Evidence for a cytoplasmic microprocessor of pri-miRNAs

Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029, USA.
RNA (Impact Factor: 4.94). 05/2012; 18(7):1338-46. DOI: 10.1261/rna.032268.112
Source: PubMed

ABSTRACT microRNAs (miRNAs) represent a class of noncoding RNAs that fine-tune gene expression through post-transcriptional silencing. While miRNA biogenesis occurs in a stepwise fashion, initiated by the nuclear microprocessor, rare noncanonical miRNAs have also been identified. Here we characterize the molecular components and unique attributes associated with the processing of virus-derived cytoplasmic primary miRNAs (c-pri-miRNAs). RNA in situ hybridization and inhibition of cellular division demonstrated a complete lack of nuclear involvement in c-pri-miRNA cleavage while genetic studies revealed that maturation still relied on the canonical nuclear RNase III enzyme, Drosha. The involvement of Drosha was mediated by a dramatic relocalization to the cytoplasm following virus infection. Deep sequencing analyses revealed that the cytoplasmic localization of Drosha does not impact the endogenous miRNA landscape during infection, despite allowing for robust synthesis of virus-derived miRNAs in the cytoplasm. Taken together, this research describes a unique function for Drosha in the processing of highly structured cytoplasmic RNAs in the context of virus infection.

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Available from: Benjamin R Tenoever, Nov 03, 2014
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    • "Alternatively, miRNA biosynthesis may be globally downregulated. Shapiro et al. demonstrated that upon infection with an RNA virus, Drosha is dramatically relocalized to the cytoplasm [84]. Others also reported global dampening of miRNA expression in productive viral infections, perhaps in an effort to enhance the immediate interferon response [41], [42]. "
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    PLoS ONE 08/2014; 9(8):e104770. DOI:10.1371/journal.pone.0104770 · 3.23 Impact Factor
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    • "It is commonly believed that negative-sense RNA paramyxoviruses do not produce viral miRNAs, as their replication does not occur in the nuclear compartment, where miRNA biogenesis is initialized.22,23 However, several other sncRNAs are known to be generated by alternative pathways, and some RNA viruses produce virus-derived sncRNAs.8,24,25 To confirm the sequencing data of hMPV-derived sncRNAs, we used northern blot to detect the two hMPV-derived sncRNAs. "
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    ABSTRACT: Small noncoding RNAs (sncRNAs), such as microRNAs (miRNA), virus-derived sncRNAs, and more recently identified tRNA-derived RNA fragments, are critical to posttranscriptional control of genes. Upon viral infection, host cells alter their sncRNA expression as a defense mechanism, while viruses can circumvent host defenses and promote their own propagation by affecting host cellular sncRNA expression or by expressing viral sncRNAs. Therefore, characterizing sncRNA profiles in response to viral infection is an important tool for understanding host-virus interaction, and for antiviral strategy development. Human metapneumovirus (hMPV), a recently identified pathogen, is a major cause of lower respiratory tract infections in infants and children. To investigate whether sncRNAs play a role in hMPV infection, we analyzed the changes in sncRNA profiles of airway epithelial cells in response to hMPV infection using ultrahigh-throughput sequencing. Of the cloned sncRNAs, miRNA was dominant in A549 cells, with the percentage of miRNA increasing in a time-dependent manner after the infection. In addition, several hMPV-derived sncRNAs and corresponding ribonucleases for their biogenesis were identified. hMPV M2-2 protein was revealed to be a key viral protein regulating miRNA expression. In summary, this study revealed several novel aspects of hMPV-mediated sncRNA expression, providing a new perspective on hMPV-host interactions.
    Molecular Therapy - Nucleic Acids 05/2014; 3(5):e163. DOI:10.1038/mtna.2014.18 · 4.51 Impact Factor
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    • "This would be a novel mechanism, by which cytoplasmic pri-miRNA transcripts function in a negative feedback-back loop to regulate miRNA function. Another possibility is that pri-miRNAs are processed in the cytoplasm, similar to Drosha-mediated processing of viral pri-miRNAs that was shown to take place in the cytoplasm [45]. In these cells relocalization of Drosha to the cytoplasm and the subsequent pri-miRNA processing was triggered by viral infection. "
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    ABSTRACT: Processing of miRNAs occurs simultaneous with the transcription and splicing of their primary transcripts. For the small subset of exonic miRNAs it is unclear if the unspliced and/or spliced transcripts are used for miRNA biogenesis. We assessed endogenous levels and cellular location of primary transcripts of three exonic miRNAs. The ratio between unspliced and spliced transcripts varied markedly, i.e. >1 for BIC, <1 for pri-miR-146a and variable for pri-miR-22. Endogenous unspliced transcripts were located almost exclusively in the nucleus and thus available for miRNA processing for all three miRNAs. Endogenous spliced pri-miRNA transcripts were present both in the nucleus and in the cytoplasm and thus only partly available for miRNA processing. Overexpression of constructs containing the 5' upstream exonic or intronic sequence flanking pre-miR-155 resulted in strongly enhanced miR-155 levels, indicating that the flanking sequence does not affect processing efficiency. Exogenously overexpressed full-length spliced BIC transcripts were present both in the nucleus and in the cytoplasm, were bound by the Microprocessor complex and resulted in enhanced miR-155 levels. We conclude that both unspliced and spliced transcripts of exonic miRNAs can be used for pre-miRNA cleavage. Splicing and cytoplasmic transport of spliced transcripts may present a mechanism to regulate levels of exonic microRNAs.
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