Poly(U) and polyadenylation termination signals are interchangeable for terminating the expression of shRNA from a pol II promoter

doi:10.1016/j.bbrc.2004.08.128

Biochemical and Biophysical Research Communications

Volume 323, Issue 2, 15 October 2004, Pages 573-578

https://doi.org/10.1016/j.bbrc.2004.08.128 Get rights and content

Abstract

Most short hairpin RNA (shRNA)-expressing vectors use RNA polymerase III promoters, as expression of shRNAs from RNA polymerase II promoters is not well understood, due to the lack of defined transcription initiation sites and functional localization of the transcription termination signal. Here we describe a modified cytomegavirus (CMV) promoter, an RNA polymerase II promoter, to express shRNAs with only four overhangs at the 5′ end. The expression of shRNAs from the modified CMV promoter was terminated by the transcription termination, the polyadenylation, or the poly(U) termination signal. These results demonstrated that the poly(U) and polyadenylation termination signals are interchangeable for terminating the expression of shRNAs from the CMV promoter and result in similar efficacies in inhibiting endogenous target genes in mammalian cells.

Section snippets

Materials and methods

Plasmids. A construct containing the cytomegavirus (CMV) promoter, either the polyadenylation signal (AATAAA) or the poly(U) termination signal (TTTTT), and the shRNA gene cloning sites, SacII and HindIII, was PCR-amplified with forward and reverse primers, using the pEGFP-N1 vector containing the CMV promoter as a template. For cloning purposes, the forward sequence, 5′-ACGTCTAGATAGTTATTAATAGTAATCAA-3′, was flanked with XbaI at the 5′ end (underlined), and the reverse sequence, 5′-GGCGAATTC

RNAi-mediated gene silencing by the CMV promoter is compatible with that of the U6 promoter

We constructed a CMV pol II promoter to express shRNAs that silenced the endogenous target gene (Fig. 1A). The transcription initiation site was merged into the cloning site (SacII) of the vector, which starts from the first G of the cloning site. This arrangement not only simplified the process of subcloning, but also reduced the overhangs at the 5′ end to four nucleotides, thereby increasing the effects of RNAi. To terminate the transcription of shRNAs from the CMV promoter, the

Discussion

RNA polymerase II promoters were used to drive the expression of long hairpin RNA cleaved by Dicer into siRNAs that silence target gene expression [8], [9], [10]. However, expression of long hairpin RNA in mammalian cells induces the interferon response, thereby limiting their usefulness. Long hairpin RNA has also been used in organisms with weak or absent interferon responses. These long hairpin expression systems have effectively silenced target genes in several different organisms, including

Acknowledgments

The authors are grateful to Kristen Lum for a critical review of the manuscript and Susan Chou for assistance with experiments. This work was supported by Public Health Services Grant CA66746 to R.C. from the National Cancer Institute.

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View full text

Poly(U) and polyadenylation termination signals are interchangeable for terminating the expression of shRNA from a pol II promoter

Abstract

Section snippets

Materials and methods

RNAi-mediated gene silencing by the CMV promoter is compatible with that of the U6 promoter

Discussion

Acknowledgments

Biochem. Biophys. Res. Commun.

Curr. Biol.

Cell. Signal.

Curr. Opin. Genet. Dev.

J. Biol. Chem.

J. Biol. Chem.

Cell

Mol. Cell

Analyt. Biochem.

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Nature

An RNA-based information superhighway in plants

Science

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

Nature

Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems

Proc. Natl. Acad. Sci. USA.

A system for stable expression of short interfering RNAs in mammalian cells

Science

Gene silencing using micro-RNA designed hairpins

RNA

Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells

Genes Dev.

Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes

Nat. Genet.