Since the ﬁrst application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNAs (shRNAs) for targeted gene silencing has become a benchmark technology. shRNAs spontaneously form hairpin structures that are recognized by the cellular RNAi machinery and are processed to form active siRNAs. Like siRNAs, shRNAs may be transfected as plasmid vectors encoding shRNAs transcribed by RNA pol III or modified pol II promoters, but can also be delivered into mammalian cells through infection of the cell with virally produced vectors. Although siRNA and shRNA ultimately utilize a similar cellular mechanism (RISC), the choice of which method to use depends on several factors such as cell type, time demands, and the need for transient versus stable integration. Although siRNAs are an undeniably effective tool for probing gene function in mammalian cells, their suppressive effects are by definition of limited duration.
A small hairpin RNA or short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. Once integrated into host genome, shRNAs can be transcribed from RNA polymerase III promoters in vivo, thus permitting the construction of continuous cell lines or transgenic animals in which RNAi enforces stable and heritable gene silencing. Generally, shRNA are synthesized in the nucleus of cells, further processed and transported to the cytoplasm and then incorporated into the RNA-interfering silencing complex (RISC) for activity. shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover. However, it requires use of an expression vector, which has the potential to cause side effects in medicinal applications.
Expression of shRNA in cells can incorporate different promoters and is accomplished by delivery of plasmids or through viral or bacterial vectors. Once the plasmid or vector has integrated into the host genome, the shRNA is then transcribed by either RNA polymerase II or III through RNA polymerase II or III promoters on the expression cassette. These initial precursors mimics pri-microRNA (pri-miRNA) and contains a hairpin like stem-loop structure that is processed in the nucleus by a complex containing the RNase III enzyme Drosha and the double-stranded RNA-binding domain protein DGCR8. The processed primary transcript is the pre-shRNA molecule. And then the pre-shRNA is transported from nucleus to the cytoplasm by exportin 5, a Ran-GTP-dependent transport mechanism.
Fig 1. Schematic of the shRNA mediated RNA interference pathway.
Fig 2. Schematic representation of shRNA structure.
In the cytoplasm the pre-shRNA is loaded onto another RNase III complex containing the RNase III enzyme Dicer and TRBP/PACT where the loop of the hairpin is processed off to form a 20-25nt double-stranded siRNA with 2nt 3' overhangs at each end. The mature shRNA in the Dicer/TRBP/PACT complex are associated with Argonaute protein containing RISC complex and provide RNA interference function either through mRNA cleavage and degradation, or through translational suppression via p-bodies. Generally, the sense strand is degraded, the antisense strand directs RISC to mRNA that has a complementary sequence. In the case of perfect complementarily, RISC cleaves the mRNA. In the case of imperfect complementarily, RISC represses translation of the mRNA. In both of these cases, the shRNA leads to target gene silencing, without any change to the gene itself.
Pre-shRNA has been found to be part of the RISC loading complex (RLC); thus, pre-shRNA may potentially directly associate with RLC rather than through a two steps process via a different Dicer/TRBP/PACT complex. After loading onto RLC and sense strand departure; both siRNA and shRNA in the RISC, in principle, should behave the same. The argonaute family of proteins is the major component of RISC (RNA-interfering silencing complex) . Within the Argonaute family of proteins, only Ago2 contains the endonuclease activity necessary to cleave and release the sense strand of the double-stranded stem. The remaining three members of Argonaute family, Ago1, Ago3 and Ago4, which do not have identiﬁable endonuclease activity, are also assembled into RISC and presumably function through a cleavage-independent manner.
As an RNAi technique, shRNA has its own advantages, as well as some problems. a. High transduction efficiency vector systems such as lentiviral shRNA vectors can be used to treat populations of cells, and for many studies, data can be acquired directly, without the need for cloning. b. Due to efficient transduction and high shRNA expression from many shRNA vector systems, experiments with shRNA vectors are generally scalable and highly consistent when repeated. c. Some vector systems, such as regular plasmid shRNA vectors used in transient transfections or piggyBac-based shRNA vectors, can be removed from cells, making the knockdown reversible. Disadvantages: a. Incomplete loss of gene function due to remaining functional mRNA. b. Some vector systems, such as regular plasmid transfection, have transient effects, rather than mediating permanent knockdown.
The simplicity of siRNA manufacturing and transient nature of the effect per dose are optimally suited for certain medical disorders (i.e. viral injections). However, using the endogenous processing machinery, optimized shRNA constructs allow for high potency and sustainable effects using low copy numbers resulting in fewer off-target effects, particularly if embedded in a miRNA scaffold. Advantages of shRNA over siRNA include the ability to use viral vectors for delivery to overcome the difficulty of transfecting certain cell types, the option to control shRNA expression using inducible promoters, and the ability to co-express them with a reporter gene. The target specificity that siRNAs and shRNAs provide has made them promising in medical applications as both therapeutic and diagnostic tools.
1. Paddison et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. GENES & DEVELOPMENT. 16:948–958.
2. D.D. Rao et al. siRNA vs. shRNA: Similarities and differences. Advanced Drug Delivery Reviews. 61 (2009) 746–759.
3. L.S. Lambeth and C.A. Smith. Short Hairpin RNA-Mediated Gene Silencing. Methods in Molecular Biology. 2013; 942:205-32.
4. Moore et al. Smith. Short Hairpin RNA (sh RNA): Design, Delivery, and Assessment of Gene Knockdown. Methods Mol Biol. 2010; 629:141–158.