miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay

microRNAs (miRNAs) regulate gene expression through translational repression and/or messenger RNA (mRNA) deadenylation and decay. Because translation, deadenylation, and decay are closely linked processes, it is important to establish their ordering and thus to define the molecular mechanism of silencing. We have investigated the kinetics of these events in miRNA-mediated gene silencing by using a Drosophila S2 cell-based controllable expression system and show that mRNAs with both natural and engineered 3' untranslated regions with miRNA target sites are first subject to translational inhibition, followed by effects on deadenylation and decay. We next used a natural translational elongation stall to show that miRNA-mediated silencing inhibits translation at an early step, potentially translation initiation.     

Target protectors reveal dampening and balancing of Nodal agonist and antagonist by miR-430

MicroRNAs (miRNAs) repress hundreds of target messenger RNAs (mRNAs), but the physiological roles of specific miRNA-mRNA interactions remain largely elusive. We report that zebrafish microRNA-430 (miR-430) dampens and balances the expression of the transforming growth factor-beta (TGF-beta) Nodal agonist squint and the TGF-beta Nodal antagonist lefty. To disrupt the interaction of specific miRNA-mRNA pairs, we developed target protector morpholinos complementary to miRNA binding sites in target mRNAs. Protection of squint or lefty mRNAs from miR-430 resulted in enhanced or reduced Nodal signaling, respectively. Simultaneous protection of squint and lefty or absence of miR-430 caused an imbalance and reduction in Nodal signaling. These findings establish an approach to analyze the in vivo roles of specific miRNA-mRNA pairs and reveal a requirement for miRNAs in dampening and balancing agonist/antagonist pairs.     

Decapping and decay of messenger RNA occur in cytoplasmic processing bodies

A major pathway of eukaryotic messenger RNA (mRNA) turnover begins with deadenylation, followed by decapping and 5' to 3' exonucleolytic decay. We provide evidence that mRNA decapping and 5' to 3' degradation occur in discrete cytoplasmic foci in yeast, which we call processing bodies (P bodies). First, proteins that activate or catalyze decapping are concentrated in P bodies. Second, inhibiting mRNA turnover before decapping leads to loss of P bodies; however, inhibiting turnover at, or after, decapping, increases the abundance and size of P bodies. Finally, mRNA degradation intermediates are localized to P bodies. These results define the flux of mRNAs between polysomes and P bodies as a critical aspect of cytoplasmic mRNA metabolism and a possible site for regulation of mRNA degradation.     

miR-221功能研究进展

miR-221是由内源性发夹(Hairpin)结构转录产物衍生出来的一类长21～28个核苷酸的小分子非编码RNA(non-coding RNA,ncRNA)。通过碱基互补配对原则与靶基因mRNA结合,从而在转录水平引起靶基因mRNA的降解或者抑制其翻译成蛋白。miR-221是成簇分布的miRNA,近来研究表明,miR-221参与调控细胞增殖、凋亡、造血、肿瘤发生等一系列生物进程。文章介绍了miR-221功能研究的进展,为进一步研究miR-221提供了理论基础。     

斑马鱼miR-30e对血细胞生成的作用机制研究

microRNA(简写为miRNA)是一类内源基因编码的单链RNA分子,参与转录后基因的表达调控,在血液生成过程中起着重要作用.为研究miR-30e对鱼类血液生成的作用机制,以斑马鱼Danio rerio作为模式生物,针对青藏高原裂腹鱼血液组织中高表达的miR-30e,利用生物信息软件预测斑马鱼miR-30e的靶基因,...     

Translational operator of mRNA on the ribosome: how repressor proteins exclude ribosome binding

The ribosome of Thermus thermophilus was cocrystallized with initiator transfer RNA (tRNA) and a structured messenger RNA (mRNA) carrying a translational operator. The path of the mRNA was defined at 5.5 angstroms resolution by comparing it with either the crystal structure of the same ribosomal complex lacking mRNA or with an unstructured mRNA. A precise ribosomal environment positions the operator stem-loop structure perpendicular to the surface of the ribosome on the platform of the 30S subunit. The binding of the operator and of the initiator tRNA occurs on the ribosome with an unoccupied tRNA exit site, which is expected for an initiation complex. The positioning of the regulatory domain of the operator relative to the ribosome elucidates the molecular mechanism by which the bound repressor switches off translation. Our data suggest a general way in which mRNA control elements must be placed on the ribosome to perform their regulatory task.     

Inhibition of translational initiation by Let-7 MicroRNA in human cells

MicroRNAs (miRNAs) are approximately 21-nucleotide-long RNA molecules regulating gene expression in multicellular eukaryotes. In metazoa, miRNAs act by imperfectly base-pairing with the 3' untranslated region of target messenger RNAs (mRNAs) and repressing protein accumulation by an unknown mechanism. We demonstrate that endogenous let-7 microribonucleoproteins (miRNPs) or the tethering of Argonaute (Ago) proteins to reporter mRNAs in human cells inhibit translation initiation. M(7)G-cap-independent translation is not subject to repression, suggesting that miRNPs interfere with recognition of the cap. Repressed mRNAs, Ago proteins, and miRNAs were all found to accumulate in processing bodies. We propose that localization of mRNAs to these structures is a consequence of translational repression.     

Movement of eukaryotic mRNAs between polysomes and cytoplasmic processing bodies

Eukaryotic cells contain nontranslating messenger RNA concentrated in P-bodies, which are sites where the mRNA can be decapped and degraded. We present evidence that mRNA molecules within yeast P-bodies can also return to translation. First, inhibiting delivery of new mRNAs to P-bodies leads to their disassembly independent of mRNA decay. Second, P-bodies decline in a translation initiation-dependent manner during stress recovery. Third, reporter mRNAs concentrate in P-bodies when translation initiation is blocked and resume translation and exit P-bodies when translation is restored. Fourth, stationary phase yeast have large P-bodies containing mRNAs that reenter translation when growth resumes. The reciprocal movement of mRNAs between polysomes and P-bodies is likely to be important in the control of mRNA translation and degradation. Moreover, the presence of related proteins in P-bodies and maternal mRNA storage granules suggests this mechanism is widely adapted for mRNA storage.     

Structural roles for human translation factor eIF3 in initiation of protein synthesis

Protein synthesis in mammalian cells requires initiation factor eIF3, a approximately 750-kilodalton complex that controls assembly of 40S ribosomal subunits on messenger RNAs (mRNAs) bearing either a 5'-cap or an internal ribosome entry site (IRES). Cryo-electron microscopy reconstructions show that eIF3, a five-lobed particle, interacts with the hepatitis C virus (HCV) IRES RNA and the 5'-cap binding complex eIF4F via the same domain. Detailed modeling of eIF3 and eIF4F onto the 40S ribosomal subunit reveals that eIF3 uses eIF4F or the HCV IRES in structurally similar ways to position the mRNA strand near the exit site of 40S, promoting initiation complex assembly.     

Control of mRNA decay by heat shock-ubiquitin-proteasome pathway

Cytokine and proto-oncogene messenger RNAs (mRNAs) are rapidly degraded through AU-rich elements in the 3' untranslated region. Rapid decay involves AU-rich binding protein AUF1, which complexes with heat shock proteins hsc70-hsp70, translation initiation factor eIF4G, and poly(A) binding protein. AU-rich mRNA decay is associated with displacement of eIF4G from AUF1, ubiquitination of AUF1, and degradation of AUF1 by proteasomes. Induction of hsp70 by heat shock, down-regulation of the ubiquitin-proteasome network, or inactivation of ubiquitinating enzyme E1 all result in hsp70 sequestration of AUF1 in the perinucleus-nucleus, and all three processes block decay of AU-rich mRNAs and AUF1 protein. These results link the rapid degradation of cytokine mRNAs to the ubiquitin-proteasome pathway.     

Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay

No-go decay (NGD) is one of several messenger RNA (mRNA) surveillance systems dedicated to the removal of defective mRNAs from the available pool. Two interacting factors, Dom34 and Hbs1, are genetically implicated in NGD in yeast. Using a reconstituted yeast translation system, we show that Dom34:Hbs1 interacts with the ribosome to promote subunit dissociation and peptidyl-tRNA drop-off. Our data further indicate that these recycling activities are shared by the homologous translation termination factor complex eRF1:eRF3, suggesting a common ancestral function. Because Dom34:Hbs1 activity exhibits no dependence on either peptide length or A-site codon identity, we propose that this quality-control system functions broadly to recycle ribosomes throughout the translation cycle whenever stalls occur.     

动物病毒microRNA的研究进展

MicroRNAs(miRNAs)是一种含有约22～25个核苷酸的非编码单链RNA分子,广泛存在于人类及其他各种生物中。它通过与靶mRNA特异性的碱基互补配对,引起靶mRNA降解或者抑制其翻译,从而调节基因的转录后表达水平。病毒microRNA是新发现的一类miRNA,综述了近年来病毒microRNA的产生、作用机制、生物学功能及其在动物上的研究进展。     

miR-210-5p对南极独角雪冰鱼心脏发育的作用机制研究

microRNA为短链非编码RNA,通过与靶基因3'UTR序列互补在转录后水平发挥作用。已有研究表明,microRNA在心脏发育过程中起着重要的调控作用。南极冰鱼因体内缺乏功能性血红细胞,其心脏出现了补偿性增生。前期的研究提示,独角雪冰鱼心脏中特异表达的microRNAs可能与冰鱼心脏的补偿性增生相关。本研究针对南极冰鱼心脏中高表达的miR-210-5p,运用斑马鱼显微注射、靶基因预测等手段研究了miR-210-5p对心脏发育的作用机制。结果表明:斑马鱼胚胎注射miR-210-5p后,出现心包膜水肿,心脏发育畸形等现象。qRT-PCR分析显示,过表达miR-210-5p的斑马鱼胚胎中,心脏发育相关的标志性基因bmp4、smad1、gata6以及tbx2b的表达水平下调。Western blotting分析发现,bmp/smad通路中的BMP2、BMP4、SMAD1、GATA6以及TBX2B的蛋白表达水平也显著下调。通过对tbx20基因3'UTR的靶基因生物信息学预测,以及对其进行GFP荧光表达分析,发现tbx20基因可能是miR-210-5p的一个靶基因。由此推测,miR-210-5p可能通过抑制tbx20基因的表达,并调控bmp/smad通路以抑制冰鱼心脏发育。     

反义抑制miR-202引起斑马鱼早期胚胎发育异常研究

为了探讨miR-202在鱼类胚胎发育中的功能,采用实时定量反转录PCR技术和整体原位杂交技术检测了miR-202在斑马鱼胚胎发育阶段的表达。结果发现miR-202是母源性分子并在斑马鱼胚胎发育过程中持续表达,尤其在早期胚胎发育阶段表达水平较高。在此基础上,采用基因沉默技术在斑马鱼受精卵中显微注射miR-202的反义锁核苷酸,实时荧光定量反转录PCR技术和整体原位杂交技术结果显示miR-202反义锁核苷酸可以显著下调斑马鱼胚胎中miR-202表达水平,同时发现反义抑制miR-202后胚胎发育停滞在4 hpf时左右。共同注射miR-202前体可以部分挽救反义抑制miR-202后导致的胚胎发育停滞。本研究证明miR-202在斑马鱼胚胎发育过程中起着重要的调控作用,其功能是斑马鱼胚胎早期发育所必需的。为进一步探索miR-202在鱼类胚胎发育过程的功能奠定了基础。