A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA.
Processing of primary microRNA (pri-miRNA) stem-loops by the Drosha-DGCR8 complex is the initial step in miRNA maturation and crucial for miRNA function. Nonetheless, the underlying mechanism that determines the Drosha cleavage site of pri-miRNAs has remained unclear. Two prevalent but seemingly conflicting models propose that Drosha-DGCR8 anchors to and directs cleavage a fixed distance from either the basal single-stranded (ssRNA) or the terminal loop. However, recent studies suggest that the basal ssRNA and/or the terminal loop may influence the Drosha cleavage site dependent upon the sequence/structure of individual pri-miRNAs. Here, using a panel of closely related pri-miRNA variants, we further examine the role of pri-miRNA structures on Drosha cleavage site selection in cells. Our data reveal that both the basal ssRNA and terminal loop influence the Drosha cleavage site within three pri-miRNAs, the Simian Virus 40 (SV40) pri-miRNA, pri-miR-30a, and pri-miR-16. In addition to the flanking ssRNA regions, we show that an internal loop within the SV40 pri-miRNA stem strongly influences Drosha cleavage position and efficiency. We further demonstrate that the positions of the internal loop, basal ssRNA, and the terminal loop of the SV40 pri-miRNA cooperatively coordinate Drosha cleavage position and efficiency. Based on these observations, we propose that the pri-miRNA stem, defined by internal and flanking structural elements, guides the binding position of Drosha-DGCR8, which consequently determines the cleavage site. This study provides mechanistic insight into pri-miRNA processing in cells that has numerous biological implications and will assist in refining Drosha-dependent shRNA design.
Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712-0162, USA.
James M
JM
Burke
Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712-0162, USA.
Demetra P
DP
Kelenis
Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712-0162, USA.
Rodney P
RP
Kincaid
Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712-0162, USA.
Christopher S
CS
Sullivan
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011506:Proteins / Q000378:metabolism; D059367:RNA Cleavage / Q000235:genetics; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*; D013539:Simian virus 40 / Q000235:genetics
C474043:DGCR8 protein, human; D035683:MicroRNAs; D011506:Proteins; D012322:RNA Precursors; D012367:RNA, Viral; D016601:RNA-Binding Proteins; C517918:DROSHA protein, human; D043244:Ribonuclease III
DGCR8; Drosha; RNAi; miRNA; pri-miRNA; shRNA