MicroRNAs with a nucleolar location

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
RNA (Impact Factor: 4.94). 08/2009; 15(9):1705-15. DOI: 10.1261/rna.1470409
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There is increasing evidence that noncoding RNAs play a functional role in the nucleus. We previously reported that the microRNA (miRNA), miR-206, is concentrated in the nucleolus of rat myoblasts, as well as in the cytoplasm as expected. Here we have extended this finding. We show by cell/nuclear fractionation followed by microarray analysis that a number of miRNAs can be detected within the nucleolus of rat myoblasts, some of which are significantly concentrated there. Pronounced nucleolar localization is a specific phenomenon since other miRNAs are present at only very low levels in the nucleolus and occur at much higher levels in the nucleoplasm and/or the cytoplasm. We have further characterized a subset of these miRNAs using RT-qPCR and in situ hybridization, and the results suggest that some miRNAs are present in the nucleolus in precursor form while others are present as mature species. Furthermore, we have found that these miRNAs are clustered in specific sites within the nucleolus that correspond to the classical granular component. One of these miRNAs is completely homologous to a portion of a snoRNA, suggesting that it may be processed from it. In contrast, the other nucleolar-concentrated miRNAs do not show homology with any annotated rat snoRNAs and thus appear to be present in the nucleolus for other reasons, such as modification/processing, or to play roles in the late stages of ribosome biosynthesis or in nonribosomal functions that have recently been ascribed to the granular component of the nucleolus.

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Available from: Joan Ritland, Oct 05, 2015
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    • "Other examples of miRNAs found in the nucleus are miR-709, miR-690, miR-30e (Tang et al., 2012), and miR-122 (Földes-Papp et al., 2009). miRNAs can also be found in the nucleolus as precursor forms, like miR-494 and miR-664, and as mature miRNAs, like miR-21, miR-1, miR-351, miR-206 (Politz et al., 2006, 2009), and miR-320 (Marcon et al., 2008). Another intriguing subcellular localization of miRNAs is mitochondria, where they may modulate apoptosis processes in a coordinated way (Kren et al., 2009). "
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    ABSTRACT: Microribonucleic acids, best known as microRNAs or miRNAs, are small, non-coding RNAs with important regulatory roles in eukaryotic cells. Here, I present a broad review on highly relevant but generally non-depicted features of miRNAs, among which stand out the non-conventional miRNA seed sites, the unusual messenger RNA (mRNA) target regions, the non-canonical miRNA-guided mechanisms of gene expression regulation, and the recently identified new class of miRNA ligands. Furthermore, I address the miRNA uncommon genomic location, transcription, and subcellular localization. Altogether, these unusual features and roles place the miRNA system as a very diverse, complex, and intriguing biological mechanism.
    Frontiers in Genetics 09/2014; 5:337. DOI:10.3389/fgene.2014.00337
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    • "The RNase P RNA assists in the 5′ processing of tRNA in the nucleolus [14]. At least one microRNA (miRNA) has been reported in the nucleolus of rat myoblasts [15], [16], and several nucleolar miRNAs were demonstrated in HeLa cells in a recent study [17]. Besides rRNA and ribosome biogenesis, a number of other functions that involve RNP assemblies have been associated with the nucleolus. "
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    ABSTRACT: Small non-coding RNAs represent RNA species that are not translated to proteins, but which have diverse and broad functional activities in physiological and pathophysiological states. The knowledge of these small RNAs is rapidly expanding in part through the use of massive parallel (deep) sequencing efforts. We present here the first deep sequencing of small RNomes in subcellular compartments with particular emphasis on small RNAs (sRNA) associated with the nucleolus. The vast majority of the cellular, cytoplasmic and nuclear sRNAs were identified as miRNAs. In contrast, the nucleolar sRNAs had a unique size distribution consisting of 19-20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA). Sequences from 47 sdRNAs were identified, which mapped to both 5' and 3' ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads mapping to SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as comprising both 20 and 25 nt RNAs. A novel 120 nt SNORD44 form was also identified. The expression of the SNORD44 sdRNA and 120 nt form was independent of Dicer/Drosha-mediated processing pathways but was dependent on the box C/D snoRNP proteins/sno-ribonucleoproteins fibrillarin and NOP58. The 120 nt SNORD44-derived RNA bound to fibrillarin suggesting that C/D sno-ribonucleoproteins are involved in regulating the stability or processing of SNORD44. This study reveals sRNA cell-compartment specific expression and the distinctive unique composition of the nucleolar sRNAs.
    PLoS ONE 09/2014; 9(9):e107519. DOI:10.1371/journal.pone.0107519 · 3.23 Impact Factor
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    • "RNA was extracted from either the nucleus, cytoplasm or whole cells using Trizol (Invitrogen) according to the manufacturer’s instructions. Purity of the nuclear and cytoplasmic RNA was confirmed by comparing the abundance of snoRNA or Y1 cytoplasmic RNA using quantitative PCR (qPCR) as previously described [26,29]. "
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    ABSTRACT: Background MicroRNAs (miRNAs) are coordinators of cellular differentiation, including granulopoiesis. Although differential expression of many miRNAs is associated with the maturation of granulocytes, analysis of differentially expressed miRNAs and their cellular localization across all stages of granulopoiesis, starting from hemopoietic stems cells, is not well characterized. Methods We analyzed whole cell miRNA and mRNA expression during granulopoiesis using Taqman low-density and Affymetrix arrays respectively. We also performed nuclear and cytoplasmic fractionation followed by Taqman low-density array and/or quantitative PCR to identify nuclear-enriched miRNAs in hemopoietic stem/progenitor cells, promyelocytes, myelocytes, granulocytes and several hemopoietic cell lines. Anti-correlation between the expression of miRNA and target pairs was used to determine putative miRNA targets. Results Analyses of our array data revealed distinct clusters of differentially expressed miRNAs that are specific to promyelocytes and granulocytes. While the roles of many of these miRNAs in granulopoiesis are not currently known, anti-correlation of the expression of miRNA/mRNA target pairs identified a suite of novel target genes. Clusters of miRNAs (including members of the let-7 and miR-17-92 families) are downregulated in hemopoietic stem/progenitor cells, potentially allowing the expression of target genes known to facilitate stem cell proliferation and homeostasis. Additionally, four miRNAs (miR-709, miR-706, miR-690 and miR-467a*) were found to be enriched in the nucleus of myeloid cells and multiple hemopoietic cell lines compared to other miRNAs, which are predominantly cytoplasmic-enriched. Both miR-709 and miR-706 are nuclear-enriched throughout granulopoiesis and have putative binding sites of extensive complementarity downstream of pri-miRNAs. Nuclear enrichment of miR-467a* is specific to hemopoietic stem/progenitors and promyelocytes. These miRNAs are also nuclear-enriched in other hemopoietic cell lines, where nuclear sequestering may fine-tune the expression of cytoplasmic mRNA targets. Conclusions Overall, we have demonstrated differentially expressed miRNAs that have not previously been associated with hemopoietic differentiation and provided further evidence of regulated nuclear-enrichment of miRNAs. Further studies into miRNA function in granulocyte development may shed light on fundamental aspects of regulatory RNA biology and the role of nuclear miRNAs.
    Journal of Hematology & Oncology 05/2014; 7(1):42. DOI:10.1186/1756-8722-7-42 · 4.81 Impact Factor
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