title: Identification of tri-phosphatase activity in the biogenesis of retroviral microRNAs and RNAP III-generated shRNAs.
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.
authors.affiliation: 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.
authors.forename: James M
authors.initials: JM
authors.lastname: Burke
authors.affiliation: 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.
authors.forename: Clovis R
authors.initials: CR
authors.lastname: Bass
authors.affiliation: 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.
authors.forename: Rodney P
authors.initials: RP
authors.lastname: Kincaid
authors.affiliation: 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.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Sullivan
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D001909:Leukemia Virus, Bovine / Q000235:genetics*; D035683:MicroRNAs / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000032:analysis; D012320:RNA Polymerase III / Q000378:metabolism*; D012322:RNA Precursors / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000378:metabolism; D058727:RNA, Small Untranslated / Q000737:chemistry / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D010710:Phosphates; D012322:RNA Precursors; D034741:RNA, Small Interfering; D058727:RNA, Small Untranslated; D012320:RNA Polymerase III; D043244:Ribonuclease III; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase