microRNA-Mediated Messenger RNA Deadenylation Contributes to Translational Repression in Mammalian Cells

Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3′ untranslated region (UTR) of messenger RNA (mRNA) reducing its translation and stability. They also commonly induce shortening of the mRNA 3′ poly(A) tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3′ UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A) tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A) tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work, these results suggest a dual role of deadenylation in miRNA function: it contributes to translational repression as well as mRNA decay and is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs.     

miR319在植物器官发育中的调控作用

microRNAs(miRNAs)是一类内源性的、21~25个碱基长度的小分子非编码RNA,它通过指导剪切或者抑制翻译等方式调节植物基因的表达,参与调控植物生长发育各个方面。大量研究表明,miR319通过靶向TCPs转录因子控制植物叶、花等器官的生长命运,并参与调控部分激素生物合成和信号传导通路,在植物发育过程中发挥重要生物学功能。文章综述了miR319在植物叶形态建成、生长发育以及叶衰老和花器官发育等过程中的重要调控作用。     

A Role for the Poly(A)-binding Protein Pab1p in PUF Protein-mediated Repression

PUF proteins regulate translation and mRNA stability throughout eukaryotes. Using a cell-free translation assay, we examined the mechanisms of translational repression of PUF proteins in the budding yeast Saccharomyces cerevisiae. We demonstrate that the poly(A)-binding protein Pab1p is required for PUF-mediated translational repression for two distantly related PUF proteins: S. cerevisiae Puf5p and Caenorhabditis elegans FBF-2. Pab1p interacts with oligo(A) tracts in the HO 3′-UTR, a target of Puf5p, to dramatically enhance the efficiency of Puf5p repression. Both the Pab1p ability to activate translation and interact with eukaryotic initiation factor 4G (eIF4G) were required to observe maximal repression by Puf5p. Repression was also more efficient when Pab1p was bound in close proximity to Puf5p. Puf5p may disrupt translation initiation by interfering with the interaction between Pab1p and eIF4G. Finally, we demonstrate two separable mechanisms of translational repression employed by Puf5p: a Pab1p-dependent mechanism and a Pab1p-independent mechanism.     

Genetic interactions of Drosophila melanogaster arrest reveal roles for translational repressor Bruno in accumulation of Gurken and activity of Delta

arrest mutants have pleiotropic phenotypes, ranging from an early arrest of oogenesis to irregular embryonic segmentation defects. One function of arrest is in translational repression of oskar mRNA; this biochemical activity is presumed to be involved in other functions of arrest. To identify genes that could provide insight into how arrest contributes to translational repression or that may be targets for arrest-dependent translational control, we screened deficiency mutants for dominant modification of the arrest phenotype. Only four of the many deficiencies tested, which cover approximately 30% of the genome, modified the starting phenotype. One enhancer, identified fortuitously, is the Star gene. Star interaction with arrest results in excess Gurken protein, supporting the model that gurken is a target of repression. Two modifiers were mapped to individual genes. One is Lk6, which encodes a protein kinase predicted to regulate the rate-limiting initiation factor eIF4E. The second is Delta. The interaction between arrest and Delta mimics the phenotype of homozygous Delta mutants, suggesting that arrest could positively control Delta activity. Indeed, arrest mutants have significantly reduced levels of Delta protein at the interface of germline and follicle cells.     

microRNA在肌肉发育中的功能研究进展

microRNA(miRNA)是一类非编码的小RNA分子,它通过对靶mRNA的翻译抑制和降解对基因表达起负调节作用。现在人们已经清楚地知道miRNA参与了增殖、分化、凋亡、发育等许多生物过程。一些miRNA在肌肉中特异表达,参与肌肉发育。该文重点介绍了参与肌肉发育的miRNA。已有证据表明肌肉miRNA在肌肉的增殖和分化过程中起了重要的调节作用,miRNA的调节异常和肌肉疾病有关。因此,miRNA是一类新的肌肉调控因子,它有可能成为畜禽肉产量提高和肌肉相关疾病治疗的新型靶标。     

Tristetraprolin Recruits Eukaryotic Initiation Factor 4E2 To Repress Translation of AU-Rich Element-Containing mRNAs

Tristetraprolin (TTP) regulates the expression of AU-rich element-containing mRNAs through promoting the degradation and repressing the translation of target mRNA. While the mechanism for promoting target mRNA degradation has been extensively studied, the mechanism underlying translational repression is not well established. Here, we show that TTP recruits eukaryotic initiation factor 4E2 (eIF4E2) to repress target mRNA translation. TTP interacted with eIF4E2 but not with eIF4E. Overexpression of eIF4E2 enhanced TTP-mediated translational repression, and downregulation of endogenous eIF4E2 or overexpression of a truncation mutant of eIF4E2 impaired TTP-mediated translational repression. Overexpression of an eIF4E2 mutant that lost the cap-binding activity also impaired TTP''s activity, suggesting that the cap-binding activity of eIF4E2 is important in TTP-mediated translational repression. We further show that TTP promoted eIF4E2 binding to target mRNA. These results imply that TTP recruits eIF4E2 to compete with eIF4E to repress the translation of target mRNA. This notion is supported by the finding that downregulation of endogenous eIF4E2 increased the production of tumor necrosis factor alpha (TNF-α) protein without affecting the mRNA levels in THP-1 cells. Collectively, these results uncover a novel mechanism by which TTP represses target mRNA translation.     

MiRNA expression in the eye

MiRNAs are a newly discovered class of small noncoding RNAs that regulate gene expression by translational repression and mRNA degradation. It has become evident that miRNAs are involved in many important biological processes, including tissue differentiation and development. The role of miRNAs in the eye is beginning to be explored following their recent detection by miRNA expression analyses. Many of the target genes for these ocular miRNAs remain undefined. This review summarizes the current information about ocular miRNA expression. Future research should focus on the function of ocular miRNAs in eye development.     

Global analysis of microRNA target gene expression reveals the potential roles of microRNAs in maintaining tissue identity

Background  

MicroRNAs are non-coding small RNAs of ~22 nucleotides that regulate the gene expression by base-paring with target mRNAs, leading to mRNA cleavage or translational repression. It is currently estimated that microRNAs account for ~ 1% of predicted genes in higher eukaryotic genomes and that up to 30% of genes might be regulated by microRNAs. However, only very few microRNAs have been functionally characterized and the general functions of microRNAs are not globally studied.     

Cytoplasmic HYL1 modulates miRNA-mediated translational repression

MicroRNAs (miRNAs) control various biological processes by repressing target mRNAs. In plants, miRNAs mediate target gene repression via both mRNA cleavage and translational repression. However, the mechanism underlying this translational repression is poorly understood. Here, we found that Arabidopsis thaliana HYPONASTIC LEAVES1 (HYL1), a core component of the miRNA processing machinery, regulates miRNA-mediated mRNA translation but not miRNA biogenesis when it localized in the cytoplasm. Cytoplasmic HYL1 localizes to the endoplasmic reticulum and associates with ARGONAUTE1 (AGO1) and ALTERED MERISTEM PROGRAM1. In the cytoplasm, HYL1 monitors the distribution of AGO1 onto polysomes, binds to the mRNAs of target genes, represses their translation, and partially rescues the phenotype of the hyl1 null mutant. This study uncovered another function of HYL1 and provides insight into the mechanism of plant gene regulation.

The nuclear miRNA biogenesis factor HYL1 also localizes to the cytoplasm to modulate miRNA-mediated translational repression.     

miRNA靶基因的筛选方法及其相关网络资源

微小RNA（microRNA,miRNA）是一类广泛存在于动植物中,大小约22 nt的单链非编码小分子RNA.它通过与靶mRNA 3′末端非翻译区（untranslational region, UTR）结合,使mRNA降解或翻译抑制.大量的研究结果表明,miRNA在动植物的生长发育、细胞分化、细胞增殖与凋亡、肿瘤发生等诸多环节发挥着重要的调节作用.目前,miRNA靶基因的筛选方法主要有生物信息学筛选和实验方法两大类,且在互联网上拥有大量相关的网络共享资源.本文通过对现有miRNA靶基因的筛选方法及其相关网络资源进行整理与比较分析,以有效指导并辅助miRNA领域的研究.     

Differential translational regulation of IRE-containing mRNAs in Drosophila melanogaster by endogenous IRP and a constitutive human IRP1 mutant

Insects, like vertebrates, express iron regulatory proteins (IRPs) that may regulate proteins in cellular iron storage and energy metabolism. Two mRNAs, an unspliced form of ferritin H mRNA and succinate dehydrogenase subunit b (SDHb) mRNA, are known to comprise an iron responsive element (IRE) in their 5'-untranslated region making them susceptible to translational repression by IRPs at low iron levels. We have investigated the effect of wild-type human IRP1 (hIRP1) and the constitutively active mutant hIRP1-S437 in transgenic Drosophila melanogaster. Endogenous Drosophila IRE-binding activity was readily detected in gel retardation assays. However, translational repression assessed by polysome gradients was only visible for unspliced IRE-containing ferritin H mRNA, but not for SDHb mRNA. Upon expression of exogenous hIRP1-S437 both mRNAs were strongly repressed. This correlated with a diminished survival rate of adult flies with hIRP1 and complete lethality with hIRP1-S437. We conclude that constitutive IRP1 expression is deleterious to fly survival, probably due to the essential function of SDHb or proteins encoded by yet unidentified target mRNAs.     

AU-rich-element-dependent translation repression requires the cooperation of tristetraprolin and RCK/P54

AU-rich elements (AREs), residing in the 3' untranslated region (UTR) of many labile mRNAs, are important cis-acting elements that modulate the stability of these mRNAs by collaborating with trans-acting factors such as tristetraprolin (TTP). AREs also regulate translation, but the underlying mechanism is not fully understood. Here we examined the function and mechanism of TTP in ARE-mRNA translation. Through a luciferase-based reporter system, we used knockdown, overexpression, and tethering assays in 293T cells to demonstrate that TTP represses ARE reporter mRNA translation. Polyribosome fractionation experiments showed that TTP shifts target mRNAs to lighter fractions. In murine RAW264.7 macrophages, knocking down TTP produces significantly more tumor necrosis factor alpha (TNF-α) than the control, while the corresponding mRNA level has a marginal change. Furthermore, knockdown of TTP increases the rate of biosynthesis of TNF-α, suggesting that TTP can exert effects at translational levels. Finally, we demonstrate that the general translational repressor RCK may cooperate with TTP to regulate ARE-mRNA translation. Collectively, our studies reveal a novel function of TTP in repressing ARE-mRNA translation and that RCK is a functional partner of TTP in promoting TTP-mediated translational repression.     

动植物miRNA的生物合成、作用机理及其功能

microRNAs(miRNAs)是生物体内源长度约为20—23个核苷酸的非编码小RNA,通过与靶mRNA的互补配对而在转录后水平上对基因的表达进行负调控,导致mRNA的降解或翻译抑制。到目前为止,已报道有几千种miRNA存在于动物、植物、真菌等多细胞真核生物中,进化上高度保守。在植物和动物中,miRNA虽然都是通过与其靶基因的相互作用来调节基因表达,进而调控生物体的生长发育,但miRNA执行这种调控作用的机理却不尽相同。同时miRNA在动植物体内的形成过程也存在很多的不同之处。本文综述了动植物miRNA的生物合成、作用机理、生物功能等方面的研究进展。     

MicroRNAs in tomato plants

MicroRNAs (miRNAs) are a specialized class of small silencing RNAs that regulate gene expression in eukaryotes. In plants, miRNAs negatively regulate target mRNAs containing a highly complementary sequence by either mRNA cleavage or translational repression. As a model plant to study fleshy fruit ripening, miRNA studies in tomato have made great progress recently. MiRNAs were predicted to be involved in nearly all biological processes in tomato, particularly development, differentiation, and biotic and abiotic stress responses. Surprisingly, several miRNAs were verified to be involved in tomato fruit ripening and senescence. Recent studies suggest that miRNAs are related to host-virus interactions, which raises the possibility that miRNAs can be used as diagnostic markers for response to virus infection in tomato plants. In this review, we summarize our current knowledge systematically and advance future directions for miRNA research in tomato.