DcpS scavenger decapping enzyme can modulate pre-mRNA splicing

The human scavenger decapping enzyme, DcpS, functions to hydrolyze the resulting cap structure following cytoplasmic mRNA decay yet is, surprisingly, a nuclear protein by immunofluorescence. Here, we show that DcpS is a nucleocytoplasmic shuttling protein that contains separable nuclear import and Crm-1-dependent export signals. We postulated that the presence of DcpS in both cellular compartments and its ability to hydrolyze cap structure may impact other cellular events dependent on cap-binding proteins. An shRNA-engineered cell line with markedly diminished DcpS levels led to a corresponding reduction in cap-proximal intron splicing of a reporter minigene and endogenous genes. The impaired cap catabolism and resultant imbalanced cap concentrations were postulated to sequester the cap-binding complex (CBC) from its normal splicing function. In support of this explanation, DcpS efficiently displaced the nuclear cap-binding protein Cbp20 from cap structure, and complementation with Cbp20 reversed the reduced splicing, indicating that modulation of splicing by DcpS is mediated through Cbp20. Our studies demonstrate that the significance of DcpS extends beyond its well-characterized role in mRNA decay and involves a broader range of functions in RNA processing including nuclear pre-mRNA splicing.     

The DHH1/RCKp54 family of helicases: An ancient family of proteins that promote translational silencing

Translational control is a vital aspect of gene expression. Message specific translational repressors have been known for decades. Recent evidence, however, suggests that a general machinery exists that dampens the translational capacity of the majority of mRNAs. This activity has been best ascribed to a conserved family of RNA helicases called the DHH1/RCKp54 family. The function of these helicases is to promote translational silencing. By transitioning mRNA into quiescence, DHH1/RCKp54 helicases promote either mRNA destruction or storage. In this review we describe the known roles of these helicases and propose a mechanistic model to explain their mode of action. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.     

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.     

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.     

Processing bodies are not required for mammalian nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality-control mechanism that recognizes and degrades mRNAs with premature termination codons (PTCs). In yeast, PTC-containing mRNAs are targeted to processing bodies (P-bodies), and yeast strains expressing an ATPase defective Upf1p mutant accumulate P-bodies. Here we show that in human cells, an ATPase-deficient UPF1 mutant and a fraction of UPF2 and UPF3b accumulate in cytoplasmic foci that co-localize with P-bodies. Depletion of the P-body component Ge-1, which prevents formation of microscopically detectable P-bodies, also impairs the localization of mutant UPF1, UPF2, and UPF3b in cytoplasmic foci. However, the accumulation of the ATPase-deficient UPF1 mutant in P-bodies is independent of UPF2, UPF3b, or SMG1, and the ATPase-deficient UPF1 mutant can localize into the P-bodies independent of its phosphorylation status. Most importantly, disruption of P-bodies by depletion of Ge-1 affects neither the mRNA levels of PTC-containing reporter genes nor endogenous NMD substrates. Consistent with the recently reported decapping-independent SMG6-mediated endonucleolytic decay of human nonsense mRNAs, our results imply that microscopically detectable P-bodies are not required for mammalian NMD.     

Crystal structures of human DcpS in ligand-free and m7GDP-bound forms suggest a dynamic mechanism for scavenger mRNA decapping

Eukaryotic cells utilize DcpS, a scavenger decapping enzyme, to degrade the residual cap structure following 3'-5' mRNA decay, thereby preventing the premature decapping of the capped long mRNA and misincorporation of methylated nucleotides in nucleic acids. We report the structures of DcpS in ligand-free form and in a complex with m7GDP. apo-DcpS is a symmetric dimer, strikingly different from the asymmetric dimer observed in the structures of DcpS with bound cap analogues. In contrast, and similar to the m7GpppG-DcpS complex, DcpS with bound m7GDP is an asymmetric dimer in which the closed state appears to be the substrate-bound complex, whereas the open state mimics the product-bound complex. Comparisons of these structures revealed conformational changes of both the N-terminal swapped-dimeric domain and the cap-binding pocket upon cap binding. Moreover, Tyr273 in the cap-binding pocket displays remarkable conformational changes upon cap binding. Mutagenesis and biochemical analysis suggest that Tyr273 seems to play an important role in cap binding and product release. Examination of the crystallographic B-factors indicates that the N-terminal domain in apo-DcpS is inherently flexible, and in a dynamic state ready for substrate binding and product release.     

Multiple binding of repressed mRNAs by the P-body protein Rck/p54

Translational repression is achieved by protein complexes that typically bind 3' UTR mRNA motifs and interfere with the formation of the cap-dependent initiation complex, resulting in mRNPs with a closed-loop conformation. We demonstrate here that the human DEAD-box protein Rck/p54, which is a component of such complexes and central to P-body assembly, is in considerable molecular excess with respect to cellular mRNAs and enriched to a concentration of 0.5 mM in P-bodies, where it is organized in clusters. Accordingly, multiple binding of p54 proteins along mRNA molecules was detected in vivo. Consistently, the purified protein bound RNA with no sequence specificity and high nanomolar affinity. Moreover, bound RNA molecules had a relaxed conformation. While RNA binding was ATP independent, relaxing of bound RNA was dependent on ATP, though not on its hydrolysis. We propose that Rck/p54 recruitment by sequence-specific translational repressors leads to further binding of Rck/p54 along mRNA molecules, resulting in their masking, unwinding, and ultimately recruitment to P-bodies. Rck/p54 proteins located at the 5' extremity of mRNA can then recruit the decapping complex, thus coupling translational repression and mRNA degradation.     

Cytoplasmic mRNA for human triosephosphate isomerase is immune to nonsense-mediated decay despite forming polysomes

Nonsense codons between position 14 within the first exon and position 193 within the penultimate exon of the human gene for triosephosphate isomerase reduce mRNA abundance to 25% of normal. The reduction in abundance is due to the decay of newly synthesized mRNA that copurifies with nuclei. TPI mRNA that copurifies with cytoplasm is immune to decay. We show here that immunity is not due to the failure of nonsense-containing mRNA to form polysomes. This finding indicates that cytoplasmic mRNA, in contrast to nucleus-associated mRNA, may have lost one or more factors that are required for nonsense-mediated decay or gained one or more factors that confer immunity to nonsense-mediated decay.     

mRNA降解在植物发育以及抗性反应中的作用

植物mRNA的降解对于维持其生化和细胞学的功能都是必需的,而且这种降解要根据植物发育和外界环境的变化进行及时的调整。与酵母和哺乳动物相比,人们对植物mRNA的降解机制了解较少。对近几年来该领域的研究进展进行总结,包括参与植物mRNA降解的酶类和基因芯片技术的应用,以及mRNA降解的生物学意义等。     

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.     

单核细胞趋化蛋白诱导蛋白-1的研究进展

单核细胞趋化蛋白诱导蛋白-1（MCPIP1）是最近发现的一类具有免疫调节作用的CCCH型锌指家族分子。MCPIP1可被LPS、IL-1β或MCP-1等多种炎性因子刺激表达,可通过下调炎症因子（如IL-6、IL-12p40等）表达,从而负向调控炎症过程。MCPIP1的作用机制主要是作为RNA酶调节某些炎性因子mRNA或pre-miRNA的降解。此外,MCPIP1也可作为去泛素化酶靶向TNF受体相关蛋白（TRAFs）成员,从而负向调控JNK和NF-κB信号活化。将从MCPIP1分子的发现、基因和蛋白质结构、生物学功能、表达调控以及临床应用前景等几个方面进行阐述。     

The tRNA Splicing Endonuclease Complex Cleaves the Mitochondria-localized CBP1 mRNA

The tRNA splicing endonuclease (Sen) complex is located on the mitochondrial outer membrane and splices precursor tRNAs in Saccharomyces cerevisiae. Here, we demonstrate that the Sen complex cleaves the mitochondria-localized mRNA encoding Cbp1 (cytochrome b mRNA processing 1). Endonucleolytic cleavage of this mRNA required two cis-elements: the mitochondrial targeting signal and the stem-loop 652–726-nt region. Mitochondrial localization of the Sen complex was required for cleavage of the CBP1 mRNA, and the Sen complex cleaved this mRNA directly in vitro. We propose that the Sen complex cleaves the CBP1 mRNA, which is co-translationally localized to mitochondria via its mitochondrial targeting signal.     

mRNA transport,translation, and decay in adult mammalian central nervous system axons

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