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An approach to genomewide screens of expressed small interfering RNAs in mammalian cells.

ABSTRACT: To facilitate the construction of large genomewide libraries of small interfering RNAs (siRNAs), we have developed a dual promoter system (pDual) in which a synthetic DNA encoding a gene-specific siRNA sequence is inserted between two different opposing polymerase III promoters, the mouse U6 and human H1 promoters. Upon transfection into mammalian cells, the sense and antisense strands of the duplex are transcribed by these two opposing promoters from the same template, resulting in a siRNA duplex with a uridine overhang on each 3' terminus. A single-step PCR protocol has been developed by using this dual promoter system that allows the production of siRNA expression cassettes in a high-throughput manner. We have shown that siRNAs transcribed by either the dual promoter vector or siRNA expression cassettes can induce strong and gene-specific suppression of both endogenous genes and ectopically expressed genes in mammalian cells. Furthermore, we have constructed an arrayed siRNA expression cassette library that targets >8000 genes with two siRNA sequences per gene. A high-throughput screen of this library has revealed both known and unique genes involved in the NF-kappaB signaling pathway.

SUBMITTER: Zheng L

PROVIDER: S-EPMC314151 | biostudies-literature | 2004 Jan

REPOSITORIES: biostudies-literature

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An approach to genomewide screens of expressed small interfering RNAs in mammalian cells.

Zheng Lianxing L Liu Jun J Batalov Sergei S Zhou Demin D Orth Anthony A Ding Sheng S Schultz Peter G PG

Proceedings of the National Academy of Sciences of the United States of America 20031219 1

To facilitate the construction of large genomewide libraries of small interfering RNAs (siRNAs), we have developed a dual promoter system (pDual) in which a synthetic DNA encoding a gene-specific siRNA sequence is inserted between two different opposing polymerase III promoters, the mouse U6 and human H1 promoters. Upon transfection into mammalian cells, the sense and antisense strands of the duplex are transcribed by these two opposing promoters from the same template, resulting in a siRNA dupl ...[more]

PMID: 14688408

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Project description:BACKGROUND: The discovery of RNA interference phenomenon (RNAi) and understanding of its mechanisms has revolutionized our views on many molecular processes in the living cell. Among the other, RNAi is involved in silencing of transposable elements and in inhibition of virus infection in various eukaryotic organisms. Recent experimental studies demonstrate few cases of viral replication suppression via complementary interactions between the mammalian small RNAs and viral transcripts. PRESENTATION OF THE HYPOTHESIS: It was found that >50% of the human genome is transcribed in different cell types and that these transcripts are mainly not associated with known protein coding genes, but represent non-coding RNAs of unknown functions. We propose a hypothesis that mammalian DNAs encode thousands RNA motifs that may serve for antiviral protection. We also presume that the evolutional success of some groups of genomic repeats and, in particular, of transposable elements (TEs) may be due to their ability to provide antiviral RNA motifs to the host organism. Intense genomic repeat propagation into the genome would inevitably cause bidirectional transcription of these sequences, and the resulting double-stranded RNAs may be recognized and processed by the RNA interference enzymatic machinery. Provided that these processed target motifs may be complementary to viral transcripts, fixation of the repeats into the host genome may be of a considerable benefit to the host. It fits with our bioinformatical data revealing thousands of 21-28 bp long motifs identical between human DNA and human-pathogenic adenoviral and herpesviral genomes. Many of these motifs are transcribed in human cells, and the transcribed part grows proportionally to their lengths. Many such motifs are included in human TEs. For example, one 23 nt-long motif that is a part of human abundant Alu retrotransposon, shares sequence identity with eight human adenoviral genomes. TESTING THE HYPOTHESIS: This hypothesis could be tested on various mammalian species and viruses infecting mammalian cells. IMPLICATIONS OF THE HYPOTHESIS: This hypothesis proposes that mammalian organisms may use their own genomes as sources of thousands of putative interfering RNA motifs that can be recruited to repress intracellular pathogens like proliferating viruses. REVIEWERS: This article was reviewed by Eugene V. Koonin, Valerian V. Dolja and Yuri V. Shpakovski.

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An approach to genomewide screens of expressed small interfering RNAs in mammalian cells.

Publications

An approach to genomewide screens of expressed small interfering RNAs in mammalian cells.

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