New perspectives on neoplasia and the RNA world

Key tenets of modern biology are the central place of protein in cell regulation and the flow of genetic information from DNA to RNA to protein. However, it is becoming increasingly apparent that genomes are much more complex than hitherto thought with remarkably complex regulatory systems. The notion that the fraction of the genome involved in coding protein is all that matters is increasingly being questioned as the roles of non-coding RNA (ncRNA) in cellular systems becomes recognised. The RNA world, including microRNA (miRNA), small inhibitory RNA (siRNA) and other RNA species, are now recognised as being crucial for the regulation of chromatin structure, gene expression, mRNA processing and splicing, mRNA stability and translational control. Furthermore such ncRNA systems may be perturbed in disease states and most notably in neoplasia, including in haematological malignancies. Here the burgeoning evidence for a role of miRNA in neoplasia is reviewed and the importance of understanding the RNA world emphasised. Copyright (c) 2005 John Wiley & Sons, Ltd.

Nidovirales: evolving the largest RNA virus genome.

This review focuses on the monophyletic group of animal RNA viruses united in the order Nidovirales. The order includes the distantly related coronaviruses, toroviruses, and roniviruses, which possess the largest known RNA genomes (from 26 to 32 kb) and will therefore be called ‘large’ nidoviruses in this review. They are compared with their arterivirus cousins, which also belong to the Nidovirales despite having a much smaller genome (13–16 kb). Common and unique features that have been identified for either large or all nidoviruses are outlined. These include the nidovirus genetic plan and genome diversity, the composition of the replicase machinery and virus particles, virus-specific accessory genes, the mechanisms of RNA and protein synthesis, and the origin and evolution of nidoviruses with small and large genomes. Nidoviruses employ single-stranded, polycistronic RNA genomes of positive polarity that direct the synthesis of the subunits of the replicative complex, including the RNA-dependent RNA polymerase and helicase. Replicase gene expression is under the principal control of a ribosomal frameshifting signal and a chymotrypsin-like protease, which is assisted by one or more papain-like proteases. A nested set of subgenomic RNAs is synthesized to express the 3'-proximal ORFs that encode most conserved structural proteins and, in some large nidoviruses, also diverse accessory proteins that may promote virus adaptation to specific hosts. The replicase machinery includes a set of RNA-processing enzymes some of which are unique for either all or large nidoviruses. The acquisition of these enzymes may have improved the low fidelity of RNA replication to allow genome expansion and give rise to the ancestors of small and, subsequently, large nidoviruses.

Human coronavirus 229E nonstructural protein 13: characterization of duplex-unwinding, nucleoside triphosphatase, and RNA 5'-triphosphatase activities.

The human coronavirus 229E (HCoV-229E) replicase gene-encoded nonstructural protein 13 (nsp13) contains an N-terminal zinc-binding domain and a C-terminal superfamily 1 helicase domain. A histidine-tagged form of nsp13, which was expressed in insect cells and purified, is reported to unwind efficiently both partial-duplex RNA and DNA of up to several hundred base pairs. Characterization of the nsp13-associated nucleoside triphosphatase (NTPase) activities revealed that all natural ribonucleotides and nucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed most efficiently. Using the NTPase active site, HCoV-229E nsp13 also mediates RNA 5'-triphosphatase activity, which may be involved in the capping of viral RNAs.

The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5'-to-3' polarity.

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.

RNA expression analysis from formalin fixed paraffin embedded tissues

Formalin fixation and paraffin embedding (FFPE) is the most commonly used method worldwide for tissue storage. This method preserves the tissue integrity but causes extensive damage to nucleic acids stored within the tissue. As methods for measuring gene expression such as RT-PCR and microarray are adopted into clinical practice there is an increasing necessity to access the wealth of information locked in the Formalin fixation and paraffin embedding archives. This paper reviews the progress in this field and discusses the unique opportunities that exist for the application of these techniques in the development of personalized medicine.

The interference effects of non-rotated versus counter-rotated trials in visuomotor adaptation

An isometric torque-production task was used to investigate interference and retention in adaptation to multiple visuomotor environments. Subjects produced isometric flexion-extension and pronation-supination elbow torques to move a cursor to acquire targets as quickly as possible. Adaptation to a 30 degrees counter-clockwise (CCW) rotation (task A), was followed by a period of rest (control), trials with no rotation (task B0), or trials with a 60 degrees clockwise (CW) rotation (task B60). For all groups, retention of task A was assessed 5 h later. With initial training, all groups reduced the angular deviation of cursor paths early in the movements, indicating feedforward adaptation. For the control group, performance at commencement of the retest was significantly better than that at the beginning of the initial learning. For the B0 group, performance in the retest of task A was not dissimilar to that at the start of the initial learning, while for the B60 group retest performance in task A was markedly worse than initially observed. Our results indicate that close juxtaposition of two visuomotor environments precludes improved retest performance in the initial environment. Data for the B60 group, specifically larger angular errors upon retest compared with initial exposures, are consistent with the presence of anterograde interference. Furthermore, full interference occurred even when the visuomotor environment encountered in the second task was not rotated (B0). This latter novel result differs from those obtained for force field learning, where interference does not occur when task B does not impose perturbing forces, i.e., when B consists of a null field (Brashers-Krug et al., Nature 382:252-255, 1996). The results are consistent with recent proposals suggesting different interference mechanisms for visuomotor (kinematic) compared to force field (dynamic) adaptations, and have implications for the use of washout trials when studying interference between multiple visuomotor environments.