Functional Selection of shRNA Loops from Randomized Retroviral Libraries

Stig Mølgaard Rask Jensen,Alexander Schmitz,Jørgen Kjems,Jesper Bertram Bramsen,Finn Skou Pedersen,John J Rossi

doi:10.1371/journal.pone.0043095

Stig Mølgaard Rask Jensen, Alexander Schmitz + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0043095

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Journal: PloS one	Publication Date: Aug 17, 2012
Citations: 41	License type: CC BY 4.0

Affiliation: Aarhus University

Abstract

Gene silencing by RNA interference (RNAi) can be achieved by the ectopic expression of tailored short hairpin RNAs (shRNAs) which after export to the cytoplasm are processed by Dicer and incorporated into the RNA induced silencing complex (RISC). Design rules for shRNAs have been the focus of several studies, but only a few reports have turned the attention to the sequence of the loop-region. In this work we selected high-functional and low-functional shRNA loops from retroviral hairpin-loop-libraries in an RNAi reporter assay. The procedure revealed a very significant and stem sequence-dependent effect of the loop on shRNA function and although neither strong consensus loop sequence nor structural motifs could be identified, a preferred loop sequence (5′-UGUGCUU-3′) was found to support robust knock down with little stem sequence dependency. These findings will serve as a guide for designing shRNAs with improved knock down capacity.

Highlights

The phenomenon of RNA interference (RNAi) in mammals is usually initiated through either the production of microRNAs, which control endogenous mRNA stability or translation levels [1,2,3] or by the production of small interfering RNAs from double stranded RNA of either exogenous or endogenous origin [4,5]. miRNAs are transcribed as primary miRNA transcripts, which are co-transcriptionally cleaved into precursor miRNAs by the microprocessor complex comprised of DGCR8 and the RNaseIII enzyme, Drosha
ShRNA loops are more likely to influence the short hairpin RNAs (shRNAs) processing by the RNAi machinery in the cytoplasm and RNA hairpins are described as loop-sequence-specific substrates of double stranded RNA binding domains [28]
A most likely candidate for differential shRNA recognition is the DicerdsRBD as the double stranded RNA binding domains (dsRBDs) in other cytoplasmic proteins TRBP and PACT seem rather implicated in protein-protein interactions [29,30,31]

Summary

Introduction

The phenomenon of RNAi in mammals is usually initiated through either the production of microRNAs (miRNAs), which control endogenous mRNA stability or translation levels [1,2,3] or by the production of small interfering RNAs (siRNAs) from double stranded RNA (dsRNA) of either exogenous or endogenous origin [4,5]. miRNAs are transcribed as primary miRNA transcripts (primiRNA), which are co-transcriptionally cleaved into precursor miRNAs (pre-miRNAs) by the microprocessor complex comprised of DGCR8 and the RNaseIII enzyme, Drosha. The pre-miRNAs are small irregular hairpin structures that are exported by the Exportin5/RanGTP complex to the cytoplasm where they are recognised and processed by the RNaseIII enzyme Dicer in conjunction with TRBP, PACT, and Ago, reviewed by Rana et al [6]. The siRNAs are naturally either generated from long exogenous dsRNAs such as viral RNAs or from endogenous convergent transcripts from overlapping genes [5]. Dicer cleaves these dsRNAs into short ,21 bp siRNAs which enter RISC and induce degradation of their innate origin RNA (reviewed in [6])

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