By Mehdi Banan, Ph.D.
Science Content/Business Development Analyst

Small interfering RNAs (siRNAs) are 21-23 bp double-stranded RNA molecules that elicit gene-specific silencing in mammalian cells. siRNA-mediated gene silencing can be achieved in one of two ways. In the first, siRNAs are synthesized in vitro and introduced into cells to transiently suppress gene expression. In the second, siRNAs are expressed in vivo from vectors--an approach that can be used to stably express siRNAs in cells or transgenic animals.

To date, most siRNA expression vectors have been engineered to drive siRNA transcription from polymerase III (pol III) transcription units (1). Pol III transcription units are particularly suited for hairpin siRNA expression, since they deploy a short AT rich transcription termination site that leads to the addition of 2 bp overhangs (UU) to hairpin siRNAs--a feature that is important for siRNA function. Pol III expression vectors have recently been used to create siRNA-expressing transgenic mice (also referred to as "knockdown" mice) (2,3). A drawback of the ubiquitously expressed pol III promoters, however, is that they do not allow for tissue-specific siRNA expression.

In the June 2003 issue of Genes & Development, Shinagawa and Ishii report on a novel scheme that could be used to express siRNAs in a tissue-specific manner (4). In their method, Shinagawa and Ishii first expressed long double-stranded RNAs (dsRNAs) from a CMV (pol II) promoter in the nuclei of cell lines and transgenic mice. The long dsRNAs were then processed into siRNAs in the nucleus (by Dicer). The siRNAs subsequently exited the nucleus and mediated gene-specific silencing. Presumably, this scheme can also be used in conjunction with tissue-specific (pol II) promoters to create tissue-specific knockdown mice.

What about the antiviral response?

A concern with expression of long (>30 bp) dsRNAs in mammalian cells is their ability to activate the antiviral interferon response--a response that is accompanied by nonspecific gene silencing. In the interferon response, long dsRNAs activate protein kinase R (PKR), which phosphorylates and inactivates eIF2a, leading to a general inhibition of protein synthesis. In addition, long dsRNAs activate 2', 5'-oligoadenylate synthetase and RNase L, leading to a general degradation of RNA molecules.

Previous reports had suggested that the interferon response takes place after long dsRNAs are transferred to the cytoplasm (5). Therefore Shinagawa and Ishii reasoned that by restricting the expression of long hairpin RNAs to the nucleus, the interferon response could be avoided. Export of RNA from the nucleus to cytoplasm occurs after RNA capping, splicing, and poly(A) tailing. The authors hypothesized that by preventing these events, long dsRNAs would stay in the nucleus. To this end, they created a vector called pDECAP [for Deletion of Cap structure and poly(A)]. In order to abolish capping, a cis-acting ribozyme coding region was cloned downstream of the hairpin dsRNA template. Poly(A) addition was prevented by addition of a pol II transcriptional pause site (and not a poly(A) addition site) downstream of the dsRNA template.

The Results

The authors first showed that siRNAs created by the above approach could be used for gene-specific silencing in 293T cells. The pDECAP vector expressing a dsRNA to the firefly luciferase gene, for example, effectively silenced the firefly luciferase gene (which was expressed from a co-transfected expression vector). This dsRNA, however, did not affect the expression of the sea pansy luciferase control. Moreover, a pDECAP vector expressing a hairpin dsRNA to the endogenous ski gene reduced Ski protein expression levels. It, however, did not affect the expression levels of the related protein, Sno (sno has 60% homology with ski at the dsRNA region). The authors also used the pDECAP vector to create ski knockdown mice. Interestingly, the phenotype of the ski "knockdown" embryos was similar to that of ski "knockout" embryos--in both cases, the embryos had defects in neural tube and eye formation.

Why not express siRNAs directly from tissue-specific promoters?

Use of pol II transcription units to drive siRNA expression has rarely been reported, presumably because pol II transcription termination sites utilize a poly(A) addition site. The addition of a poly(A) tail to siRNAs would, in all likelihood, abolish their function. Functional siRNAs, however, have been expressed in cell lines from expression units containing a CMV promoter and a minimal poly(A) cassette, which presumably avoids poly(A) tailing (6). It may therefore be possible to express siRNAs from tissue-specific promoters by utilizing a similar approach.

It is likely that for tissue-specific siRNA expression, all of the above approaches will be investigated. Whether expression of siRNAs in a tissue-specific manner can be done directly (i.e. from tissue-specific promoters) or indirectly (i.e. by expressing long dsRNAs from tissue-specific promoters, as reported by Shinagawa and Ishii) remains to be seen. Either way, such a possibility would greatly expand the range of loss-of-function experiments that could be performed in mice.

References

1. Tuschl T. (2002). Expanding small RNA interference. Nature Biotechnol. 20: 446-448.
2. Carmell MA, Zhang L, Conklin DS, Hannon GJ, and Rosenquist TA (2003). Germline transmission of RNAi in mice. Nature Struct. Biol. 10(2): 91-92.
3. Kunath T, Gish G, Lickert H, Jones N, Pawson T, and Rossant J (2003). Transgenic RNA interference in ES cells-derived embryos recapitulates a genetic null phenotype. Nature Biotechnol. 21:559-561.
4. Shinagawa T and Ishii S. (2003). Generation of Ski-knockdown mice by expressing a long double-strand RNA from an RNA polymerase II promoter. Genes & Dev. 17:1340-1345.
5. Stark GR, Kerr IM, Williams BR, Silverman RH, and Schreiber RD. (1998). How cells respond to interferons. Annu. Rev. Biochem. 67:227-264.
6. Xia H, Mao Q, Paulson HL, and Davidson BL. (2002). siRNA-mediated gene silencing in vitro and in vivo. Nature Biotech. 20:1006-1010.