RNA chaperones encoded by RNA viruses

RNAs are functionally diverse macromolecules whose proper functions rely strictly upon their correct tertiary structures. However, because of their high structural flexibility, correct folding of RNAs is challenging and slow. Therefore, cells and viruses encode a variety of RNA remodeling proteins, including helicases and RNA chaperones. In RNA viruses, these proteins are believed to play pivotal roles in all the processes involving viral RNAs during the life cycle. RNA helicases have been studied extensively for decades, whereas RNA chaperones, particularly virus-encoded RNA chaperones, are often overlooked. This review describes the activities of RNA chaperones encoded by RNA viruses, particularly the ones identified and characterized in recent years, and the functions of these proteins in different steps of viral life cycles, and presents an overview of this unique group of proteins.     

Enzymatic cleavage of RNA by RNA

The catalytic activity of E. coli RNase P, an enzyme essential for tRNA biosynthesis in vivo, resides in the RNA subunit of the enzyme. This RNA, which has all the properties of a classical enzyme, can cleave precursor tRNAs in vitro in the total absence of proteins.     

Enzymatic cleavage of RNA by RNA

The discovery and characterization of the catalytic RNA subunit of the enzyme ribonuclease P ofEscherichia coli is described.Nobel lecture given on December 8, 1989, by Professor Sidney Altman, and published in LES PRIX NOBEL 1989, printed in Sweden by Norstedts Tryckeri, Stockholm, Sweden, 1990, republished here with the permission of the Nobel Foundation, the copyright holder.     

Catalysis by RNA

Until the discovery of catalytic RNA, the notion that all enzymes are proteins had seemed incontrovertible. Now the existence of RNA enzymes has been confirmed in a variety of contexts. What is known about the chemistry of RNA-catalyzed reactions is reviewed below, with particular attention to the self-splicing rRNA intron of Tetrahymena thermophila and the processing of pre-tRNA molecules by RNase P.     

RNA interference in mammalian cells by chemically-modified RNA

RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.     

Suppression of RNA interference by adenovirus virus-associated RNA

We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3' strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.     

Completion of RNA synthesis by viral RNA replicases

How the 5′-terminus of the template affects RNA synthesis by viral RNA replicases is poorly understood. Using short DNA, RNA and RNA–DNA chimeric templates that can direct synthesis of replicase products, we found that DNA templates tend to direct the synthesis of RNA products that are shorter by 1 nt in comparison to RNA templates. Template-length RNA synthesis was also affected by the concentration of nucleoside triphosphates, the identity of the bases at specific positions close to the 5′-terminus and the C2′-hydroxyl of a ribose at the third nucleotide from the 5′-terminal nucleotide. Similar requirements are observed with two bromoviral replicases, but not with a recombinant RNA-dependent RNA polymerase. These results begin to define the interactions needed for the viral replicase to complete synthesis of viral RNA.     

Nuclear architecture by RNA

The dynamic organization of the cell nucleus into subcompartments with distinct biological activities represents an important determinant of cell function. Recent studies point to a crucial role of RNA as an architectural factor for shaping the genome and its nuclear environment. Here, we outline general principles by which RNA organizes functionally different nuclear subcompartments in mammalian cells. RNA is a structural component of mobile DNA-free nuclear bodies like paraspeckles or Cajal bodies, and is involved in establishing specific chromatin domains. The latter group comprises largely different structures that require RNA for the formation of active or repressive chromatin compartments with respect to gene expression as well as separating boundaries between these.     

Virus Specific RNA in Cells transformed by RNA Tumour Viruses

Virus specific RNA comprises 5% of the nuclear RNA and 0.5–1.0% of the cytoplasmic RNA of cells transformed by murine sarcoma viruses. Even cryptically transformed cells which possess no detectable virus or viral proteins synthesize detectable amounts of viral RNA.     

De novo RNA synthesis catalyzed by HCV RNA-dependent RNA polymerase

The 65 kDa RNA-dependent RNA polymerase (NS5B), encoded by the hepatitis C virus (HCV) genome, is a key component involved in viral replication. Here we provide the direct evidence that purified HCV polymerase catalyzed de novo RNA synthesis in a primer-independent manner using homopolymers and HCV RNA as templates. The enzyme could utilize both polyC and polyU as templates for de novo RNA synthesis, suggesting that NS5B specifically recognized pyrimidine bases for initiation. More importantly, NS5B also catalyzed de novo RNA synthesis with an HCV RNA template; the resulting nascent RNA products, smaller than the template used, contained ATP as the first nucleotide. These results indicate that the newly synthesized RNAs did not result from template self-priming and suggest that a replication initiation site in the HCV RNA genome is a uridylate.