无义介导的mRNA降解机制及其在单基因遗传病中的作用

无义介导的mRNA降解(Nonsense-mediated mRNA decay,NMD)是一种广泛存在于真核生物细胞中的mRNA质量监控机制。该机制通过识别和降解含有提前终止密码子(Premature translational-termination codon,PTC)的转录产物防止有潜在毒性的截短蛋白的产生。据估计,约1/3的遗传性疾病是由提前终止密码子引起的,而NMD作用通常会改变某些遗传病的临床症状或遗传方式。文章主要综述了人体细胞中NMD对底物的识别及其作用机制,并以几种单基因遗传病为例探讨其对这些疾病表型的影响,表明NMD作用机制的进一步揭示将有助于单基因遗传病发病机制的阐明及治疗方法的改进。     

Lsm1 promotes genomic stability by controlling histone mRNA decay

Lsm1 forms part of a cytoplasmic protein complex, Lsm1-7-Pat1, involved in the degradation of mRNAs. Here, we show that Lsm1 has an important role in promoting genomic stability in Saccharomyces cerevisiae. Budding yeast cells lacking Lsm1 are defective in recovery from replication-fork stalling and show DNA damage sensitivity. Here, we identify histone mRNAs as substrates of the Lsm1-7-Pat1 complex in yeast, and show that abnormally high amounts of histones accumulate in lsm1Δ mutant cells. Importantly, we show that the excess of histones is responsible for the lsm1Δ replication-fork instability phenotype, since sensitivity of lsm1Δ cells to drugs that stall replication forks is significantly suppressed by a reduction in histone gene dosage. Our results demonstrate that improper histone stoichiometry leads to genomic instability and highlight the importance of regulating histone mRNA decay in the tight control of histone levels in yeast.     

Bioinformatic identification of candidate cis-regulatory elements involved in human mRNA polyadenylation

Polyadenylation is an essential step for the maturation of almost all cellular mRNAs in eukaryotes. In human cells, most poly(A) sites are flanked by the upstream AAUAAA hexamer or a close variant, and downstream U/GU-rich elements. In yeast and plants, additional cis elements have been found to be located upstream of the poly(A) site, including UGUA, UAUA, and U-rich elements. In this study, we have developed a computer program named PROBE (Polyadenylation-Related Oligonucleotide Bidimensional Enrichment) to identify cis elements that may play regulatory roles in mRNA polyadenylation. By comparing human genomic sequences surrounding frequently used poly(A) sites with those surrounding less frequently used ones, we found that cis elements occurring in yeast and plants also exist in human poly(A) regions, including the upstream U-rich elements, and UAUA and UGUA elements. In addition, several novel elements were found to be associated with human poly(A) sites, including several G-rich elements. Thus, we suggest that many cis elements are evolutionarily conserved among eukaryotes, and human poly(A) sites have an additional set of cis elements that may be involved in the regulation of mRNA polyadenylation.     

Alternative polyadenylation sites of human endothelial nitric oxide synthase mRNA

The mRNA 3'-untranslated region (3'-UTR) has been shown to have important roles in the regulation of mRNA function. In this study, we investigated the human endothelial nitric oxide synthase (eNOS) 3'-UTR to evaluate its potential regulatory role. 3'-RACE analysis revealed that the human eNOS mRNA has multiple alternative polyadenylation sites. Apart from the proximal site (418bp downstream of the stop codon), we identified two additional distal sites approximately 770 and 1478bp downstream of the stop codon. In addition, Northern analysis showed that the usage of these sites differed among human tissues. Further, amounts of these eNOS mRNAs were changed during growth of cultured human aortic endothelial cells; mRNAs with long 3'-UTRs decreased more rapidly than total mRNA, as cells approached confluency. Thus, the 3'-UTRs of human eNOS results from alternative polyadenylation sites and differ across tissues and during cell growth.     

Roles of polyadenylation and nucleolytic cleavage in the filamentous phage mRNA processing and decay pathways in Escherichia coli.

To define basic features of mRNA processing and decay in Escherichia coli, we have examined a set of mRNAs encoded by the filamentous phage f1 that have structures typical of bacterial mRNAs. They bear a stable hairpin stem-loop on the 3' end left from rho-independent termination and are known to undergo processing by RNase E. A small percentage of the f1 mRNAs were found to bear poly(A) tails that were attached to heterogeneous positions near the common 3' end. In a poly(A) polymerase-deficient host, the later-appearing processed mRNAs were stabilized, and a novel small RNA accumulated. This approximately 125-nt RNA proved to arise via RNase E cleavage from the 3'-terminal region of the mRNAs bearing the terminator. Normally ribosomes translating gene VIII appear to protect this cleavage site from RNase E, so that release of the fragment from the mRNAs occurs very slowly. The data presented define additional steps in the f1 mRNA processing and decay pathways and clarify how features of the pathways are used in establishing and maintaining the persistent filamentous phage infection. Although the primary mode of decay is endonucleolytic cleavage generating a characteristic 5' --> 3' wave of products, polyadenylation is involved in part in degradation of the processed mRNAs and is required for turnover of the 125-nt mRNA fragment. The results place polyadenylation at a later rather than an initiating step of decay. They also provide a clear illustration of how stably structured RNA 3' ends act as barriers to 3' --> 5' exonucleolytic mRNA decay.     

Saccharomyces cerevisiae Ebs1p is a putative ortholog of human Smg7 and promotes nonsense-mediated mRNA decay

The Smg proteins Smg5, Smg6 and Smg7 are involved in nonsense-mediated RNA decay (NMD) in metazoans, but no orthologs have been found in the budding yeast Saccharomyces cerevisiae. Sequence alignments reveal that yeast Ebs1p is similar in structure to the human Smg5-7, with highest homology to Smg7. We demonstrate here that Ebs1p is involved in NMD and behaves similarly to human Smg proteins. Indeed, both loss and overexpression of Ebs1p results in stabilization of NMD targets. However, Ebs1-loss in yeast or Smg7-depletion in human cells only partially disrupts NMD and in the latter, Smg7-depletion is partially compensated for by Smg6. Ebs1p physically interacts with the NMD helicase Upf1p and overexpressed Ebs1p leads to recruitment of Upf1p into cytoplasmic P-bodies. Furthermore, Ebs1p localizes to P-bodies upon glucose starvation along with Upf1p. Overall our findings suggest that NMD is more conserved in evolution than previously thought, and that at least one of the Smg5-7 proteins is conserved in budding yeast.     

The apolipoprotein B mRNA editing complex performs a multifunctional cycle and suppresses nonsense-mediated decay

The C to U editing of apolipoprotein B (apoB) mRNA is mediated by a minimal complex composed of an RNA-binding cytidine deaminase (APOBEC1) and a complementing specificity factor (ACF). This editing generates a premature termination codon and a truncated open reading frame. We demonstrate that the APOBEC1-ACF holoenzyme mediates a multifunctional cycle. The atypical APOBEC1 nuclear localization signal is involved in RNA binding and is used to import ACF into the nucleus as cargo. APOBEC1 alone induces nonsense-mediated decay (NMD). The APOBEC1-ACF complex edits and remains associated with the edited RNA to protect it from NMD. The APOBEC1 nuclear export signal is involved in the export of ACF and the edited apoB mRNA together, to the site of translation.     

Heterogeneity of polyadenylation site of mRNA coding for human serum albumin

Human fetal liver cDNA was cloned in pBR322 vector by dG:dC-tailing method. The cDNA library was screened for liver-specific clones by means of differential hybridization. Human fetal liver and human kidney cDNAs were used as hybridization probes. Application of this procedure revealed twenty five liver-specific clones among about one thousand recombinants analysed. These clones represent cDNAs corresponding abundant mRNAs. Eighteen clones were identified as encoding serum albumin. Two different mRNA polyadenylation sites were found in four sequenced plasmids. Cleavage/polyadenylation site in two plasmids, pHA1 and pHA12, is situated fifteen nucleotides downstream the AATAAA signal; in two other plasmids, pHA8 and pHA25, this site is ten nucleotides downstream the same signal.     

Chunghyuldan activates NOS mRNA expression and suppresses VCAM-1 mRNA expression in human endothelial cells

Chunghyuldan (CHD), a combinatorial drug that has antihyperlipidemic and anti-inflammatory activities, has been shown to improve arterial stiffness and inhibit stroke recurrence in clinical study. To understand the molecular basis of CHD's clinical effects, we explored its effect on cell proliferation and expression of nitric oxide synthase (NOS) and vascular cell adhesion molecule (VCAM-1) in human umbilical vein endothelial cells (HUVECs). Cell number counting and [3H]thymidine incorporation assay demonstrated that nontoxic doses of CHD have an inhibitory effect on DNA synthesis and suppress cell cycle progression of HUVECs. CHD treatment led to a marked induction of NO production through up-regulation of NOS mRNA expression in a dose- and time-dependent manner, whereas it suppressed VCAM-1 expression. CHD inhibition of VCAM-1 expression was totally blocked by pretreatment with the NO synthesis inhibitor L-NMMA, whereas pretreatment with the NO donor DETA-NO further decreased VCAM-1 level in CHD-treated HUVECs, indicating that VCAM-1 regulation by CHD is mediated through increased NO synthesis by CHD. In addition, TNF-alpha-mediated VCAM-1 activation was substantially impeded by CHD treatment. Collectively, our data suggest that anti-inflammatory or anti-hyperlipidemic effects of CHD might be associated with its ability to activate NO production and suppress VCAM-1 expression in human endothelial cells.     

Translation and mRNA decay

Summary 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 nearby 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.     

Cytoplasmic foci are sites of mRNA decay in human cells

Understanding gene expression control requires defining the molecular and cellular basis of mRNA turnover. We have previously shown that the human decapping factors hDcp2 and hDcp1a are concentrated in specific cytoplasmic structures. Here, we show that hCcr4, hDcp1b, hLsm, and rck/p54 proteins related to 5'-3' mRNA decay also localize to these structures, whereas DcpS, which is involved in cap nucleotide catabolism, is nuclear. Functional analysis using fluorescence resonance energy transfer revealed that hDcp1a and hDcp2 interact in vivo in these structures that were shown to differ from the previously described stress granules. Our data indicate that these new structures are dynamic, as they disappear when mRNA breakdown is abolished by treatment with inhibitors. Accumulation of poly(A)(+) RNA in these structures, after RNAi-mediated inactivation of the Xrn1 exonuclease, demonstrates that they represent active mRNA decay sites. The occurrence of 5'-3' mRNA decay in specific subcellular locations in human cells suggests that the cytoplasm of eukaryotic cells may be more organized than previously anticipated.