Abstract
We have previously reported an antisense technology, ‘snoMEN vectors’, for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.
Highlights
SnoMEN vectors provide a form of antisense technology for modulating the expression of target genes based upon complementary base pairing interactions, analogous to the more familiar siRNA/shRNA vector systems[1]
This snoMEN vector encoded three snoMEN, which each targeted different regions of the pri-miR21 sequence. These three snoMEN RNAs are each encoded within separate introns of the same RNA pol II transcript, which encodes the mCherry protein that acts as a fluorescent protein (FP) marker in transfected cells
In this study we report the successful knock-down of Micro RNAs (miRNAs) in human cells using snoMEN vectors targeted to miRNA primary transcripts
Summary
SnoMEN (snoRNA Modulator of gene ExpressioN) vectors provide a form of antisense technology for modulating the expression of target genes based upon complementary base pairing interactions, analogous to the more familiar siRNA/shRNA vector systems[1]. The snoMEN vector technology is created by manipulation of the human box C/D small nucleolar RNA (snoRNA) HBII-180C. This class of snoRNAs contain an internal sequence (M box) that can be altered to make it complementary to RNA targets. Box C/D snoRNAs are named after a common RNA motif in this subfamily that serves as a binding site for a group of box C/D proteins, including NOP56, NOP58, 15.5K and the highly conserved protein fibrillarin, which has the specific 2’-O-methylase activity. Most snoRNAs are encoded within intron sequences, either located in the primary transcripts of protein coding genes, or in dedicated transcripts containing tandem arrays of multiple snoRNAs. Endogenous snoRNAs are highly abundant nuclear RNAs that are efficiently processed from primary transcripts. Processing and delivery of snoMEN RNAs is efficient and not prone to saturation of the host cell processing machinery when snoMEN are expressed from exogenous vectors
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