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Short hairpin RNAs (shRNAs) are effective in generating stable repression of gene expression. RNA polymerase III (RNAP III) type III promoters (U6 or H1) are typically used to drive shRNA expression. While useful for some knockdown applications, the robust expression of U6/H1-driven shRNAs can induce toxicity and generate heterogeneous small RNAs with undesirable off-target effects. Additionally, typical U6/H1 promoters encompass the majority of the ∼270 base pairs (bp) of vector space required for shRNA expression. This can limit the efficacy and/or number of delivery vector options, particularly when delivery of multiple gene/shRNA combinations is required. Here, we develop a compact shRNA (cshRNA) expression system based on retroviral microRNA (miRNA) gene architecture that uses RNAP III type II promoters. We demonstrate that cshRNAs coded from as little as 100 bps of total coding space can precisely generate small interfering RNAs (siRNAs) that are active in the RNA-induced silencing complex (RISC). We provide an algorithm with a user-friendly interface to design cshRNAs for desired target genes. This cshRNA expression system reduces the coding space required for shRNA expression by >2-fold as compared to the typical U6/H1 promoters, which may facilitate therapeutic RNAi applications where delivery vector space is limiting., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)