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Results for “MicroRNA, (miRNA)”

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PubMed2006

MIWI associates with translational machinery and PIWI-interacting RNAs (piRNAs) in regulating spermatogenesis.

Noncoding small RNAs have emerged as important regulators of gene expression at both transcriptional and posttranscriptional levels. Particularly, microRNA (miRNA)-mediated translational repression involving PIWI/Argonaute family proteins has been widely recognized as a novel mechanism of gene regulation. We previously reported that MIWI, a murine PIWI family member, is required for initiating spermiogenesis, a process that transforms round spermatids into mature sperm. MIWI is a cytoplasmic protein present in spermatocytes and round spermatids, and it is required for the expression of its target mRNAs involved in spermiogenesis. Most recently, we discovered a class of noncoding small RNAs called PIWI-interacting RNAs (piRNAs) that are abundantly expressed during spermiogenesis in a MIWI-dependent fashion. Here, we show that MIWI associates with both piRNAs and mRNAs in cytosolic ribonucleoprotein and polysomal fractions. As polysomes increase in early spermiogenesis, MIWI increases in polysome fractions. Moreover, MIWI associates with the mRNA cap-binding complex. Interestingly, MIWI is required for the expression of not only piRNAs but also a subset of miRNAs, despite the presence of Dicer. These results suggest that MIWI has a complicated role in the biogenesis and/or maintenance of two distinct types of small RNAs. Together, our results indicate that MIWI, a PIWI subfamily protein, uses piRNA as the major, but not exclusive, binding partner, and it is associated with translational machinery.

Animals↗

PubMed2006

Analysis of short RNAs in the malaria parasite and its red blood cell host.

RNA interference (RNAi) is an RNA degradation process that involves short, double-stranded RNAs (dsRNA) as sequence specificity factors. The natural function of the RNAi machinery is to generate endogenous short double-stranded RNAs to regulate gene expression. It has been shown that treatment of Plasmodium falciparum, the etiologic agent of malaria, with dsRNA induces degradation of the corresponding microRNA (miRNA), yet typical RNAi-associated genes have not been identifiable in the parasite genome. To clarify this discrepancy we set out to clone short RNAs from P. falciparum-infected red blood cells and from purified parasites. We did not find any short RNA that was not a rRNA or tRNA fragment. Indeed, only known human miRNAs were isolated in parasite preparations indicating that very few if any short RNAs exist in P. falciparum. This suggests a different mechanism than classical RNAi in observations of dsRNA-mediated degradation. Of the human miRNAs identified, the human miRNA mir-451 accumulates at a very high level in both infected and healthy red blood cells. Interestingly, mir-451 was not detectable in a series of immortalised cell lines representing progenitor stages of all major blood lineages, suggesting that mir-451 may play a role in the differentiation of erythroid cells.

Animals↗

PubMed2026

Whole-transcriptome RNA sequencing and ceRNA network analyses provide novel insights into the antibacterial immune response of Hippocampus abdominalis against Vibrio harveyi.

Long non-coding RNAs (lncRNAs) stand as newly-arisen molecular types that exert regulatory effects, able to operate as competitive endogenous RNAs (ceRNAs) to engage microRNAs (miRNAs) in interaction, resulting in the recovery of target mRNA expression and activity. Increasing evidences indicate that the ceRNA network affects various biological processes in mammals, including development, cellular differentiation, metabolism, immune response, and disease pathogenesis. In teleost fish, the lncRNA-miRNA-mRNA regulatory networks have been reported occasionally. However, up to now, the roles of lncRNAs in the big-belly seahorse (Hippocampus abdominalis) remains unclear. In this study, we reported for the first time, via whole-transcriptome RNA sequencing, the lncRNA mediated ceRNA regulatory network in Vibrio harveyi-infected H. abdominalis. A total of 4197 differentially expressed mRNAs (DE-mRNAs), 1317 DE-lncRNAs, and 183 DE-miRNAs were identified. Furthermore, the crosstalk between miRNAs and lncRNAs as well as between miRNAs and mRNAs was inferred based on the negative correlations between miRNAs and their target lncRNAs/mRNAs. A core immune associated lncRNA-miRNA-mRNA putative regulatory network was thus constructed, comprising 211 lncRNA-miRNA and 224 mRNA-miRNA pairs. In conclusion, our findings provide an integrative overview of the ceRNA regulatory networks on the underlying immune responses to V. harveyi infection in the big-belly seahorse, and offer a solid theoretical foundation for the comparative immunological research of teleost fish.

Animals↗

PubMed2004

Overexpression of exportin 5 enhances RNA interference mediated by short hairpin RNAs and microRNAs.

Plasmids or viral vectors that express short hairpin RNAs (shRNAs) have emerged as important tools for the stable inhibition of specific genes by RNA interference. shRNAs are structural and functional homologs of pre-microRNAs, intermediates in the production of endogenously encoded microRNAs (miRNAs). Therefore, overexpressed shRNAs could inhibit miRNA function by competing for a limiting level of one or more factors involved in miRNA biogenesis or function. Here, we demonstrate that overexpressed shRNAs can saturate the activity of endogenous Exportin 5, a factor required for nuclear export of both shRNAs and pre-miRNAs. While shRNA overexpression can therefore inhibit miRNA function, simultaneous overexpression of Exportin 5 reverses this effect. Moreover, Exportin 5 overexpression can significantly enhance RNA interference mediated by shRNAs. These data have implications for the future clinical utilization of shRNAs and also provide a simple method to enhance RNA interference by shRNAs in culture.

Active Transport, Cell Nucleus↗

PubMed2005

Involvement of microRNA in AU-rich element-mediated mRNA instability.

AU-rich elements (AREs) in the 3' untranslated region (UTR) of unstable mRNAs dictate their degradation. An RNAi-based screen performed in Drosophila S2 cells has revealed that Dicer1, Argonaute1 (Ago1) and Ago2, components involved in microRNA (miRNA) processing and function, are required for the rapid decay of mRNA containing AREs of tumor necrosis factor-alpha. The requirement for Dicer in the instability of ARE-containing mRNA (ARE-RNA) was confirmed in HeLa cells. We further observed that miR16, a human miRNA containing an UAAAUAUU sequence that is complementary to the ARE sequence, is required for ARE-RNA turnover. The role of miR16 in ARE-RNA decay is sequence-specific and requires the ARE binding protein tristetraprolin (TTP). TTP does not directly bind to miR16 but interacts through association with Ago/eiF2C family members to complex with miR16 and assists in the targeting of ARE. miRNA targeting of ARE, therefore, appears to be an essential step in ARE-mediated mRNA degradation.

3' Untranslated Regions↗

PubMed2006

Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues.

MicroRNAs (miRNAs) are a non-coding family of genes involved in post-transcriptional gene regulation. These transcripts are associated with cell proliferation, cell differentiation, cell death and carcinogenesis. We analysed the miRNA expression profiles in 25 pairs of hepatocellular carcinoma (HCC) and adjacent non-tumorous tissue (NT) and nine additional chronic hepatitis (CH) specimens using a human miRNA microarray. Targets and references samples were co-hybridized to a microarray containing whole human mature and precursor miRNA sequences. Whereas three miRNAs exhibited higher expression in the HCC samples than that in the NT samples, five miRNAs demonstrated lower expression in the HCC samples than in the NT samples (P<0.0001). Classification of samples as HCC or NT by using support vector machine algorithms based on these data provided an overall prediction accuracy of 97.8% (45/46). In addition, the expression levels of four miRNAs were inversely correlated with the degree of HCC differentiation (P<0.01). A comparison of CH and liver cirrhosis samples revealed significantly different pattern of miRNA expression (P<0.01). There were no differences, however, between hepatitis B-positive and hepatitis C-positive samples. This information may help clarify the molecular mechanisms involved in the progression of liver disease, potentially serving as a diagnostic tool of HCC.

Adult↗

PubMed2002

A microRNA in a multiple-turnover RNAi enzyme complex.

In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. Here we show that, in human cell extracts, the miRNA let-7 naturally enters the RNAi pathway, which suggests that only the degree of complementarity between a miRNA and its RNA target determines its function. Human let-7 is a component of a previously identified, miRNA-containing ribonucleoprotein particle, which we show is an RNAi enzyme complex. Each let-7-containing complex directs multiple rounds of RNA cleavage, which explains the remarkable efficiency of the RNAi pathway in human cells.

Adenosine Triphosphate↗

PubMed2005

MicroRNA expression in zebrafish embryonic development.

MicroRNAs (miRNAs) are small noncoding RNAs, about 21 nucleotides in length, that can regulate gene expression by base-pairing to partially complementary mRNAs. Regulation by miRNAs can play essential roles in embryonic development. We determined the temporal and spatial expression patterns of 115 conserved vertebrate miRNAs in zebrafish embryos by microarrays and by in situ hybridizations, using locked-nucleic acid-modified oligonucleotide probes. Most miRNAs were expressed in a highly tissue-specific manner during segmentation and later stages, but not early in development, which suggests that their role is not in tissue fate establishment but in differentiation or maintenance of tissue identity.

Animals↗

PubMed2004

Microarray-based, high-throughput gene expression profiling of microRNAs.

MicroRNAs (miRNAs) are small regulatory RNAs that serve fundamental biological roles across eukaryotic species. We describe a new method for high-throughput miRNA detection. The technique is termed the RNA-primed, array-based Klenow enzyme (RAKE) assay, because it involves on-slide application of the Klenow fragment of DNA polymerase I to extend unmodified miRNAs hybridized to immobilized DNA probes. We used RAKE to study human cell lines and brain tumors. We show that the RAKE assay is sensitive and specific for miRNAs and is ideally suited for rapid expression profiling of all known miRNAs. RAKE offers unique advantages for specificity over northern blots or other microarray-based expression profiling platforms. Furthermore, we demonstrate that miRNAs can be isolated and profiled from formalin-fixed paraffin-embedded tissue, which opens up new opportunities for analyses of small RNAs from archival human tissue. The RAKE assay is theoretically versatile and may be used for other applications, such as viral gene profiling.

Cell Line↗

PubMed2006

Mouse microRNA profiles determined with a new and sensitive cloning method.

MicroRNAs (miRNAs) are noncoding RNA molecules of 21 to 24 nt that regulate the expression of target genes in a post-transcriptional manner. Although evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation and pathogenesis of human diseases, extensive miRNA profiling in cells or tissues has been hampered by the lack of sensitive cloning methods. Here we describe a highly efficient profiling method, termed miRNA amplification profiling (mRAP), as well as its application both to mouse embryos at various developmental stages and to adult mouse organs. A total of 77,436 Small-RNA species was sequenced, with 11,776 of these sequences found to match previously described miRNAs. With the use of a newly developed computational prediction algorithm, we further identified 229 independent candidates for previously unknown miRNAs. The expression of some of these candidate miRNAs was confirmed by northern blot analysis and whole-mount in situ hybridization. Our data thus indicate that the total number of miRNAs in vertebrates is larger than previously appreciated and that the expression of these molecules is tightly controlled in a tissue- and developmental stage-specific manner.

Animals↗

PubMed2006

Highly specific gene silencing by artificial microRNAs in Arabidopsis.

Plant microRNAs (miRNAs) affect only a small number of targets with high sequence complementarity, while animal miRNAs usually have hundreds of targets with limited complementarity. We used artificial miRNAs (amiRNAs) to determine whether the narrow action spectrum of natural plant miRNAs reflects only intrinsic properties of the plant miRNA machinery or whether it is also due to past selection against natural miRNAs with broader specificity. amiRNAs were designed to target individual genes or groups of endogenous genes. Like natural miRNAs, they had varying numbers of target mismatches. Previously determined parameters of target selection for natural miRNAs could accurately predict direct targets of amiRNAs. The specificity of amiRNAs, as deduced from genome-wide expression profiling, was as high as that of natural plant miRNAs, supporting the notion that extensive base pairing with targets is required for plant miRNA function. amiRNAs make an effective tool for specific gene silencing in plants, especially when several related, but not identical, target genes need to be downregulated. We demonstrate that amiRNAs are also active when expressed under tissue-specific or inducible promoters, with limited nonautonomous effects. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (http://wmd.weigelworld.org).

Arabidopsis↗

PubMed2004

Identification of virus-encoded microRNAs.

RNA silencing processes are guided by small RNAs that are derived from double-stranded RNA. To probe for function of RNA silencing during infection of human cells by a DNA virus, we recorded the small RNA profile of cells infected by Epstein-Barr virus (EBV). We show that EBV expresses several microRNA (miRNA) genes. Given that miRNAs function in RNA silencing pathways either by targeting messenger RNAs for degradation or by repressing translation, we identified viral regulators of host and/or viral gene expression.

Animals↗

PubMed2006

MicroRNA protocols. Perspectives.

The discoveries of microRNAs (miRNAs), a class of noncoding RNAs that can regulate gene expression by translational repression, have opened a new avenue on gene modulation in mammalian cells. Conceivably, this highly effective method of modulation of gene expression would be exploited for numerous prospectives, including human, therapeutics. This chapter offers some perspective, with emphasis on areas that can be further developed.

Animals↗

PubMed2004

Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step.

RNA silencing suppressors from different plant viruses are structurally diverse. In addition to inhibiting the antiviral silencing response to condition susceptibility, many suppressors are pathogenicity factors that cause disease or developmental abnormalities. Here, unrelated suppressors from multiple viruses were shown to inhibit microRNA (miRNA) activities and trigger an overlapping series of severe developmental defects in transgenic Arabidopsis thaliana. This suggests that interference with miRNA-directed processes may be a general feature contributing to pathogenicity of many viruses. A normally labile intermediate in the miRNA biogenesis/RNA-induced silencing complex (RISC) assembly pathway, miRNA*, accumulated specifically in the presence of suppressors (P1/HC-Pro, p21, or p19) that inhibited miRNA-guided cleavage of target mRNAs. Both p21 and p19, but not P1/HC-Pro, interacted with miRNA/miRNA* complexes and hairpin RNA-derived short interfering RNAs (siRNAs) in vivo. In addition, p21 bound to synthetic miRNA/miRNA* and siRNA duplexes in vitro. We propose that several different suppressors act by distinct mechanisms to inhibit the incorporation of small RNAs into active RISCs.

Arabidopsis↗

PubMed2006

HIV-1 Tat interaction with Dicer: requirement for RNA.

Dicer is an RNase III which processes two classes of cellular small RNAs: the microRNAs (miRNA) and short interfering RNAs (siRNA). Previously, we observed that over-expressed HIV-1 Tat protein can suppress the processing of small RNAs inside cells. Here, we have investigated the requirements for Tat interaction with Dicer. We report that Tat-Dicer interaction depends on RNA, requires the helicase domain of Dicer, and is independent of Tat's transactivation domain.

Argonaute Proteins↗

PubMed2006

Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54.

RNA interference is triggered by double-stranded RNA that is processed into small interfering RNAs (siRNAs) by Dicer enzyme. Endogenously, RNA interference triggers are created from small noncoding RNAs called microRNAs (miRNAs). RNA-induced silencing complexes (RISC) in human cells can be programmed by exogenously introduced siRNA or endogenously expressed miRNA. siRNA-programmed RISC (siRISC) silences expression by cleaving a perfectly complementary target mRNA, whereas miRNA-induced silencing complexes (miRISC) inhibits translation by binding imperfectly matched sequences in the 3' UTR of target mRNA. Both RISCs contain Argonaute2 (Ago2), which catalyzes target mRNA cleavage by siRISC and localizes to cytoplasmic mRNA processing bodies (P-bodies). Here, we show that RCK/p54, a DEAD box helicase, interacts with argonaute proteins, Ago1 and Ago2, in affinity-purified active siRISC or miRISC from human cells; directly interacts with Ago1 and Ago2 in vivo, facilitates formation of P-bodies, and is a general repressor of translation. Disrupting P-bodies by depleting Lsm1 did not affect RCK/p54 interactions with argonaute proteins and its function in miRNA-mediated translation repression. Depletion of RCK/p54 disrupted P-bodies and dispersed Ago2 throughout the cytoplasm but did not significantly affect siRNA-mediated RNA functions of RISC. Depleting RCK/p54 released general, miRNA-induced, and let-7-mediated translational repression. Therefore, we propose that translation repression is mediated by miRISC via RCK/p54 and its specificity is dictated by the miRNA sequence binding multiple copies of miRISC to complementary 3' UTR sites in the target mRNA. These studies also suggest that translation suppression by miRISC does not require P-body structures, and location of miRISC to P-bodies is the consequence of translation repression.

3' Untranslated Regions↗

PubMed2004

Human MicroRNA targets.

MicroRNAs (miRNAs) interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. The specific function of most mammalian miRNAs is unknown. We have predicted target sites on the 3' untranslated regions of human gene transcripts for all currently known 218 mammalian miRNAs to facilitate focused experiments. We report about 2,000 human genes with miRNA target sites conserved in mammals and about 250 human genes conserved as targets between mammals and fish. The prediction algorithm optimizes sequence complementarity using position-specific rules and relies on strict requirements of interspecies conservation. Experimental support for the validity of the method comes from known targets and from strong enrichment of predicted targets in mRNAs associated with the fragile X mental retardation protein in mammals. This is consistent with the hypothesis that miRNAs act as sequence-specific adaptors in the interaction of ribonuclear particles with translationally regulated messages. Overrepresented groups of targets include mRNAs coding for transcription factors, components of the miRNA machinery, and other proteins involved in translational regulation, as well as components of the ubiquitin machinery, representing novel feedback loops in gene regulation. Detailed information about target genes, target processes, and open-source software for target prediction (miRanda) is available at http://www.microrna.org. Our analysis suggests that miRNA genes, which are about 1% of all human genes, regulate protein production for 10% or more of all human genes.

3' Untranslated Regions↗

PubMed2005

SERRATE coordinates shoot meristem function and leaf axial patterning in Arabidopsis.

Leaves of flowering plants are determinate organs produced by pluripotent structures termed shoot apical meristems. Once specified, leaves differentiate an adaxial (upper) side specialized for light capture, and an abaxial (lower) side specialized for gas exchange. A functional relationship between meristem activity and the differentiation of adaxial leaf fate has been recognized for over fifty years, but the molecular basis of this interaction is unclear. In Arabidopsis thaliana, activity of the class I KNOX (KNOTTED1-like homeobox) genes SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP) is required for meristem function but excluded from leaves, whereas members of the HD-Zip III (class III homeodomain leucine zipper) protein family function to promote both meristem activity and adaxial leaf fate. Here we show that the zinc-finger protein SERRATE acts in a microRNA (miRNA) gene-silencing pathway to regulate expression of the HD-Zip III gene PHABULOSA (PHB) while also limiting the competence of shoot tissue to respond to KNOX expression. Thus, SERRATE acts to coordinately regulate meristem activity and leaf axial patterning.

Alleles↗