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.

Getting to the root of neuronal signalling in plant-parasitic nematodes using RNA interference

A variety of genes expressed in preparasitic second-stage juveniles (J2) of plant-parasitic nematodes appear to be vulnerable to RNA interference (RNAi) in vitro by coupling double-stranded (ds)RNA soaking with the artificial stimulation of pharyngeal pumping. Also, there is mounting evidence that the in planta generation of nematode-specific double-stranded RNAs (dsRNAs) has real utility in the control of these pests. Although neuronally-expressed genes in Caenorhabditis elegans are commonly refractory to RNAi, we have discovered that neuronally-expressed genes in plant-parasitic nematodes are highly susceptible to RNAi and that silencing can be induced by simple soaking procedures without the need for pharyngeal stimulation. Since most front-line anthelmintics that are used for the control of nematode parasites of animals and humans act to disrupt neuromuscular coordination, we argue that intercellular signalling processes associated with neurons have much appeal as targets for transgenic plant-based control strategies for plant-parasitic nematodes. FMRFamide-like peptides (FLPs) are a large family of neuropeptides which are intimately associated with neuromuscular regulation, and our studies on flp gene function in plant-parasitic nematodes have revealed that their expression is central to coordinated locomotory activities. We propose that the high level of conservation in nervous systems across nematodes coupled with the RNAi-susceptibility of neuronally-expressed genes in plant-parasitic nematodes provides a valuable research tool which could be used to interrogate neuronal signalling processes in nematodes.

FACT facilitates chromatin transcription by RNA polymerases I and III

Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle. The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III and facilitates their transcription in cells. Our findings indicate that, beyond the established role in Pol II transcription, FACT has physiological functions in chromatin transcription by all three nuclear RNA Pols. Our data also imply that local chromatin dynamics influence transcription of the active rRNA genes by Pol I and of Pol III-transcribed genes.

Regulation of cyclin D1 RNA stability by SNIP1

Cyclin D1 expression represents one of the key mitogen-regulated events during the G1 phase of the cell cycle, whereas Cyclin D1 overexpression is frequently associated with human malignancy. Here, we describe a novel mechanism regulating Cyclin D1 levels. We find that SNIP1, previously identified as a regulator of Cyclin D1 expression, does not, as previously thought, primarily function as a transcriptional coactivator for this gene. Rather, SNIP1 plays a critical role in cotranscriptional or posttranscriptional Cyclin D1 mRNA stability. Moreover, we show that the majority of nucleoplasmic SNIP1 is present within a previously undescribed complex containing SkIP, THRAP3, BCLAF1, and Pinin, all proteins with reported roles in RNA processing and transcriptional regulation. We find that this complex, which we have termed the SNIP1/SkIP–associated RNA-processing complex, is coordinately recruited to both the 3' end of the Cyclin D1 gene and Cyclin D1 RNA. Significantly, SNIP1 is required for the further recruitment of the RNA processing factor U2AF65 to both the Cyclin D1 gene and RNA. This study shows a novel mechanism regulating Cyclin D1 expression and offers new insight into the role of SNIP1 and associated proteins as regulators of proliferation and cancer.