RNA-dependent RNA polymerase

Abstract: A gene expression system based on bacteriophage T7 RNA polymerase has been developed. T7 RNA polymerase is highly selective for its own promoters, which do not occur naturally in Escherichia coli. A relatively small amount of T7 RNA polymerase provided from a cloned copy of T7 gene 1 is sufficient to direct high-level transcription from a T7 promoter in a multicopy plasmid. Such transcription can proceed several times around the plasmid without terminating, and can be so active that transcription by E. coli RNA polymerase is greatly decreased. When a cleavage site for RNase III is introduced, discrete RNAs of plasmid length can accumulate. The natural transcription terminator from T7 DNA also works effectively in the plasmid. Both the rate of synthesis and the accumulation of RNA directed by T7 RNA polymerase can reach levels comparable with those for ribosomal RNAs in a normal cell. These high levels of accumulation suggest that the RNAs are relatively stable, perhaps in part because their great length and/or stem-and-loop structures at their 3' ends help to protect them against exonucleolytic degradation. It seems likely that a specific mRNA produced by T7 RNA polymerase can rapidly saturate the translational machinery of E. coli, so that the rate of protein synthesis from such an mRNA will depend primarily on the efficiency of its translation. When the mRNA is efficiently translated, a target protein can accumulate to greater than 50% of the total cell protein in three hours or less. We have used two ways to deliver active T7 RNA polymerase to the cell; infection by a lambda derivative that carries gene 1, or induction of a chromosomal copy of gene 1 under control of the lacUV5 promoter. When gene 1 is delivered by infection, very toxic target genes can be maintained silent in the cell until T7 RNA polymerase is introduced, when they rapidly become expressed at high levels. When gene 1 is resident in the chromosome, even the very low basal levels of T7 RNA polymerase present in the uninduced cell can prevent the establishment of plasmids carrying toxic target genes, or make the plasmid unstable.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract: A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

Abstract: Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 21- and 22-nt RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III–like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3 ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA–protein complex.

Abstract: The innate immune system senses viral infection by recognizing a variety of viral components (including double-stranded (ds)RNA) and triggers antiviral responses. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I, also known as Ddx58) and MDA5 (melanoma-differentiation-associated gene 5, also known as Ifih1 or Helicard) have been implicated in viral dsRNA recognition. In vitro studies suggest that both RIG-I and MDA5 detect RNA viruses and polyinosine-polycytidylic acid (poly(I:C)), a synthetic dsRNA analogue. Although a critical role for RIG-I in the recognition of several RNA viruses has been clarified, the functional role of MDA5 and the relationship between these dsRNA detectors in vivo are yet to be determined. Here we use mice deficient in MDA5 (MDA5-/-) to show that MDA5 and RIG-I recognize different types of dsRNAs: MDA5 recognizes poly(I:C), and RIG-I detects in vitro transcribed dsRNAs. RNA viruses are also differentially recognized by RIG-I and MDA5. We find that RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus, whereas MDA5 is critical for picornavirus detection. Furthermore, RIG-I-/- and MDA5-/- mice are highly susceptible to infection with these respective RNA viruses compared to control mice. Together, our data show that RIG-I and MDA5 distinguish different RNA viruses and are critical for host antiviral responses.