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Results for “MicroRNA, (miRNA)”

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PubMed2003

A biochemical framework for RNA silencing in plants.

RNA silencing phenomena were first discovered in plants, yet only the RNA interference pathway in animals has been subject to biochemical analysis. Here, we extend biochemical analysis to plant RNA silencing. We find that standard wheat germ extract contains Dicer-like enzymes that convert double-stranded RNA (dsRNA) into two classes of small interfering RNAs, as well as an RNA-dependent RNA polymerase activity that can convert exogenous single-stranded RNA into dsRNA. In this plant embryo extract, an endogenous microRNA (miRNA) that lacks perfect complementarity to its RNA targets nonetheless acts as a small interfering RNA. The miRNA guides an endonuclease to cleave efficiently wild-type Arabidopsis PHAVOLUTA mRNA, but not a dominant mutant previously shown to perturb leaf development. This finding supports the view that plant miRNAs direct RNAi and that miRNA-specified mRNA destruction is important for proper plant development. Thus, endonuclease complexes guided by small RNAs are a common feature of RNA silencing in both animals and plants.

Arabidopsis↗

PubMed2005

Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs.

Although hundreds of distinct animal microRNAs (miRNAs) are known, the specific biological functions of only a handful are understood at present. Here, we demonstrate that three different families of Drosophila miRNAs directly regulate two large families of Notch target genes, including basic helix-loop-helix (bHLH) repressor and Bearded family genes. These miRNAs regulate Notch target gene activity via GY-box (GUCUUCC), Brd-box (AGCUUUA), and K-box (cUGUGAUa) motifs. These are conserved sites in target 3'-untranslated regions (3'-UTRs) that are complementary to the 5'-ends of miRNAs, or "seed" regions. Collectively, these motifs represent >40 miRNA-binding sites in Notch target genes, and we show all three classes of motif to be necessary and sufficient for miRNA-mediated regulation in vivo. Importantly, many of the validated miRNA-binding sites have limited pairing to miRNAs outside of the "box:seed" region. Consistent with this, we find that seed-related miRNAs that are otherwise quite divergent can regulate the same target sequences. Finally, we demonstrate that ectopic expression of several Notch-regulating miRNAs induces mutant phenotypes that are characteristic of Notch pathway loss of function, including loss of wing margin, thickened wing veins, increased bristle density, and tufted bristles. Collectively, these data establish insights into miRNA target recognition and demonstrate that the Notch signaling pathway is a major target of miRNA-mediated regulation in Drosophila.

3' Untranslated Regions↗

PubMed2005

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.

Argonaute Proteins↗

PubMed2006

Methods for analyzing microRNA expression and function during hematopoietic lineage differentiation.

MicroRNAs (miRNAs), an abundant class of approx 22-nucleotide (nt) small RNAs that control gene expression at the posttranscriptional level, may play important roles during normal hematopoiesis and leukemogenesis. This chapter focuses on the methods and strategies for dissecting miRNA function during hematopoietic lineage differentiation. We describe a modified miRNA cloning method and expression analysis approach for determining miRNA expression during hematopoietic lineage differentiation. We illustrate a retroviral vector and a general strategy for the ectopic expression of miRNAs in hemato-poietic stem/progenitor cells. We discuss in vitro and in vivo functional assays that can be used to examine the roles of miRNAs during hematopoietic lineage differentiation. The methods and principles described here should also be applicable to study the roles of miRNAs in the differentiation and function of nonhematopoietic cell types.

Animals↗

PubMed2006

The phylogenetic distribution of metazoan microRNAs: insights into evolutionary complexity and constraint.

How complex body plans evolved in animals such as fruit flies and vertebrates, as compared to the relatively simple jellyfish and sponges, is not known, given the similarity of developmental genetic repertoires shared by all these taxa. Here, we show that a core set of 18 microRNAs (miRNAs), non-coding RNA molecules that negatively regulate the expression of protein-coding genes, are found only in protostomes and deuterostomes and not in sponges or cnidarians. Because many of these miRNAs are expressed in specific tissues and/or organs, miRNA-mediated regulation could have played a fundamental evolutionary role in the origins of organs such as brain and heart--structures not found in cnidarians or sponges--and thus contributed greatly to the evolution of complex body plans. Furthermore, the continuous acquisition and fixation of miRNAs in various animal groups strongly correlates both with the hierarchy of metazoan relationships and with the non-random origination of metazoan morphological innovations through geologic time.

Animals↗

PubMed2004

Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs.

Here we describe a set of endogenous short interfering RNAs (siRNAs) in Arabidopsis, some of which direct the cleavage of endogenous mRNAs. These siRNAs correspond to both sense and antisense strands of a noncoding RNA (At2g27400) that apparently is converted to double-stranded RNA and then processed in 21 nt increments. These siRNAs differ from previously described regulatory small RNAs in two respects. First, they require components of the cosuppression pathway (RDR6 and SGS3) and also components of the microRNA (miRNA) pathway (AGO1, DCL1, HEN1, and HYL1) but not components needed for heterochromatic siRNAs (DCL3 and RDR2), another class of endogenous plant siRNAs. Second, these siRNAs repress the expression of genes that have little overall resemblance to the genes from which they originate, a characteristic previously reported only for miRNAs. The identification of this silencing pathway provides yet another dimension to posttranscriptional mRNA regulation in plants.

Arabidopsis↗

PubMed2006

Conserved microRNA characteristics in mammals.

Short hairpin RNAs (shRNAs) and short interfering RNAs (siRNAs) probably enter different stages of the microRNA (miRNA) pathway for depletion of mRNA and suppression of protein translation. Primary and secondary structural characteristics that are shared between endogenous primary miRNA transcripts (pri-miRNAs) may contribute toward efficient biogenesis and potent silencing. This study investigates known miRNA transcripts for characteristics that are conserved between miRNAs and that distinguish them from random hairpins with similar lengths. The primary structure is conserved, as demonstrated by a significant presence or absence of certain bases at specific positions in the miRNA precursors and their flanking regions. The secondary structure is also conserved between miRNAs, as internal loops and bulges commonly appear in specific positions in the miRNA stem. The conservation of base-pairing continues past the mature duplex and 13 bases into the primary stem, with no detectable conservation of secondary structure beyond this region. Based on these observations, we have designed a hairpin construct that incorporates the most important characteristics present in endogenous miRNAs. Preliminary experiments suggest that this construct may rescue the efficacy of shRNA triggers that cannot be used with a miR-30-based hairpin, and vice versa.

Animals↗

PubMed2005

MicroRNA identification based on sequence and structure alignment.

MOTIVATION: MicroRNAs (miRNA) are approximately 22 nt long non-coding RNAs that are derived from larger hairpin RNA precursors and play important regulatory roles in both animals and plants. The short length of the miRNA sequences and relatively low conservation of pre-miRNA sequences restrict the conventional sequence-alignment-based methods to finding only relatively close homologs. On the other hand, it has been reported that miRNA genes are more conserved in the secondary structure rather than in primary sequences. Therefore, secondary structural features should be more fully exploited in the homologue search for new miRNA genes. RESULTS: In this paper, we present a novel genome-wide computational approach to detect miRNAs in animals based on both sequence and structure alignment. Experiments show this approach has higher sensitivity and comparable specificity than other reported homologue searching methods. We applied this method on Anopheles gambiae and detected 59 new miRNA genes. AVAILABILITY: This program is available at http://bioinfo.au.tsinghua.edu.cn/miralign. SUPPLEMENTARY INFORMATION: Supplementary information is available at http://bioinfo.au.tsinghua.edu.cn/miralign/supplementary.htm.

Algorithms↗

PubMed2026

The investigation of human cerebrospinal fluid exosome in spinal cord injury.

Spinal cord injury (SCI) leads to severe neurological and functional impairments, yet reliable biomarkers for assessing injury severity and predicting recovery remain limited. Cerebrospinal fluid (CSF) is in direct contact with the central nervous system and provides a valuable source for detecting molecular changes after SCI. Although exosomal microRNAs (miRNAs) and proteins are increasingly recognized as mediators of intercellular communication, the role of human CSF exosomes in SCI has not been systematically investigated. To identify exosome-based biomarkers and potential therapeutic targets, we analyzed CSF and serum exosomes from patients with acute SCI using RNA sequencing and proteomic profiling. Weighted gene co-expression network analysis identified 6 gene modules significantly associated with injury severity and neurological recovery at 3 months. Proteomic analysis revealed a 5-protein panel that distinguished complete from incomplete SCI and a 4-protein panel that predicted neurological improvement. Additionally, 15 CSF-specific and 9 serum-specific exosomal miRNAs were identified independent of injury severity. Among 10 tested miRNAs associated with neurological recovery, 7 regulated astrocyte proliferation, and 6 promoted neurite extension and synapse formation. Overall, this study provides a comprehensive characterization of CSF exosomal miRNAs and proteins in human SCI and identifies molecular signatures associated with injury severity and recovery.

Humans↗

PubMed2005

Reciprocal expression of lin-41 and the microRNAs let-7 and mir-125 during mouse embryogenesis.

In C. elegans, heterochronic genes control the timing of cell fate determination during development. Two heterochronic genes, let-7 and lin-4, encode microRNAs (miRNAs) that down-regulate a third heterochronic gene lin-41 by binding to complementary sites in its 3'UTR. let-7 and lin-4 are conserved in mammals. Here we report the cloning and sequencing of mammalian lin-41 orthologs. We find that mouse and human lin-41 genes contain predicted conserved complementary sites for let-7 and the lin-4 ortholog, mir-125, in their 3'UTRs. Mouse lin-41 (Mlin-41) is temporally expressed in developing mouse embryos, most dramatically in the limb buds. Mlin-41 is down-regulated during mid-embryogenesis at the time when mouse let-7c and mir-125 RNA levels are up-regulated. Our results suggest that mammalian lin-41 is temporally regulated by miRNAs in order to direct key developmental events such as limb formation.

Amino Acid Sequence↗

PubMed2007

Emerging role for microRNAs in acute promyelocytic leukemia.

Hematopoiesis is highly controlled by lineage-specific transcription factors that, by interacting with specific DNA sequences, directly activate or repress specific gene expression. These transcription factors have been found mutated or altered by chromosomal translocations associated with leukemias, indicating their role in the pathogenesis of these malignancies. The post-genomic era, however, has shown that transcription factors are not the only key regulators of gene expression. Epigenetic mechanisms such as DNA methylation, posttranslational modifications of histones, remodeling of nucleosomes, and expression of small regulatory RNAs all contribute to the regulation of gene expression and determination of cell and tissue specificity. Deregulation ofthese epigenetic mechanisms cooperates with genetic alterations to the establishment and progression of tumors. MicroRNAs (miRNAs) are negative regulators of the expression of genes involved in development, differentiation, proliferation, and apoptosis. Their expression appears to be tissue-specific and highly regulated according to the cell's developmental lineage and stage. Interestingly, miRNAs expressed in hematopoietic cells have been found mutated or altered by chromosomal translocations associated with leukemias. The expression levels of a specific miR-223 correlate with the differentiation fate of myeloid precursors. The activation of both pathways of transcriptional regulation by the myeloid lineage-specific transcription factor C/EBPalpha (CCAAT/enhancer-binding protein-alpha), and posttranscriptional regulation by miR-223 appears essential for granulocytic differentiation and clinical response of acute promyelocytic leukemia (APL) blasts to all-trans retinoic acid (ATRA). Together, this evidence underlies transcription factors, chromatin remodeling, and miRNAs as ultimate determinants for the correct organization of cell type-specific gene arrays and hematopoietic differentiation, therefore providing new targets for the diagnosis and treatment of leukemias.

CCAAT-Enhancer-Binding Protein-alpha↗

PubMed2005

The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development.

MicroRNAs (miRNAs) are an abundant class of gene regulatory molecules (reviewed in refs 1, 2). Although computational work indicates that miRNAs repress more than a third of human genes, their roles in vertebrate development are only now beginning to be determined. Here we show that miR-196 acts upstream of Hoxb8 and Sonic hedgehog (Shh) in vivo in the context of limb development, thereby identifying a previously observed but uncharacterized inhibitory activity that operates specifically in the hindlimb. Our data indicate that miR-196 functions in a fail-safe mechanism to assure the fidelity of expression domains that are primarily regulated at the transcriptional level, supporting the idea that many vertebrate miRNAs may function as a secondary level of gene regulation.

Animals↗

PubMed2025

Genome-wide analysis of host-encoded microRNAs modulating SARS-CoV-2 infection.

Viruses exploit cellular machinery to complete their replication cycle. Furthering our understanding of this process provides insight into the mechanism of virus replication and potential targets for antiviral therapeutics. Genome-wide CRISPR screens have identified cellular pathways important in the SARS-COV-2 infection process, including vesicular traffic, lipid homeostasis and PI3K signalling. Functional genomics-driven analysis of host-encoded microRNAs (miRNAs) impacting SARS-CoV-2 infection would provide further unbiased and discovery-driven insight into the host-pathogen interface. Here we present findings from genome-wide complementary miRNA mimic and inhibitor screens performed in a bio-safety level (BSL)-4 laboratory using a combination of high-throughput robotics, high-content imaging and novel data analysis pipelines. This dataset has identified both miRNA promoters and inhibitors of SARS-CoV-2 replication which may be used by researchers to further explore therapeutic targets against SARS-CoV-2 and the host factors influencing COVID pathogenesis.

MicroRNAs↗

PubMed2003

The microRNAs of Caenorhabditis elegans.

MicroRNAs (miRNAs) are an abundant class of tiny RNAs thought to regulate the expression of protein-coding genes in plants and animals. In the present study, we describe a computational procedure to identify miRNA genes conserved in more than one genome. Applying this program, known as MiRscan, together with molecular identification and validation methods, we have identified most of the miRNA genes in the nematode Caenorhabditis elegans. The total number of validated miRNA genes stands at 88, with no more than 35 genes remaining to be detected or validated. These 88 miRNA genes represent 48 gene families; 46 of these families (comprising 86 of the 88 genes) are conserved in Caenorhabditis briggsae, and 22 families are conserved in humans. More than a third of the worm miRNAs, including newly identified members of the lin-4 and let-7 gene families, are differentially expressed during larval development, suggesting a role for these miRNAs in mediating larval developmental transitions. Most are present at very high steady-state levels-more than 1000 molecules per cell, with some exceeding 50,000 molecules per cell. Our census of the worm miRNAs and their expression patterns helps define this class of noncoding RNAs, lays the groundwork for functional studies, and provides the tools for more comprehensive analyses of miRNA genes in other species.

Animals↗

PubMed2006

Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues.

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes. In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent non-neoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor. Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.

Cluster Analysis↗

PubMed2026

Identification of Freezing-Responsive microRNAs and Their Targets in Chinese Jujube by Small RNA and Degradome Sequencing.

The jujube tree fruit remains a primary fruit in northern China, yet its geographical distribution and yield are significantly constrained by freezing stress during winter. Numerous studies have highlighted the pivotal regulatory function of microRNAs (miRNAs) in plant responses to low-temperature stress. Nevertheless, the specific miRNAs involved in the response to low temperatures and their associated gene networks in Ziziphus jujuba Mill are not well understood. In this investigation, we utilized high-throughput sequencing to analyze small RNA libraries from branches subjected to temperatures of 4 °C and -30 °C. Our analysis identified a total of 342 miRNAs, comprising 123 known miRNAs and 219 novel miRNAs. The differential expression analysis revealed that under low-temperature conditions, 177 miRNAs underwent significant changes. Among them, specific upregulation of miR319 in the less cold-resistant variety and miR6483 in sensitive variety was observed. By employing degradome sequencing, we identified a total of 1551 target genes corresponding to 3059 unique miRNA target interaction pairs involving 299 miRNAs. Functional analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways indicated that these target genes are primarily associated with transcriptional regulation, metabolic pathways, and genetic information processing. Through a comprehensive analysis, we pinpointed 11 genes corresponding to 9 miRNAs that are implicated in jujube tree cold stress, and 7 target genes of 7 miRNAs were confirmed by 5'-RACE analysis. These miRNAs are likely to exert crucial regulatory functions in the context of jujube tree cold stress. This study is the first to systematically identify miRNAs and their target genes in the response of Ziziphus jujuba Mill to low-temperature stress, which provides important resources for in-depth analysis of the molecular mechanism of jujube tree cold resistance and for cold-resistant breeding.

Ziziphus↗

PubMed2005

MicroRNA expression profiles classify human cancers.

Recent work has revealed the existence of a class of small non-coding RNA species, known as microRNAs (miRNAs), which have critical functions across various biological processes. Here we use a new, bead-based flow cytometric miRNA expression profiling method to present a systematic expression analysis of 217 mammalian miRNAs from 334 samples, including multiple human cancers. The miRNA profiles are surprisingly informative, reflecting the developmental lineage and differentiation state of the tumours. We observe a general downregulation of miRNAs in tumours compared with normal tissues. Furthermore, we were able to successfully classify poorly differentiated tumours using miRNA expression profiles, whereas messenger RNA profiles were highly inaccurate when applied to the same samples. These findings highlight the potential of miRNA profiling in cancer diagnosis.

Animals↗

PubMed2004

The Drosha-DGCR8 complex in primary microRNA processing.

RNase III proteins play key roles in microRNA (miRNA) biogenesis. The nuclear RNase III Drosha cleaves primary miRNAs (pri-miRNAs) to release hairpin-shaped pre-miRNAs that are subsequently cut by the cytoplasmic RNase III Dicer to generate mature miRNAs. While Dicer (class III) and other simple RNase III proteins (class I) have been studied intensively, the class II enzyme Drosha remains to be characterized. Here we dissected the action mechanism of human Drosha by generating mutants and by characterizing its new interacting partner, DGCR8. The basic action mechanism of Drosha was found to be similar to that of human Dicer; the RNase III domains A and B form an intramolecular dimer and cleave the 3' and 5' strands of the stem, respectively. Human Drosha fractionates at approximately 650 kDa, indicating that Drosha functions as a large complex. In this complex, Drosha interacts with DGCR8, which contains two double-stranded RNA (dsRNA)-binding domains. By RNAi and biochemical reconstitution, we show that DGCR8 may be an essential component of the pri-miRNA processing complex, along with Drosha. Based on these results, we propose a model for the action mechanism of class II RNase III proteins.

Blotting, Northern↗