RNA-dependent RNA polymerase

About: RNA-dependent RNA polymerase is a research topic. Over the lifetime, 13904 publications have been published within this topic receiving 767954 citations. The topic is also known as: RdRp & RNA replicase.

RNA-directed DNA methylation.

Abstract: Double-stranded RNAs (dsRNAs) and their 'diced' small RNA products can guide key developmental and defense mechanisms in eukaryotes. Some RNA-directed mechanisms act at a post-transcriptional level to degrade target messenger RNAs. However, dsRNA-derived species can also direct changes in the chromatin structure of DNA regions with which they share sequence identity. For example, plants use such RNA species to lay down cytosine methylation imprints on identical DNA sequences, providing a fundamental mark for the formation of transcriptionally silent heterochromatin. Thus, RNA can feed backwards to modulate the accessibility of information stored in the DNA of cognate genes. RNA triggers for DNA methylation can come from different sources, including invasive viral, transgene or transposon sequences, and in some cases are derived from single-stranded RNA precursors by RNA-dependent RNA polymerases. The mechanism by which RNA signals are translated into DNA methylation imprints is currently unknown, but two plant-specific types of cytosine methyltransferase have been implicated in this process. RNA can also direct heterochromatin formation in fission yeast and Drosophila, but in these organisms the process occurs in the absence of DNA methylation.

Site-specific binding of a PPR protein defines and stabilizes 5′ and 3′ mRNA termini in chloroplasts

Abstract: Chloroplast mRNA populations are characterized by overlapping transcripts derived by processing from polycistronic precursors. The mechanisms and functional significance of these processing events are poorly understood. We describe a pentatricopeptide repeat (PPR) protein, PPR10, whose binding defines mRNA segments derived from two transcription units in maize chloroplasts. PPR10 interacts in vivo and in vitro with two intergenic RNA regions of similar sequence. The processed 5 0 and 3 0 RNA termini in these regions overlap by approximately 25 nucleotides. The PPR10-binding sites map precisely to these overlapping sequences, and PPR10 is required specifically for the accumulation of RNAs with these termini. These findings show that PPR10 serves as a barrier to RNA decay from either the 5 0 or 3 0 direction and that a bound protein provides an alternative to an RNA hairpin as a barrier to 3 0 exonucleases. The results imply that protein ‘caps’ at both 5 0 and 3 0 ends can define the termini of chloroplast mRNA segments. These results, together with recent insights into bacterial RNA decay, suggest a unifying model for the biogenesis of chloroplast transcript populations and for the determinants of chlo

Ribozyme-catalyzed transcription of an active ribozyme.

Abstract: A critical event in the origin of life is thought to have been the emergence of an RNA molecule capable of replicating a primordial RNA "genome." Here we describe the evolution and engineering of an RNA polymerase ribozyme capable of synthesizing RNAs of up to 95 nucleotides in length. To overcome its sequence dependence, we recombined traits evolved separately in different ribozyme lineages. This yielded a more general polymerase ribozyme that was able to synthesize a wider spectrum of RNA sequences, as we demonstrate by the accurate synthesis of an enzymatically active RNA, a hammerhead endonuclease ribozyme. This recapitulates a central aspect of an RNA-based genetic system: the RNA-catalyzed synthesis of an active ribozyme from an RNA template.

RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells

Abstract: RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects >270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both a messenger RNA and replication template, making it an attractive target for the study of RNA interference. Double-stranded small interfering RNA (siRNA) molecules designed to target the HCV genome were introduced through electroporation into a human hepatoma cell line (Huh-7) that contained an HCV subgenomic replicon. Two siRNAs dramatically reduced virus-specific protein expression and RNA synthesis to levels that were 90% less than those seen in cells treated with negative control siRNAs. These same siRNAs protected naive Huh-7 cells from challenge with HCV replicon RNA. Treatment of cells with synthetic siRNA was effective >72 h, but the duration of RNA interference could be extended beyond 3 weeks through stable expression of complementary strands of the interfering RNA by using a bicistronic expression vector. These results suggest that a gene-therapeutic approach with siRNA could ultimately be used to treat HCV.

Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome.

Abstract: Hepadnaviruses, as well as other pararetroviruses, express their pol (P) gene product unfused to the preceding core gene implying that these retroelements have developed a mechanism for initiating assembly and replication that is principally different from the one used by retroviruses and retrotransposons. We have analysed this mechanism for the human hepatitis B virus by using a newly developed, highly sensitive detection method based upon radiolabelling of the P protein at newly introduced target sites for protein kinase A. The results obtained demonstrate that polymerase encapsidation depends on the concomittant encapsidation of the HBV RNA pregenome and that packaging of the viral RNA, in turn, depends on the presence of P protein. Loss of P protein encapsidation by mutations inactivating the HBV RNA encapsidation signal epsilon could be compensated by trans-complementation with recombinant RNA molecules carrying the epsilon sequence. Thus, in contrast to retroviral replication, the interaction of the hepadnaviral P protein and the RNA genome at its packaging signal appears to be crucial for initiating the formation of replication-competent nucleocapsids. Furthermore, RNA control of P protein packaging stringently limits the number of polymerase molecules that can be encapsidated.