真核生物翻译过程中的mRNA质量控制

成熟mRNA的合成是一个复杂的过程,往往会产生错误.原核和真核细胞都在多水平进化出了mRNA监视机制,以保证mRNA的质量,甚至在翻译起始之后.真核生物胞质中有4种翻译依赖性的mRNA质量监视机制:无意义介导的降解、No-go降解、Non-stop降解和核糖体延伸介导的降解.这些机制不仅可以识别并迅速降解有缺陷的mRNA,控制mRNA质量,还都在调节基因表达方面具有重要作用,而且也与一些遗传病有关.本文主要综述了真核生物4种mRNA质量监视机制的研究进展,并对相关研究的应用前景做了展望.     

Translational control of maskin mRNA by its 3' untranslated region

BACKGROUND INFORMATION: Maskin is a member of the TACC (transforming acidic coiled-coil) domain proteins found in Xenopus laevis oocytes and embryos. It has been implicated in the co-ordination of the spindle and has been reported to mediate translational repression of cyclin B1 mRNA. RESULTS: In the present study, we report that maskin mRNA is translationally repressed at the level of initiation in stage 4 oocytes and becomes activated in stage 6 oocytes. The translational repression of maskin mRNA correlates with the presence of a short poly(A) tail on this mRNA in stage 4 oocytes. The 3'-UTR (untranslated region) of maskin can confer the translational regulation to a reporter mRNA, and so can the 3'-UTR of human TACC3. A conserved GUCU repeat element was found to repress translation in both stage 4 and stage 6 oocytes, but deletion of this element did not abrogate repression in stage 4 oocytes. UV cross-linking experiments indicated that overlapping sets of proteins bind efficiently to both the maskin and the cyclin B1 3'-UTRs. As reported previously, CPEB [CPE (cytoplasmic polyadenylation element)-binding protein] binds to the cyclin B1 3'-UTR, but its binding to the maskin 3'-UTR is minimal. By RNA affinity chromatography and MS, we identified the EDEN-BP [EDEN (embryonic deadenylation element)-binding protein] as one of the proteins binding to both the maskin and the cyclin B1 3'-UTRs. CONCLUSIONS: Maskin mRNA is translationally regulated by at least two repressor elements and an activation element. One of the repessor elements is the evolutionarily conserved GUCU repeat. EDEN-BP binds to both the maskin and cyclin B1 3'-UTRs, indicating it may be involved in the deadenylation of these mRNAs.     

A ribosome–nascent chain sensor of membrane protein biogenesis in Bacillus subtilis

Proteins in the YidC/Oxa1/Alb3 family have essential functions in membrane protein insertion and folding. Bacillus subtilis encodes two YidC homologs, one that is constitutively expressed (spoIIIJ/yidC1) and a second (yqjG/yidC2) that is induced in spoIIIJ mutants. Regulated induction of yidC2 allows B. subtilis to maintain capacity of the membrane protein insertion pathway. We here show that a gene located upstream of yidC2 (mifM/yqzJ) serves as a sensor of SpoIIIJ activity that regulates yidC2 translation. Decreased SpoIIIJ levels or deletion of the MifM transmembrane domain arrests mifM translation and unfolds an mRNA hairpin that otherwise blocks initiation of yidC2 translation. This regulated translational arrest and yidC2 induction require a specific interaction between the MifM C‐terminus and the ribosomal polypeptide exit tunnel. MifM therefore acts as a ribosome–nascent chain complex rather than as a fully synthesized protein. B. subtilis MifM and the previously described secretion monitor SecM in Escherichia coli thereby provide examples of the parallel evolution of two regulatory nascent chains that monitor different protein export pathways by a shared molecular mechanism.     

The binding interface between Bacillus stearothermophilus ribosomal protein S15 and its 5'-translational operator mRNA

The Bacillus stearothermophilus ribosomal protein S15 (BS15) binds a purine-rich three-helix junction motif in the central domain of 16S ribosomal RNA (rRNA) as well as a translational operator located in the 5'-untranslated region (5'-UTR) of its cognate messenger RNA (mRNA). An in-frame fusion between the 5'-UTR of the BS15 gene and beta-galactosidase (lacZ) was prepared, and tested for BS15-dependent translational repression of lacZ activity in Escherichia coli. The presence of BS15 in trans represses lacZ activity 24-fold. A series of detailed point mutations in BS15 were tested for their effects upon translational repression of lacZ activity. These point mutations demonstrated that the 5'-UTR-BS15 binding interface utilizes many of the same conserved amino acid residues implicated in the binding of BS15 to 16S rRNA. The data demonstrate that the S15 protein can bind to an RNA target motif based primarily upon appropriate minor groove and sugar-phosphate backbone contacts, irrespective of the specific RNA sequence.     

Confined dynamics of a ribosome‐bound nascent globin: Cone angle analysis of fluorescence depolarization decays in the presence of two local motions

We still know very little about how proteins achieve their native three‐dimensional structure in vitro and in the cell. Folding studies as proteins emerge from the mega Dalton‐sized ribosome pose special challenges due to the large size and complicated nature of the ribosome‐nascent chain complex. This work introduces a combination of three‐component analysis of fluorescence depolarization decays (including the presence of two local motions) and in‐cone analysis of diffusive local dynamics to investigate the spatial constraints experienced by a protein emerging from the ribosomal tunnel. We focus on E. coli ribosomes and an all‐α‐helical nascent globin in the presence and absence of the cotranslationally active chaperones DnaK and trigger factor. The data provide insights on the dynamic nature and structural plasticity of ribosome‐nascent chain complexes. We find that the sub‐ns motions of the N‐terminal fluorophore, reporting on the globin dynamics in the vicinity of the N terminus, are highly constrained both inside and outside the ribosomal tunnel, resulting in high‐order parameters (>0.85) and small cone semiangles (<30°). The shorter globin chains buried inside the tunnel are less spatially constrained than those of a reference sequence from a natively unfolded protein, suggesting either that the two nascent chain sequences have a different secondary structure and therefore sample different regions of the tunnel or that the tunnel undergoes local structural adjustments to accommodate the globin sequence. Longer globins emerging out of the ribosomal tunnel are also found to have highly spatially constrained slow (ns) motions. There are no observable spectroscopic changes in the absence of bound chaperones.     

Translational bypassing without peptidyl-tRNA anticodon scanning of coding gap mRNA

Half the ribosomes translating the mRNA for phage T4 gene 60 topoisomerase subunit bypass a 50 nucleotide coding gap between codons 46 and 47. The pairing of codon 46 with its cognate peptidyl-tRNA anticodon dissociates, and following mRNA slippage, peptidyl-tRNA re-pairs to mRNA at a matched triplet 5' adjacent to codon 47, where translation resumes. Here, in studies with gene 60 cassettes, it is shown that the peptidyl-tRNA anticodon does not scan the intervening sequence for potential complementarity. However, certain coding gap mutants allow peptidyl-tRNA to scan sequences in the bypassed segment. A model is proposed in which the coding gap mRNA enters the ribosomal A-site and forms a structure that precludes peptidyl-tRNA scanning of its sequence. Dissipation of this RNA structure, together with the contribution of 16S rRNA anti-Shine-Dalgarno sequence pairing with GAG, facilitates peptidyl-tRNA re-pairing to mRNA.     

MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system

Arrays of MS2 binding sites are placed into mRNAs and are commonly used to visualize the localization of mRNAs in vivo by the expression of an MS2-GFP fusion protein. In Saccharomyces cerevisiae, we observed that arrays of MS2 binding sites inhibit 5′ to 3′ degradation of the mRNA in yeast cells and lead to the accumulation of a 3′ mRNA fragment containing the MS2 binding sites. This accumulation can be dependent on the binding of the MS2 stem loops (MS2-SL) by MS2 coat proteins (MCPs). Since such decay fragments can still bind MCP-GFP, the localization of such mRNA fragments can complicate the interpretation of the localization of full-length mRNA in vivo.     

Translational regulation of ribosomal protein S15 drives characteristic patterns of protein‐mRNA epistasis

Do coding and regulatory segments of a gene co‐evolve with each‐other? Seeking answers to this question, here we analyze the case of Escherichia coli ribosomal protein S15, that represses its own translation by specifically binding its messenger RNA (rpsO mRNA) and stabilizing a pseudoknot structure at the upstream untranslated region, thus trapping the ribosome into an incomplete translation initiation complex. In the absence of S15, ribosomal protein S1 recognizes rpsO and promotes translation by melting this very pseudoknot. We employ a robust statistical method to detect signatures of positive epistasis between residue site pairs and find that biophysical constraints of translational regulation (S15‐rpsO and S1‐rpsO recognition, S15‐mediated rpsO structural rearrangement, and S1‐mediated melting) are strong predictors of positive epistasis. Transforming the epistatic pairs into a network, we find that signatures of two different, but interconnected regulatory cascades are imprinted in the sequence‐space and can be captured in terms of two dense network modules that are sparsely connected to each other. This network topology further reflects a general principle of how functionally coupled components of biological networks are interconnected. These results depict a model case, where translational regulation drives characteristic residue‐level epistasis—not only between a protein and its own mRNA but also between a protein and the mRNA of an entirely different protein.     

Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex

In eukaryotic cells, polypeptides are N glycosylated after passing through the membrane of the ER into the ER lumen. This modification is effected cotranslationally by the multimeric oligosaccharyltransferase (OST) enzyme. Here, we report the first cross-linking of an OST subunit to a nascent chain that is undergoing translocation through, or integration into, the ER membrane. A photoreactive probe was incorporated into a nascent chain using a modified Lys-tRNA and was positioned in a cryptic glycosylation site (-Q-K-T- instead of -N-K-T-) in the nascent chain. When translocation intermediates with nascent chains of increasing length were irradiated, nascent chain photocross-linking to translocon components, Sec61alpha and TRAM, was replaced by efficient photocross-linking solely to a protein identified by immunoprecipitation as the STT3 subunit of the OST. No cross-linking was observed in the absence of a cryptic sequence or in the presence of a competitive peptide substrate of the OST. As no significant nascent chain photocross-linking to other OST subunits was detected in these fully assembled translocation and integration intermediates, our results strongly indicate that the nascent chain portion of the OST active site is located in STT3.     

Birth, life and death of nascent polypeptide chains

The journey of nascent polypeptides from synthesis at the peptidyl transferase center of the ribosome ("birth") to full function ("maturity") involves multiple interactions, constraints, modifications and folding events. Each step of this journey impacts the ultimate expression level and functional capacity of the translated protein. It has become clear that the kinetics of protein translation is predominantly modulated by synonymous codon usage along the mRNA, and that this provides an active mechanism for coordinating the synthesis, maturation and folding of nascent polypeptides. Multiple quality control systems ensure that proteins achieve their native, functional form. Unproductive co-translational folding intermediates that arise during protein synthesis may undergo enhanced interaction with components of these systems, such as chaperones, and/or be subjects of co-translational degradation ("death"). This review provides an overview of our current understanding of the complex co-translational events that accompany the synthesis, maturation, folding and degradation of nascent polypeptide chains.     

Selection of random RNA fragments as method for searching for a site of regulation of translation of <Emphasis Type="Italic">E. coli</Emphasis> streptomycin mRNA by ribosomal protein S7

In E. coli cells ribosomal small subunit biogenesis is regulated by RNA-protein interactions involving protein S7. S7 initiates the subunit assembly interacting with 16S rRNA. During shift-down of rRNA synthesis level, free S7 inhibits self-translation by interacting with 96 nucleotides long specific region of streptomycin (str) mRNA between cistrons S12 and S7 (intercistron). Many bacteria do not have the extended intercistron challenging development of specific approaches for searching putative mRNA regulatory regions, which are able to interact with proteins. The paper describes application of SERF approach (Selection of Random RNA Fragments) to reveal regulatory regions of str mRNA. Set of random DNA fragments has been generated from str operon by random hydrolysis and then transcribed into RNA; the fragments being able to bind protein S7 (serfamers) have been selected by iterative rounds. S7 binds to single serfamer, 109 nucleotide long (RNA109), derived from the intercistron. After multiple copying and selection, the intercistronic mutant (RNA109) has been isolated; it has enhanced affinity to S7. RNA109 binds to the protein better than authentic intercistronic str mRNA; apparent dissociation constants are 26 +/- 5 and 60 +/- 8 nM, respectively. Location of S7 binding site on the mRNA, as well as putative mode of regulation of coupled translation of S12 and S7 cistrons have been hypothesized.     

脱腺苷酸酶与RNA代谢调控

真核细胞中，RNA 3’端poly(A)或oligo(A)的特异性水解被称为脱腺苷酸化（deadenylation）。脱腺苷酸化的执行者被称为脱腺苷酸酶（deadenylase）。绝大多数真核细胞中都存在多种脱腺苷酸酶，其中CCR4-NOT复合体和PAN2-PAN3复合体负责细胞中大多数mRNA的非特异性降解，PARN和PNLDC1等参与了特定子集mRNA的降解和多种非编码RNA的生物合成。作为RNA水平的重要调控者之一，脱腺苷酸酶参与了几乎所有细胞生命活动和多种重要生理和病理过程。在真核细胞中，脱腺苷酸酶的分子调控机制可能是：细胞中的大量RNA结合蛋白是RNA命运调控的中心分子，一方面根据RNA的状态或细胞需求识别特定的靶标RNA子集，另一方面招募特定脱腺苷酸酶，对特定子集RNA的3’端进行降解或修剪，从而调控RNA的最终命运。细胞中十余种脱腺苷酸酶同工酶、上千种RNA结合蛋白以及多种多样的翻译后修饰构成了复杂的动态分子调控网络，帮助细胞在生长、增殖、分化、应激、死亡等重要生命活动中精确维持RNA稳态或快速转换基因表达谱。     

Distinct modes of recruitment of the CCR4–NOT complex by Drosophila and vertebrate Nanos

Nanos proteins repress the expression of target mRNAs by recruiting effector complexes through non‐conserved N‐terminal regions. In vertebrates, Nanos proteins interact with the NOT1 subunit of the CCR4–NOT effector complex through a NOT1 interacting motif (NIM), which is absent in Nanos orthologs from several invertebrate species. Therefore, it has remained unclear whether the Nanos repressive mechanism is conserved and whether it also involves direct interactions with the CCR4–NOT deadenylase complex in invertebrates. Here, we identify an effector domain (NED) that is necessary for the Drosophila melanogaster (Dm) Nanos to repress mRNA targets. The NED recruits the CCR4–NOT complex through multiple and redundant binding sites, including a central region that interacts with the NOT module, which comprises the C‐terminal domains of NOT1–3. The crystal structure of the NED central region bound to the NOT module reveals an unanticipated bipartite binding interface that contacts NOT1 and NOT3 and is distinct from the NIM of vertebrate Nanos. Thus, despite the absence of sequence conservation, the N‐terminal regions of Nanos proteins recruit CCR4–NOT to assemble analogous repressive complexes.     

Translational standby sites: how ribosomes may deal with the rapid folding kinetics of mRNA

We have previously shown that stable base-pairing at a translational initiation site in Escherichia coli can inhibit translation by competing with the binding of ribosomes. When the base-pairing is not too strong, this competition is won by the ribosomes, resulting in efficient translation from a structured ribosome binding site (RBS). We now re-examine these results in the light of RNA folding kinetics and find that the window during which a folded RBS is open is generally much too short to recruit a 30S ribosomal subunit from the cytoplasm. We argue that to achieve efficient expression, a 30S subunit must already be in contact with the mRNA while this is still folded, to shift into place as soon as the structure opens. Single-stranded regions flanking the structure may constitute a standby site, to which the 30S subunit can attach non-specifically. We propose a steady-state kinetic model for the early steps of translational initiation and use this to examine various quantitative aspects of standby binding. The kinetic model provides an explanation of why the earlier equilibrium competition model predicted implausibly high 30S-mRNA affinities. Because all RNA is structured to some degree, standby binding is probably a general feature of translational initiation.     

The role of mRNA structure in the regulation of protein synthesis: Implications for studies of development

A method is described for the experimental determination of the secondary structure of RNA using enzymatic cleavage data coupled with computer analysis. The structure-specific enzymes S1 nuclease and cobra venom ribonuclease are used to locate nonpaired and basepaired nucleotides, respectively. Computer techniques that utilize the enzymatic susceptibility information to generate a minimum free-energy structure are used to obtain secondary structure models. A second method, using acrylamide-agarose gel electrophoresis, is described for the determination of the relative protein synthesis initiation rates of endlabeled eukaryotic mRNAs. These methods are applied to the rabbit globin mRNAs as an example of a general approach for relating mRNA structure and function. A discussion of the role of messenger RNA structure in the regulation of translation is included with an emphasis on studies of development.