense readthrough mRNA transcripts.

We have previously described an example of extensively A-to-G edited cDNA derived from adult heads of the fruitfly Drosophila melanogaster. In that study, the source of the predicted antisense RNA pairing strand for template recognition by dADAR editase was not identified, and the biological significance of the observed hyperediting was not known. Here, we address each of these questions. 4f-rnp and sas-10 are closely adjacent X-linked genes located on opposite DNA strands that produce convergent transcripts. We show that developmentally regulated antisense sas-10 readthrough mRNA arises by activation of an upstream promoter P2 during the late embryo stage of fly development. The sas-10 readthrough transcripts pair with 4f-rnp mRNA to form double-stranded molecules, as indicated by A-to-G editing observed in both RNA strands. It would be predicted that perfect RNA duplexes would be targeted for modification/degradation by enzyme pathways that recognize double-stranded RNAs, leading to decline in 4f-rnp mRNA levels, and this is what we observe. The observation using quantitative RT-PCR that sas-10 readthrough and 4f-rnp transcript levels are inversely related suggests a role for the antisense RNA in posttranscriptional regulation of 4f-rnp gene expression during development. Potential molecular mechanisms that could lead to this result are discussed, one of which is targeted transcript degradation via the RNAi pathway. Insofar as the dADAR editase and RNAi pathways are known to be constitutive in this system, it is likely that control of antisense RNA transcription is the rate-limiting factor. The results provide insight into roles of naturally occurring antisense RNAs in regulation of eukaryotic gene expression.

Amino Acid Sequence↗
PubMed2005

Nonsense-mediated mRNA decay factors act in concert to regulate common mRNA targets.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades mRNAs containing nonsense codons, and regulates the expression of naturally occurring transcripts. While NMD is not essential in yeast or nematodes, UPF1, a key NMD effector, is essential in mice. Here we show that NMD components are required for cell proliferation in Drosophila. This raises the question of whether NMD effectors diverged functionally during evolution. To address this question, we examined expression profiles in Drosophila cells depleted of all known metazoan NMD components. We show that UPF1, UPF2, UPF3, SMG1, SMG5, and SMG6 regulate in concert the expression of a cohort of genes with functions in a wide range of cellular activities, including cell cycle progression. Only a few transcripts were regulated exclusively by individual factors, suggesting that these proteins act mainly in the NMD pathway and their role in mRNA decay has not diverged substantially. Finally, the vast majority of NMD targets in Drosophila are not orthologs of targets previously identified in yeast or human cells. Thus phenotypic differences observed across species following inhibition of NMD can be largely attributed to changes in the repertoire of regulated genes.

Animals↗
PubMed2005

Yeast Puf3 mutants reveal the complexity of Puf-RNA binding and identify a loop required for regulation of mRNA decay.

The eukaryotic Puf proteins regulate mRNA translation and degradation by binding the 3' untranslated regions of target mRNAs. Crystal structure analysis of a human Puf bound to RNA suggested a modular mode of binding, with specific amino acids within each of eight repeat domains contacting a single nucleotide of the target RNA. Here we study the mechanism by which the yeast Puf3p binds and stimulates the degradation of COX17 mRNA. Mutation of the predicted RNA-binding positions of Puf3p to those found in Puf5p demonstrated that a single amino acid change in Puf3p abolished detectable binding to COX17. Since this amino acid position in both Puf3p and Puf5p is predicted to contact an adenine in the respective target RNAs, the amino acid in Puf3p must play a more critical role in promoting COX17 interaction. In contrast, an amino acid change in the third repeat of Puf3p, which interacts with the only divergent nucleotide between the Puf3p and Puf5p targets, had no effect on binding COX17. These results argue that a simple set of rules cannot reliably link specific amino acid positions with target specificity. Each of these amino acid changes in Puf3p enhanced binding to the Puf5p target HO RNA, suggesting a different mode of binding to this target. Finally, we identified an outer surface loop that was dispensable for binding but was required to promote both rapid deadenylation and subsequent decapping of the COX17 mRNA, most likely as a point of protein-protein interactions.

3' Untranslated Regions↗
PubMed2006

Mutational analysis of human eIF4AIII identifies regions necessary for exon junction complex formation and nonsense-mediated mRNA decay.

The exon junction complex (EJC) is deposited on mRNAs by the process of pre-mRNA splicing and is a key effector of downstream mRNA metabolism. We previously demonstrated that human eIF4AIII, which is essential for nonsense-mediated mRNA decay (NMD), constitutes at least part of the RNA-binding platform anchoring other EJC components to the spliced mRNA. To determine the regions of eIF4AIII that are functionally important for EJC formation, for binding to other EJC components, and for NMD, we now report results of an extensive mutational analysis of human eIF4AIII. Using GFP-, GST- or Flag-fusions of eIF4AIII versions containing site-specific mutations or truncations, we analyzed subcellular localizations, protein-protein interactions, and EJC formation in vivo and in vitro. We also tested whether mutant proteins could rescue NMD inhibition resulting from RNAi depletion of endogenous eIF4AIII. Motifs Ia and VI, which are conserved among the eIF4A family of RNA helicases (DEAD-box proteins), are crucial for EJC formation and NMD, as is one eIF4AIII-specific region. An additional eIF4AIII-specific motif forms part of the binding site for MLN51, another EJC core component. Mutations in the canonical Walker A and B motifs that eliminate RNA-dependent ATP hydrolysis by eIF4AIII in vitro are of no detectable consequence for EJC formation and NMD activation. Implications of these findings are discussed in the context of other recent results and a new structural model for human eIF4AIII based on the known crystal structure of Saccharomyces cerevisiae eIF4AI.

Adenosine Triphosphatases↗
PubMed2003

Stable RNA structures can repress RNA synthesis in vitro by the brome mosaic virus replicase.

A 15-nucleotide (nt) unstructured RNA with an initiation site but lacking a promoter could direct the initiation of RNA synthesis by the brome mosaic virus (BMV) replicase in vitro. However, BMV RNA with a functional initiation site but a mutated promoter could not initiate RNA synthesis either in vitro or in vivo. To explain these two observations, we hypothesize that RNA structures that cannot function as promoters could prevent RNA synthesis by the BMV RNA replicase. We documented that four different nonpromoter stem-loops can inhibit RNA synthesis from an initiation-competent RNA sequence in vitro. Destabilizing these structures increased RNA synthesis. However, RNA synthesis was restored in full only when a BMV RNA promoter element was added in cis. Competition assays to examine replicase-RNA interactions showed that the structured RNAs have a lower affinity for the replicase than do RNAs lacking stable structures or containing a promoter element. The results characterize another potential mechanism whereby the BMV replicase can specifically recognize BMV RNAs.

Base Sequence↗
PubMed2005

A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing.

In eukaryotic cells degradation of bulk mRNA in the 5' to 3' direction requires the consecutive action of the decapping complex (consisting of DCP1 and DCP2) and the 5' to 3' exonuclease XRN1. These enzymes are found in discrete cytoplasmic foci known as P-bodies or GW-bodies (because of the accumulation of the GW182 antigen). Proteins acting in other post-transcriptional processes have also been localized to P-bodies. These include SMG5, SMG7, and UPF1, which function in nonsense-mediated mRNA decay (NMD), and the Argonaute proteins that are essential for RNA interference (RNAi) and the micro-RNA (miRNA) pathway. In addition, XRN1 is required for degradation of mRNAs targeted by NMD and RNAi. To investigate a possible interplay between P-bodies and these post-transcriptional processes we depleted P-body or essential pathway components from Drosophila cells and analyzed the effects of these depletions on the expression of reporter constructs, allowing us to monitor specifically NMD, RNAi, or miRNA function. We show that the RNA-binding protein GW182 and the DCP1:DCP2 decapping complex are required for miRNA-mediated gene silencing, uncovering a crucial role for P-body components in the miRNA pathway. Our analysis also revealed that inhibition of one pathway by depletion of its key effectors does not prevent the functioning of the other pathways, suggesting a lack of interdependence in Drosophila.

Animals↗
PubMed2006

tmRNA determinants required for facilitating nonstop mRNA decay.

In bacteria, ribosomes stalled on nonstop mRNAs are rescued by tmRNA in a unique process called trans-translation. The two known tmRNA functions in trans-translation are (1) a tRNA-like function, which transfers the partially synthesized protein fragment to itself; and (2) an mRNA-like function, which enables ribosomes to resume and terminate translation on tmRNA as a surrogate template. We present evidence to demonstrate that tmRNA performs a third function, namely, facilitating the degradation of the causative defective mRNA. Our investigations have revealed the identity of key sequence determinants that promote the degradation of the nonstop mRNA. These sequence determinants are located in the distal part of the tmRNA open reading frame, encoding the ultimate, penultimate, and anti-penultimate amino acids of the peptide tag. We show that mutation of these tmRNA sequence elements has an adverse affect on the disposal of the nonstop mRNA, while leaving the tRNA and mRNA functions entirely unaffected. More significantly, specific mutations that change the nucleotide sequence of the peptide-reading frame without altering the nature or identity of the encoded amino acids still exhibit the characteristic defect in nonstop mRNA decay. In contrast, mutations in codons 3, 4, 5, and 6 of the tmRNA open reading frame do not have an adverse affect on degradation of defective mRNAs. Based on these results, we propose that tmRNA plays an important role in promoting the decay of nonstop mRNAs and that sequence elements in the distal segment of the peptide-reading frame make sequence-specific contributions that are crucial for this activity.

Mutation↗
PubMed2004

Evidence against a direct role for the Upf proteins in frameshifting or nonsense codon readthrough.

The Upf proteins are essential for nonsense-mediated mRNA decay (NMD). They have also been implicated in the modulation of translational fidelity at viral frameshift signals and premature termination codons. How these factors function in both mRNA turnover and translational control remains unclear. In this study, mono- and bicistronic reporter systems were used in the yeast Saccharomyces cerevisae to differentiate between effects at the levels of mRNA turnover and those at the level of translation. We confirm that upfDelta mutants do not affect programmed frameshifting, and show that this is also true for mutant forms of eIF1/Sui1p. Further, bicistronic reporters did not detect defects in translational readthrough due to deletion of the UPF genes, suggesting that their function in termination is not as general a phenomenon as was previously believed. The demonstration that upf sui1 double mutants are synthetically lethal demonstrates an important functional interaction between the NMD and translation initiation pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Conservation of the deadenylase activity of proteins of the Caf1 family in human.

The yeast Pop2 protein, belonging to the eukaryotic Caf1 family, is required for mRNA deadenylation in vivo. It also catalyzes poly(A) degradation in vitro, even though this property has been questioned. Caf1 proteins are related to RNase D, a feature supported by the recently published structure of Pop2. Yeast Pop2 contains, however, a divergent active site while its human homologs harbor consensus catalytic residues. Given these differences, we tested whether its deadenylase activity is conserved in the human homologs Caf1 and Pop2. Our data demonstrate that both human factors degrade poly(A) tails indicating their involvement in mRNA metabolism.

Amino Acid Sequence↗
PubMed2005

Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome.

RNA interference (RNAi) is a conserved RNA silencing pathway that leads to sequence-specific mRNA decay in response to the presence of double-stranded RNA (dsRNA). Long dsRNA molecules are first processed by Dicer into 21-22-nucleotide small interfering RNAs (siRNAs). The siRNAs are incorporated into a multimeric RNA-induced silencing complex (RISC) that cleaves mRNAs at a site determined by complementarity with the siRNAs. Following this initial endonucleolytic cleavage, the mRNA is degraded by a mechanism that is not completely understood. We investigated the decay pathway of mRNAs targeted by RISC in Drosophila cells. We show that 5' mRNA fragments generated by RISC cleavage are rapidly degraded from their 3' ends by the exosome, whereas the 3' fragments are degraded from their 5' ends by XRN1. Exosome-mediated decay of the 5' fragments requires the Drosophila homologs of yeast Ski2p, Ski3p, and Ski8p, suggesting that their role as regulators of exosome activity is conserved. Our findings indicate that mRNAs targeted by siRNAs are degraded from the ends generated by RISC cleavage, without undergoing decapping or deadenylation.

Acyltransferases↗
PubMed2005

UTP-dependent turnover of Trypanosoma brucei mitochondrial mRNA requires UTP polymerization and involves the RET1 TUTase.

Trypanosoma brucei mitochondria possess a unique RNA decay pathway in which rapid degradation of polyadenylated mRNAs is dependent on the addition of UTP, as measured by in organello pulse chase assays. To determine the mechanism by which UTP stimulates the degradation of polyadenylated RNAs, we performed in organello pulse chase assays under different conditions. Treatment of mitochondria with proteinase K revealed that UTP does not act through a receptor on the surface of the mitochondria. To determine if the UTP-stimulated RNA decay pathway is triggered by the mitochondrial energy state or ATP:UTP ratio, increasing ATP was added to a constant amount of UTP during the chase period of the assay. Results indicate that rapid turnover is responsive to UTP and not the ATP:UTP ratio. Experiments using UTP analogs demonstrate that UTP polymerization into RNAs is necessary for UTP-dependent degradation. Furthermore, experiments performed with RNAi cells indicate that the RET1 terminal uridylyl transferase (TUTase) is required for UTP-dependent decay of polyadenylated RNAs. Overall, these results show that degradation of polyadenylated RNAs in T. brucei mitochondria can occur through a unique mechanism that requires the polymerization of UTP into RNAs, presumably by the RET1 TUTase.

Animals↗
PubMed2005

Processing bodies require RNA for assembly and contain nontranslating mRNAs.

Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), wherein mRNA decay factors are concentrated and where mRNA decay can occur. However, the physical nature of P-bodies, their relationship to translation, and possible roles of P-bodies in cellular responses remain unclear. We describe four properties of yeast P-bodies that indicate that P-bodies are dynamic structures that contain nontranslating mRNAs and function during cellular responses to stress. First, in vivo and in vitro analysis indicates that P-bodies are dependent on RNA for their formation. Second, the number and size of P-bodies vary in response to glucose deprivation, osmotic stress, exposure to ultraviolet light, and the stage of cell growth. Third, P-bodies vary with the status of the cellular translation machinery. Inhibition of translation initiation by mutations, or cellular stress, results in increased P-bodies. In contrast, inhibition of translation elongation, thereby trapping the mRNA in polysomes, leads to dissociation of P-bodies. Fourth, multiple translation factors and ribosomal proteins are lacking from P-bodies. These results suggest additional biological roles of P-bodies in addition to being sites of mRNA degradation.

Cytoplasmic Granules↗
PubMed2005

Suppression of termination mutations caused by defects of the NMD machinery in Saccharomyces cerevisiae.

Among a large collection of nonsense (termination) suppressors of Saccharomyces cerevisiae, a few remained obscure for their molecular nature. Of those, a group of weak and recessive suppressors, sup111, sup112 and sup113, is of particular interest because of their dependency on [PSI+], a yeast prion. From the facts that these suppressors map at positions quite similar to the UPF2, UPF3 and UPF1 genes, respectively, and that some mutations in the UPF genes confer termination suppressor activity, we suspected that sup111, sup112 and sup113 would very well be mutant alleles of the UPF genes. We tested our speculation and found that sup113, sup111 and sup112 were in fact complemented with the wild-type alleles of UPF1, UPF2 and UPF3, respectively. We further obtained evidence that the UPF1, UPF2 and UPF3 loci of the strains carrying sup113, sup111 and sup112, respectively, had point mutations. From these results, we conclude that sup111, sup112 and sup113 are mutant alleles of UPF2, UPF3 and UPF1, respectively, and thus attribute suppressor activity of these mutations to defects in the NMD (nonsense-mediated mRNA decay) machinery.

Gene Expression Regulation, Fungal↗
PubMed2003

The protein component of bacterial ribonuclease P flickers the metal ion response to the substrate shape preference of the ribozyme.

The substrate shape specificity of the Escherichia coli ribonuclease P (RNase P) ribozyme depends on the concentration of magnesium ion. At 10 mM or more, it can cleave a hairpin substrate as well as a cloverleaf pre-transfer RNA (tRNA). The results showed, however, that the holo enzyme cleaved the hairpin substrate at low concentrations of magnesium ion. Considering that the homologous E. coli tRNAs are resistant to internal cleavage by the RNase P, the phenomena suggest that this catalytic activity might take part in the removing the mis-folded RNAs in the cell.

Catalysis↗