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Nucleic Acids Res. 2014 Dec 16;42(22):13949-62. doi: 10.1093/nar/gku1247. Epub 2014 Nov 26.

Identification of tri-phosphatase activity in the biogenesis of retroviral microRNAs and RNAP III-generated shRNAs.

Author information

1
The University of Texas at Austin, Institute for Cellular and Molecular Biology, Center for Synthetic and Systems Biology, Center for Infectious Disease and Department of Molecular Biosciences, 1 University Station A5000, Austin TX 78712-0162, USA.
2
The University of Texas at Austin, Institute for Cellular and Molecular Biology, Center for Synthetic and Systems Biology, Center for Infectious Disease and Department of Molecular Biosciences, 1 University Station A5000, Austin TX 78712-0162, USA chris_sullivan@austin.utexas.edu.

Abstract

Transcripts possessing a 5'-triphosphate are a hallmark of viral transcription and can trigger the host antiviral response. 5'-triphosphates are also found on common host transcripts transcribed by RNA polymerase III (RNAP III), yet how these transcripts remain non-immunostimulatory is incompletely understood. Most microRNAs (miRNAs) are 5'-monophosphorylated as a result of sequential endonucleolytic processing by Drosha and Dicer from longer RNA polymerase II (RNAP II)-transcribed primary transcripts. In contrast, bovine leukemia virus (BLV) expresses subgenomic RNAP III transcripts that give rise to miRNAs independent of Drosha processing. Here, we demonstrate that each BLV pre-miRNA is directly transcribed by RNAP III from individual, compact RNAP III type II genes. Thus, similar to manmade RNAP III-generated short hairpin RNAs (shRNAs), the BLV pre-miRNAs are initially 5'-triphosphorylated. Nonetheless, the derivative 5p miRNAs and shRNA-generated 5p small RNAs (sRNAs) possess a 5'-monophosphate. Our enzymatic characterization and small RNA sequencing data demonstrate that BLV 5p miRNAs are co-terminal with 5'-triphosphorylated miRNA precursors (pre-miRNAs). Thus, these results identify a 5'-tri-phosphatase activity that is involved in the biogenesis of BLV miRNAs and shRNA-generated sRNAs. This work advances our understanding of retroviral miRNA and shRNA biogenesis and may have implications regarding the immunostimulatory capacity of RNAP III transcripts.

PMID:
25428356
PMCID:
PMC4267658
DOI:
10.1093/nar/gku1247
[Indexed for MEDLINE]
Free PMC Article

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