Altering the 3′ UTR endonucleolytic cleavage site of a Chlamydomonas chloroplast mRNA affects 3′-end maturation in vitro but not in vivo

The 3 ends of chloroplast mRNAs are produced by the processing of longer precursors. The 3 ends of most plastid mRNAs are located at, or several nucleotides downstream of, stem-loop structures, which act as 3-end-processing signals and RNA stability elements. In chloroplasts of the green alga Chlamydomonas reinhardtii, 3-end maturation of atpB mRNA involves endonucleolytic cleavage of the pre-mRNA at an AU-rich site located about 10 nucleotides downstream of the stem-loop structure. This cleavage is followed by exonucleolytic resection to generate the mature 3 end. In order to define critical nucleotides of the endonucleolytic cleavage site, we mutated its sequence. Incubation of synthetic atpB pre-RNAs containing these mutations in a chloroplast protein extract resulted in the accumulation of 3-end-processed products. However, in two cases where the AU-rich sequence of this site was replaced with a GC-rich one, the 3 end of the stable processing product differed from that of the wild-type product. To examine whether these mutations affected atpB mRNA processing or accumulation in vivo, the endogenous 3 UTR was replaced with mutated sequences by biolistic transformation of Chlamydomonas chloroplasts. Analysis of the resulting strains revealed that the accumulation of atpB mRNA was approximately equal to that of wild-type cells, and that a wild-type atpB 3 end was generated. These results imply that Chlamydomonas atpB 3 processing parallels the situation with other endonucleases such as Escherichia coli RNAse E, where specific sequences are required for correct in vitro processing, but in vivo these mutations can be overcome.     

人SDCT2β 3′-非翻译区基因表达负调控序列对mRNA稳定性的影响

为探索人高亲和力钠离子依赖性二羧酸转运蛋白基因（high affinity sodium-dependent dicarboxylate transporter,SDCT2）3′端非翻译区是否在基因表达调控中起作用,首先通过生物信息学分析发现,在SDCT2βmRNA的3′端非翻译区内存在585nt的AU富含区（AU-rich region,AUR）,其中包括3个AU富含元件（AU-rich element,ARE）,然后将SDCT2β的AU富含区DNA片段插入报告基因GFP表达载体pcDNA-GFP的下游,构建pcDNA-GFP-AUR表达载体并转染HEK293、HKC和LLC-PK1细胞系,用Western blot和流式细胞仪检测细胞中GFP的表达水平.结果显示,SDCT2β的AU富含区序列可显著降低GFP的表达水平（P〈0.01）.利用放线菌素D阻断RNA转录后,每隔2h从稳定转染的HEK293细胞中提取总RNA,用RNA印迹分析GFP mRNA的稳定性.结果显示GFP-AURmRNA较GFP mRNA不稳定.这些结果提示,在SDCT2β3′非翻译区的AU富含区内存在基因表达负调控区,该区可降低mRNA的稳定性、促进mRNA的降解,从而在转录后水平调控基因的表达.     

Chlamydomonas reinhardtii as a unicellular model for circadian rhythm analysis

Chlamydomonas reinhardtii has been used as an experimental model organism for circadian rhythm research for more than 30 yr. Some of the physiological rhythms of this alga are well established, and several clock mutants have been isolated. The cloning of clock genes from these mutant strains by positional cloning is under way and should give new insights into the mechanism of the circadian clock. In a spectacular space experiment, the question of the existence of an endogenous clock vs. an exogenous mechanism has been studied in this organism. With the emergence of molecular analysis of circadian rhythms in plants in 1985, a circadian gene expression pattern of several nuclear and chloroplast genes was detected. Evidence is now accumulating that shows circadian control at the translational level. In addition, the gating of the cell cycle by the circadian clock has been analyzed. This review focuses on the different aspects of circadian rhythm research in C. reinhardtii over the past 30 yr. The suitability of Chlamydomonas as a model system in chronobiology research and the adaptive significance of the observed rhythms will be discussed.     

Chlamydomonas reinhardtii as a unicellular model for circadian rhythm analysis

Chlamydomonas reinhardtii has been used as an experimental model organism for circadian rhythm research for more than 30 yr. Some of the physiological rhythms of this alga are well established, and several clock mutants have been isolated. The cloning of clock genes from these mutant strains by positional cloning is under way and should give new insights into the mechanism of the circadian clock. In a spectacular space experiment, the question of the existence of an endogenous clock vs. an exogenous mechanism has been studied in this organism. With the emergence of molecular analysis of circadian rhythms in plants in 1985, a circadian gene expression pattern of several nuclear and chloroplast genes was detected. Evidence is now accumulating that shows circadian control at the translational level. In addition, the gating of the cell cycle by the circadian clock has been analyzed. This review focuses on the different aspects of circadian rhythm research in C. reinhardtii over the past 30 yr. The suitability of Chlamydomonas as a model system in chronobiology research and the adaptive significance of the observed rhythms will be discussed.     

mRNA结构及其稳定性的关系

mRNA结构与mRNA稳定性关系密切,mRNA稳定性与基因表达调控之间也有着紧密的联系。现从mRNA结构中所含的5'端帽结构、3'端poly(A)尾、5'非翻译区、3'非翻译区、编码区、富AU元件等方面综述了mRNA结构与mRNA稳定性之间的关系,为深入了解基因表达调控的分子机制提供理论基础。     

Tubulin mRNA instability and stabilization by protein synthesis inhibitors are reproducible in nontranslating extracts from Chlamydomonas

In Chlamydomonas rein-hardtii, flagellar amputation stimulates an induction in the synthesis of flagellar proteins which allows the cells to rapidly regenerate their flagella. The induction involves the coordinate accumulation and rapid degradation of a large number mRNAs, including those encoding the tubulins. The post-induction degradation of induced tubulin mRNAs has been shown to differ from the consti-tutive turnover pathway in two ways: (1) the rate of degradation is accelerated, and (2) degradation is prevented by inhibition of protein synthesis. In this report, it is shown that the post-induction degradation of all deflagellation-induced mRNAs examined is prevented by cycloheximide (CX), suggesting they all may be degraded via the same pathway. A cell-free decay system has been developed to investigate the degradation pathway. At least two characteristics of tubulir mRNA degradation are reproducible in these extracts: (1) endogenous α-tubulin mRNA is less stable than constitutive mRNAs in the same extract and (2) α-tubulin mRNA in extracts prepared from CX-treated cells (CX ex-tracts) is significantly more stable than it is in extracts from untreated cells (control extracts). This indicates that the mechanism by which CX blocks rapid degradation of tubulin mRNA in vivo is not simply by preventing its translation and suggests the involvement of an altered trans-factor. The difference in tubulin mRNA stability in the two extracts is maintained when the extracts are prepared under conditions that dissociate ribosomes from mRNPs, indicating intact polysome structure is not necessary. Tubulin mRNA-containing polysomes isolated from control and CX extracts are equally stable when assayed alone. However, the poly-somes from control extracts are more sensitive to exogenous RNAse treatment than are those from CX extracts, indicating a structural difference. There are no detectable differences in soluble factors that influence tubulin mRNA degradation rate between control and CX extracts; addition of excess soluble factors to either control or CX extracts does not alter the tubulin mRNA degradation in the extract, nor does a simple one-to-one combination of the two extracts result in stabilization or destabilization of the whole population of tubulin mRNAs in the mixture. The deflagellation-induced mRNAs, as a group, are shown to be particularly susceptible to a nuclease activity in extracts, inhibitable by vanadyl ribonucleoside complexes, which does not appear to attack constitutive mRNAs. It is proposed that a structural difference in the tubulin mRNPs produced in the presence and absence of CX underlies their differences in stabilities, and that a common nuclease targets the induced flagellar protein mRNAs. © 1993 Wiley-Liss, Inc.     

非编码长链RNA与基因表达调控

近年来,在小鼠全长cDNA文库大规模测序中发现一类新的转录物——非编码长链RNA(long noncoding RNA,lncRNA),引起了科学界的关注.lncRNA长度大于200个核苷酸,无蛋白质编码功能,在真核细胞基因组中被普遍转录.lncRNA种类繁多,数量庞大,占哺乳动物基因组转录物的绝大部分.相对于研究较多的非编码小RNA,lncRNA的功能目前尚不完全清楚.但越来越多的研究发现,lncRNA在多个水平调控基因的表达,在胚胎发育、物种进化、细胞分化和某些疾病如神经退行性疾病及肿瘤的发生过程中起着重要作用.本文在简要介绍lncRNA基本概念的基础上,结合当前研究成果,就lncRNA在转录水平、转录后水平和表观遗传水平调控基因表达的机制作一综述.     

Polyamines Stimulate the Level of the σ38 Subunit (RpoS) of Escherichia coli RNA Polymerase,Resulting in the Induction of the Glutamate Decarboxylase-dependent Acid Response System via the gadE Regulon

To study the physiological roles of polyamines, we carried out a global microarray analysis on the effect of adding polyamines to an Escherichia coli mutant that lacks polyamines because of deletions in the genes in the polyamine biosynthetic pathway. Previously, we have reported that the earliest response to polyamine addition is the increased expression of the genes for the glutamate-dependent acid resistance system (GDAR). We also presented preliminary evidence for the involvement of rpoS and gadE regulators. In the current study, further confirmation of the regulatory roles of rpoS and gadE is shown by a comparison of genome-wide expression profiling data from a series of microarrays comparing the genes induced by polyamine addition to polyamine-free rpoS⁺/gadE⁺ cells with genes induced by polyamine addition to polyamine-free ΔrpoS/gadE⁺ and rpoS⁺/ΔgadE cells. The results indicate that most of the genes in the E. coli GDAR system that are induced by polyamines require rpoS and gadE. Our data also show that gadE is the main regulator of GDAR and other acid fitness island genes. Both polyamines and rpoS are necessary for the expression of gadE gene from the three promoters of gadE (P1, P2, and P3). The most important effect of polyamine addition is the very rapid increase in the level of RpoS sigma factor. Our current hypothesis is that polyamines increase the level of RpoS protein and that this increased RpoS level is responsible for the stimulation of gadE expression, which in turn induces the GDAR system in E. coli.