Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination

RNA polymerase II (pol II) transcription termination requires co-transcriptional recognition of a functional polyadenylation signal, but the molecular mechanisms that transduce this signal to pol II remain unclear. We show that Yhh1p/Cft1p, the yeast homologue of the mammalian AAUAAA interacting protein CPSF 160, is an RNA-binding protein and provide evidence that it participates in poly(A) site recognition. Interestingly, RNA binding is mediated by a central domain composed of predicted -propeller-forming repeats, which occurs in proteins of diverse cellular functions. We also found that Yhh1p/Cft1p bound specifically to the phosphorylated C-terminal domain (CTD) of pol II in vitro and in a two-hybrid test in vivo. Furthermore, transcriptional run-on analysis demonstrated that yhh1 mutants were defective in transcription termination, suggesting that Yhh1p/Cft1p functions in the coupling of transcription and 3'-end formation. We propose that direct interactions of Yhh1p/Cft1p with both the RNA transcript and the CTD are required to communicate poly(A) site recognition to elongating pol II to initiate transcription termination.

Proteolytic processing of the P2/nucleocapsid cleavage site is critical for human immunodeficiency virus type 1 RNA dimer maturation

Differences in virion RNA dimer stability between mature and protease-defective (immature) forms of human immunodeficiency virus type 1 (HIV-1) suggest that maturation of the viral RNA dimer is regulated by the proteolytic processing of the HIV-1 Gag and Gag-Pol precursor proteins. However, the proteolytic processing of these proteins occurs in several steps denoted primary, secondary, and tertiary cleavage events and, to date, the processing step associated with formation of stable HIV-1 RNA dimers has not been identified. We show here that a mutation in the primary cleavage site (p2/nucleocapsid [NC]) hinders formation of stable virion RNA dimers, while dimer stability is unaffected by mutations in the secondary (matrix/capsid [CA], p1/p6) or a tertiary cleavage site (CA/p2). By introducing mutations in a shared cleavage site of either Gag or Gag-Pol, we also show that the cleavage of the p2/NC site in Gag is more important for dimer formation and stability than p2/NC cleavage in Gag-Pol. Electron microscopy analysis of viral particles shows that mutations in the primary cleavage site in Gag but not in Gag-Pol inhibit viral particle maturation. We conclude that virion RNA dimer maturation is dependent on proteolytic processing of the primary cleavage site and is associated with virion core formation.

The A-rich RNA sequences of HIV-1 pol are important for the synthesis of viral cDNA

The bias of A-rich codons in HIV-1 pol is thought to be a record of hypermutations in viral genomes that lack biological functions. Bioinformatic analysis predicted that A-rich sequences are generally associated with minimal local RNA structures. Using codon modifications to reduce the amount of A-rich sequences within HIV-1 genomes, we have reduced the flexibility of RNA sequences in pol to analyze the functional significance of these A-rich ‘structurally poor’ RNA elements in HIV-1 pol. Our data showed that codon modification of HIV-1 sequences led to a suppression of virus infectivity by 5–100-fold, and this defect does not correlate with, viral entry, viral protein expression levels, viral protein profiles or virion packaging of genomic RNA. Codon modification of HIV-1 pol correlated with an enhanced dimer stability of the viral RNA genome, which was associated with a reduction of viral cDNA synthesis both during HIV-1 infection and in a cell free reverse transcription assay. Our data provided direct evidence that the HIV-1 A-rich pol sequence is not merely an evolutionary artifact of enzyme-induced hypermutations, and that HIV-1 has adapted to rely on A-rich RNA sequences to support the synthesis of viral cDNA during reverse transcription, highlighting the utility of using ‘structurally poor’ RNA domains in regulating biological process.

Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device

This paper describes the development of a microfluidic methodology, using RNA extraction and reverse transcription PCR, for investigating expression levels of cytochrome P450 genes. Cytochrome P450 enzymes are involved in the metabolism of xenobiotics, including many commonly prescribed drugs, therefore information on their expression is useful in both pharmaceutical and clinical settings. RNA extraction, from rat liver tissue or primary rat hepatocytes, was performed using a silica-based solid-phase extraction technique. Following elution of the purified RNA, amplification of target sequences for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the cytochrome P450 gene CYP1A2, was carried out using a one-step reverse transcription PCR. Once the microfluidic methodology had been optimized, analysis of control and 3-methylcholanthrene-induced primary rat hepatocytes were used to evaluate the system. As expected, GAPDH was consistently expressed, whereas CYP1A2 levels were found to be raised in the drug-treated samples. The proposed system offers an initial platform for development of both rapid throughput analyzers for pharmaceutical drug screening and point-of-care diagnostic tests to aid provision of drug regimens, which can be tailor-made to the individual patient.

Maternal high-fat diet alters expression of pathways of growth, blood supply and arachidonic acid in rat placenta

The high fat content in Western diets probably affects placental function during pregnancy with potential consequences for the offspring in the short and long term. The aim of the present study was to compare genome-wide placental gene expression between rat dams fed a high-fat diet (HFD) and those fed a control diet for 3 weeks before conception and during gestation. Gene expression was measured by microarray and pathway analysis was performed. Gene expression differences were replicated by real-time PCR and protein expression was assessed by Western blot analysis. Placental and fetal weights at E17.25 were not altered by exposure to the maternal HFD. Gene pathways targeting placental growth, blood supply and chemokine signalling were up-regulated in the placentae of dams fed the HFD. The up-regulation in messenger RNA expression for five genes Ptgs2 (fatty acid cyclo-oxidase 2; COX2), Limk1 (LIM domain kinase 1), Pla2g2a (phospholipase A2), Itga1 (integrin α-1) and Serpine1 was confirmed by real-time PCR. Placental protein expression for COX2 and LIMK was also increased in HFD-fed dams. In conclusion, maternal HFD feeding alters placental gene expression patterns of placental growth and blood supply and specifically increases the expression of genes involved in arachidonic acid and PG metabolism. These changes indicate a placental response to the altered maternal metabolic environment.

Specific recognition of the HIV-1 genomic RNA by the Gag precursor

During assembly of HIV-1 particles in infected cells, the viral Pr55(Gag) protein (or Gag precursor) must select the viral genomic RNA (gRNA) from a variety of cellular and viral spliced RNAs. However, there is no consensus on how Pr55(Gag) achieves this selection. Here, by using RNA binding and footprinting assays, we demonstrate that the primary Pr55(Gag) binding site on the gRNA consists of the internal loop and the lower part of stem-loop 1 (SL1), the upper part of which initiates gRNA dimerization. A double regulation ensures specific binding of Pr55(Gag) to the gRNA despite the fact that SL1 is also present in spliced viral RNAs. The region upstream of SL1, which is present in all HIV-1 RNAs, prevents binding to SL1, but this negative effect is counteracted by sequences downstream of SL4, which are unique to the gRNA.

Functional genomic analysis of systemic cell division regulation in legumes

Legumes develop root nodules from pluripotent stem cells in the rootpericycle in response to mitogenic activation by a decorated chitin-likenodulation factor synthesized in Rhizobium bacteria. The soybean genes encoding the receptor for such signals were cloned using map-based cloning approaches. Pluripotent cells in the root pericycle and the outer or inner cortex undergo repeated cell divisions to initiate a composite nodule primordium that develops to a functional nitrogen-fixing nodule. The process itself is autoregulated, leading to the characteristic nodulation of the upper root system. Autoregulation of nodulation (AON) in all legumes is controlled in part by a leucine-rich repeat receptor kinase gene (GmNARK). Mutations of GmNARK, and its other legume orthologues, result in abundant nodulation caused by the loss of a yet-undefined negative nodulation repressor system. AON receptor kinases are involved in perception of a long distance, root-derived signal, to negatively control nodule proliferation. GmNARK and LjHAR1 are expressed in phloem parenchyma. GmNARK kinase domain interacts with Kinase Associated Protein Phosphatase (KAPP). NARK gene expression did not mirror biological NARK activity in nodulation control, as q-RT-PCR in soybean revealed high NARK expression in roots, root tips, leaves, petioles, stems and hypocotyls, while shoot and root apical meristems were devoid of NARK RNA. High through-put transcript analysis in soybean leaf and root indicated that major genes involved in JA synthesis or response are preferentially down-regulated in leaf but not root of wild type, but not NARK mutants, suggesting that AON signaling may in part be controlled by events relating to hormone metabolism. Ethylene and abscisic acid insensitive mutants of L. japonicus are described. Nodulation in legumes has significance to global economies and ecologies, as the nitrogen input into the biosphere allows food, feed and biofuel production without the inherent costs associated with nitrogen fertilization [1]. Nodulation involves the production of a new organ capable of nitrogen fixation [2] and as such is an excellent system to study plant – microbe interaction, plant development, long distance signaling and functional genomics of stem cell proliferation [3, 4]. Concerted international effort over the last 20 years, using a combination of induced mutagenesis followed by gene discovery (forward genetics), and molecular/biochemical approaches revealed a complex developmental pathway that ‘loans’ genetic programs from various sources and orchestrates these into a novel contribution. We report our laboratory’s contribution to the present analysis in the field.

A comparison of collection techniques for gene expression analysis of human oral taste tissue

Variability in human taste perception is associated with both genetic and environmental factors. The influence of taste receptor expression on this variability is unknown, in part, due to the difficulty in obtaining human oral tissue that enables quantitative expression measures of taste genes. In a comparison of six current techniques (Oragene RNeasy Kit, Isohelix swab, Livibrush cytobrush, tongue saliva, cheek saliva collection, and fungiform papillae biopsy), we identify the fungiform papillae biopsy is the optimal sampling technique to analyse human taste gene expression. The fungiform papillae biopsy resulted in the highest RNA integrity, enabling amplification of all the assessed taste receptor genes (TAS1R1, TAS1R2, TAS1R3, SCNN1A and CD36) and taste tissue marker genes (NCAM1, GNAT3 and PLCβ2). Furthermore, quantitative expression was observed in a subset of taste genes assessed from the saliva collection techniques (cheek saliva, tongue saliva and Oragene RNA kit). These saliva collection techniques may be useful as a non-invasive alternative sampling technique to the fungiform papillae biopsy. Identification of the fungiform papillae biopsy as the optimal collection method will facilitate further research into understanding the effect of gene expression on variability in human taste perception.

An association rule analysis framework for complex physiological and genetic data

Physiological and genetic information has been critical to the successful diagnosis and prognosis of complex diseases. In this paper, we introduce a support-confidence-correlation framework to accurately discover truly meaningful and interesting association rules between complex physiological and genetic data for disease factor analysis, such as type II diabetes (T2DM). We propose a novel Multivariate and Multidimensional Association Rule mining system based on Change Detection (MMARCD). Given a complex data set u i (e.g. u 1 numerical data streams, u 2 images, u 3 videos, u 4 DNA/RNA sequences) observed at each time tick t, MMARCD incrementally finds correlations and hidden variables that summarise the key relationships across the entire system. Based upon MMARCD, we are able to construct a correlation network for human diseases. © 2012 Springer-Verlag.

Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing

In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization.