The DExH protein NPH-II is a processive and directional motor for unwinding RNA

All aspects of cellular RNA metabolism and processing involve DExH/D proteins, which are a family of enzymes that unwind or manipulate RNA in an ATP-dependent fashion. DExH/D proteins are also essential for the replication of many viruses, and therefore provide targets for the development of therapeutics. All DExH/D proteins characterized to date hydrolyse nucleoside triphosphates and, in most cases, this activity is stimulated by the addition of RNA or DNA. Several members of the family unwind RNA duplexes in an NTP-dependent fashion in vitro; therefore it has been proposed that DExH/D proteins couple NTP hydrolysis to RNA conformational change in complex macromolecular assemblies. Despite the central role of DExH/D proteins, their mechanism of RNA helicase activity remains unknown. Here we show that the DExH protein NPH-II unwinds RNA duplexes in a processive, unidirectional fashion with a step size of roughly one-half helix turn. We show that there is a quantitative connection between ATP utilization and helicase processivity, thereby providing direct evidence that DExH/D proteins can function as molecular motors on RNA.     

A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans

In many organisms, introducing double-stranded RNA (dsRNA) causes the degradation of messenger RNA that is homologous to the trigger dsRNA--a process known as RNA interference. The dsRNA is cleaved into short interfering RNAs (siRNAs), which hybridize to homologous mRNAs and induce their degradation. dsRNAs vary in their ability to trigger RNA interference: many mRNA-targeting dsRNAs show weak phenotypes, and nearly all mRNAs of the Caenorhabditis elegans nervous system are refractory to RNA interference. C. elegans eri-1 was identified in a genetic screen for mutants with enhanced sensitivity to dsRNAs. Here we show that eri-1 encodes an evolutionarily conserved protein with domains homologous to nucleic-acid-binding and exonuclease proteins. After exposure to dsRNA or siRNAs, animals with eri-1 mutations accumulate more siRNAs than do wild-type animals. C. elegans ERI-1 and its human orthologue degrade siRNAs in vitro. In the nematode worm, ERI-1 is predominantly cytoplasmic and is expressed most highly in the gonad and a subset of neurons, suggesting that ERI-1 siRNase activity suppresses RNA interference more intensely in these tissues. Thus, ERI-1 is a negative regulator that may normally function to limit the duration, cell-type specificity or endogenous functions of RNA interference.     

Mechanisms of gene silencing by double-stranded RNA

Double-stranded RNA (dsRNA) is an important regulator of gene expression in many eukaryotes. It triggers different types of gene silencing that are collectively referred to as RNA silencing or RNA interference. A key step in known silencing pathways is the processing of dsRNAs into short RNA duplexes of characteristic size and structure. These short dsRNAs guide RNA silencing by specific and distinct mechanisms. Many components of the RNA silencing machinery still need to be identified and characterized, but a more complete understanding of the process is imminent.     

Alu sequences are processed 7SL RNA genes

7SL RNA is an abundant cytoplasmic RNA which functions in protein secretion as a component of the signal recognition particle. Alu sequences are the most abundant family of human and rodent middle repetitive DNA sequences (reviewed in ref. 2). The primary structure of human 7SL RNA consists of an Alu sequence interrupted by a 155-base pair (bp) sequence that is unique to 7SL RNA. In order to obtain information about the evolution of the Alu domain of 7SL RNA, we have determined the nucleotide sequence of a cDNA copy of Xenopus laevis 7SL RNA and of the 7SL RNA gene of Drosophila melanogaster. We find that the Xenopus sequence is 87% homologous with its human counterpart and the Drosophila 7SL RNA is 64% homologous to both the human and amphibian molecules. Despite the evolutionary distance between the species, significant blocks of homology to both the Alu and 7SL-specific portions of mammalian 7SL RNA can be found in the insect sequence. These results clearly demonstrate that the Alu sequence in 7SL RNA appeared in evolution before the mammalian radiation. We suggest that mammalian Alu sequences were derived from 7SL RNA (or DNA) by a deletion of the central 7SL-specific sequence, and are therefore processed 7SL RNA genes.     

RNA editing by cytidine insertion in mitochondria of Physarum polycephalum

A corollary of the central dogma of molecular biology is that genetic information passes from DNA to RNA by the continuous synthesis of RNA on a DNA template. The demonstration of RNA editing (the specific insertion, deletion or substitution of residues in RNA to create an RNA with a sequence different from its own template) raised the possibility that in some cases not all of the genetic information for a trait residues in the DNA template. Two different types of RNA editing have been identified in mitochondria: insertional editing represented by the extensive insertion (and occasional deletion) of uridine residues in mitochondrial RNAs of the kinetoplastid protozoa and the substitutional editing represented by the cytidine to uridine substitutions in some plant mitochondria. These editing types have not been shown to be present in the same organism and may have very different mechanisms. RNA editing of both types has been observed in nonmitochondrial systems but is not as extensive and may involve still different mechanisms. Here we report the discovery of extensive insertional RNA editing in mitochondria from an organism other than a kinetoplastid protozoan. The mitochondrial RNA apparently encoding the alpha subunit of ATP synthetase in the acellular slime mould, Physarum polycephalum, is edited at 54 sites by cytidine insertion.     

Role of RNA transcripts in replication incompatibility and copy number control in antibiotic resistance plasmid derivatives

The genes required for autonomous replication and incompatibility in the antibiotic resistance plasmids R100 and R1 have been located within a 2.5-kilobase region of the 90-kilobase genome, within which the incompatibility gene occupies a 1.3-kilobase region excluding the replication origin. We now report that three RNA species are synthesized in vitro from the 2.5-kilobase region, which R100 and R1 have in common. One, a long RNA molecule which is transcribed in the direction of DNA replication, probably acts as a messenger or a protein required for plasmid replication. The second RNA species, only 91 nucleotides long, is transcribed in the opposite direction, from a region of the DNA entirely contained within the first and known to specify incompatibility and copy control functions. The third RNA species, 150 bases long, is transcribed from a region including the replication origin; it may be a primer of DNA synthesis or, in conjunction with the second of the three RNA species, an influence in the control of replication.     

Unravelling the dynamics of RNA degradation by ribonuclease II and its RNA-bound complex

RNA degradation is a determining factor in the control of gene expression. The maturation, turnover and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a major exoribonuclease that intervenes in all of these fundamental processes; it can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. RNase II-like enzymes are found in all three kingdoms of life, but there are no structural data for any of the proteins of this family. Here we report the X-ray crystallographic structures of both the ligand-free (at 2.44 A resolution) and RNA-bound (at 2.74 A resolution) forms of Escherichia coli RNase II. In contrast to sequence predictions, the structures show that RNase II is organized into four domains: two cold-shock domains, one RNB catalytic domain, which has an unprecedented alphabeta-fold, and one S1 domain. The enzyme establishes contacts with RNA in two distinct regions, the 'anchor' and the 'catalytic' regions, which act synergistically to provide catalysis. The active site is buried within the RNB catalytic domain, in a pocket formed by four conserved sequence motifs. The structure shows that the catalytic pocket is only accessible to single-stranded RNA, and explains the specificity for RNA versus DNA cleavage. It also explains the dynamic mechanism of RNA degradation by providing the structural basis for RNA translocation and enzyme processivity. We propose a reaction mechanism for exonucleolytic RNA degradation involving key conserved residues. Our three-dimensional model corroborates all existing biochemical data for RNase II, and elucidates the general basis for RNA degradation. Moreover, it reveals important structural features that can be extrapolated to other members of this family.