Small regulatory RNAs in mammals

Mammalian cells harbor numerous small non-protein-coding RNAs, including small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), short interfering RNAs (siRNAs) and small double-stranded RNAs, which regulate gene expression at many levels including chromatin architecture, RNA editing, RNA stability, translation, and quite possibly transcription and splicing. These RNAs are processed by multistep pathways from the introns and exons of longer primary transcripts, including protein-coding transcripts. Most show distinctive temporal- and tissue-specific expression patterns in different tissues, including embryonal stem cells and the brain, and some are imprinted. Small RNAs control a wide range of developmental and physiological pathways in animals, including hematopoietic differentiation, adipocyte differentiation and insulin secretion in mammals, and have been shown to be perturbed in cancer and other diseases. The extent of transcription of non-coding sequences and the abundance of small RNAs suggests the existence of an extensive regulatory network on the basis of RNA signaling which may underpin the development and much of the phenotypic variation in mammals and other complex organisms and which may have different genetic signatures from sequences encoding proteins.

Genomic organization of human arylamine N-acetyltransferase Type I reveals alternative promoters that generate different 5'-UTR splice variants with altered translational activities

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NATI (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NATI is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5'non-coding region of NATI. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NATI expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9-11-fold in a luciferase reporter assay, while the other isoforrns were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NATI transcripts may contribute to the variation in NATI activity in vivo.

Prediction of protein B-factor profiles

The polypeptide backbones and side chains of proteins are constantly moving due to thermal motion and the kinetic energy of the atoms. The B-factors of protein crystal structures reflect the fluctuation of atoms about their average positions and provide important information about protein dynamics. Computational approaches to predict thermal motion are useful for analyzing the dynamic properties of proteins with unknown structures. In this article, we utilize a novel support vector regression (SVR) approach to predict the B-factor distribution (B-factor profile) of a protein from its sequence. We explore schemes for encoding sequences and various settings for the parameters used in SVR. Based on a large dataset of high-resolution proteins, our method predicts the B-factor distribution with a Pearson correlation coefficient (CC) of 0.53. In addition, our method predicts the B-factor profile with a CC of at least 0.56 for more than half of the proteins. Our method also performs well for classifying residues (rigid vs. flexible). For almost all predicted B-factor thresholds, prediction accuracies (percent of correctly predicted residues) are greater than 70%. These results exceed the best results of other sequence-based prediction methods. (C) 2005 Wiley-Liss, Inc.

RNAdb - a comprehensive mammalian noncoding RNA database

In recent years, there have been increasing numbers of transcripts identified that do not encode proteins, many of which are developmentally regulated and appear to have regulatory functions. Here, we describe the construction of a comprehensive mammalian noncoding RNA database (RNAdb) which contains over 800 unique experimentally studied noncoding RNAs (ncRNAs), including many associated with diseases and/or developmental processes. The database is available at http://research.imb.uq. edu.au/RNAdb and is searchable by many criteria. It includes microRNAs and snoRNAs, but not infrastructural RNAs, such as rRNAs and tRNAs, which are catalogued elsewhere. The database also includes over 1100 putative antisense ncRNAs and almost 20000 putative ncRNAs identified in high-quality murine and human cDNA libraries, with more to be added in the near future. Many of these RNAs are large, and many are spliced, some alternatively. The database will be useful as a foundation for the emerging field of RNomics and the characterization of the roles of ncRNAs in mammalian gene expression and regulation.

Mechanisms of acetohydroxyacid synthases

Acetohydroxyacid synthases are thiamin diphosphate- (ThDP-) dependent biosynthetic enzymes found in all autotrophic organisms. Over the past 4-5 years, their mechanisms have been clarified and illuminated by protein crystallography, engineered mutagenesis and detailed single-step kinetic analysis. Pairs of catalytic subunits form an intimate dimer containing two active sites, each of which lies across a dimer interface and involves both monomers. The ThDP adducts of pyruvate, acetaldehyde and the product acetohydroxyacids can be detected quantitatively after rapid quenching. Determination of the distribution of intermediates by NMR then makes it possible to calculate individual forward unimolecular rate constants. The enzyme is the target of several herbicides and structures of inhibitor-enzyme complexes explain the herbicide-enzyme interaction.

How an enzyme answers. multiple-choice questions

Acetohydroxyacid synthase (AHAS) is the first common enzyme in the pathway for the biosynthesis of branched-chain amino acids. Interest in the enzyme has escalated over the past 20 years since it was discovered that AHAS is the target of the sulfonylurea and imidazolinone herbicides. However, several questions regarding the reaction mechanism have remained unanswered, particularly the way in which AHAS I chooses' its second substrate. A new method for the detection of reaction intermediates enables calculation of the microscopic rate constants required to explain this phenomenon.

Suicide inhibition of acetohydroxyacid synthase by hydroxypyruvate

Acetohydroxyacid synthase (Ec 2.2.1.6) catalyses the thiamine diphosphate-dependent reaction between two molecules of pyruvate yielding 2-acetolactacte and CO2. The enzyme will also utilise hydroxypyruvate with a k(cat) value that is 12% of that observed with pyruvate. When hydroxypyruvate is the substrate, the enzyme undergoes progressive inactivation with kinetics that are characteristic of suicide inhibition. It is proposed that the dihydroxyethyl-thiamine diphosphate intermediate can expel a hydroxide ion forming an enol that rearranges to a bound acetyl group.

hnRNP A2, a potential ssDNA/RNA molecular adapter at the telomere

The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multi-tasking protein that acts in the cytoplasm and nucleus. We have explored the possibility that this protein is associated with telomeres and participates in their maintenance. Rat brain hnRNP A2 was shown to have two nucleic acid binding sites. In the presence of heparin one site binds single-stranded oligodeoxyribonucleotides irrespective of sequence but not the corresponding oligoribonucleotides. Both the hnRNP A2-binding cis-acting element for the cytoplasmic RNA trafficking element, A2RE, and the ssDNA telomere repeat match a consensus sequence for binding to a second sequence-specific site identified by mutational analysis. hnRNP A2 protected the telomeric repeat sequence, but not the complementary sequence, against DNase digestion: the glycine-rich domain was found to be necessary, but not sufficient, for protection. The N-terminal RRM (RNA recognition motif) and tandem RRMs of hnRNP A2 also bind the single-stranded, template-containing segment of telomerase RNA. hnRNP A2 colocalizes with telomeric chromatin in the subset of PML bodies that are a hallmark of ALT cells, reinforcing the evidence for hnRNPs having a role in telomere maintenance. Our results support a model in which hnRNP A2 acts as a molecular adapter between single-stranded telomeric repeats, or telomerase RNA, and another segment of ssDNA.

Development of an oligonucleotide-based SNP detection method on lateral flow strips using hexapet tages

Detection of point mutations or single nucleotide polymorphisms (SNPs) is important in relation to disease susceptibility or detection in pathogens of mutations determining drug resistance or host range. There is an emergent need for rapid detection methods amenable to point-of-care applications. The purpose of this study was to reduce to practice a novel method for SNP detection and to demonstrate that this technology can be used downstream of nucleic acid amplification. The authors used a model system to develop an oligonucleotide-based SNP detection system on nitrocellulose lateral flow strips. To optimize the assay they used cloned sequences of the herpes simplex virus-1 (HSV-1) DNA polymerase gene into which they introduced a point mutation. The assay system uses chimeric polymerase chain reaction (PCR) primers that incorporate hexameric repeat tags ("hexapet tags"). The chimeric sequences allow capture of amplified products to predefined positions on a lateral flow strip. These "hexapet" sequences have minimal cross-reactivity and allow specific hybridization-based capture of the PCR products at room temperature onto lateral flow strips that have been striped with complementary hexapet tags. The allele-specific amplification was carried out with both mutant and wild-type primer sets present in the PCR mix ("competitive" format). The resulting PCR products carried a hexapet tag that corresponded with either a wild-type or mutant sequence. The lateral flow strips are dropped into the PCR reaction tube, and mutant sequence and wild-type sequences diffuse along the strip and are captured at the corresponding position on the strip. A red line indicative of a positive reaction is visible after 1 minute. Unlike other systems that require separate reactions and strips for each target sequence, this system allows multiplex PCR reactions and multiplex detection on a single strip or other suitable substrates. Unambiguous visual discrimination of a point mutation under room temperature hybridization conditions was achieved with this model system in 10 minutes after PCR. The authors have developed a capture-based hybridization method for the detection and discrimination of HSV-1 DNA polymerase genes that contain a single nucleotide change. It has been demonstrated that the hexapet oligonucleotides can be adapted for hybridization on the lateral flow strip platform for discrimination of SNPs. This is the first step in demonstrating SNP detection on lateral flow using the hexapet oligonucleotide capture system. It is anticipated that this novel system can be widely used in point-of-care settings.

Recent advances in alcohol-induced adduct formation

This article presents the proceedings of a symposium presented at the ISBRA 12th World Congress on Biomedical Alcohol Research, held in Heidelberg/Mannheim, Germany, September 29 through October 2, 2004. The organizers of the symposium were Simon Worrall and Victor Preedy, and the symposium was chaired by Onni Niemelä and Geoffrey Thiele. The presentations scheduled for this symposium were (1) Adduct chemistry and mechanisms of adduct formation, by Thomas L. Freeman; (2) Malondialdehyde- acetaldehyde adducts: the 2004 update, by Geoffrey Thiele; (3) Adduct formation in the liver, by Simon Worrall; (4) Protein adducts in alcoholic cardiomyopathy, by Onni Niemelä; and (5) Alcoholic skeletal muscle myopathy: a role for protein adducts, by Victor R. Preedy.

A need for caution in the use of frontal analysis continuous capillary electrophoresis for the determination of ligand binding data

Attention is drawn to a need for caution in the determination of binding data for protein-polyelectrolyte interactions by frontal analysis continuous capillary electrophoresis (FACCE). Because the method is valid only for systems involving comigration of complex(es) and slower-migrating reactant, establishing conformity with that condition is clearly a prerequisite for its application. However, that requirement has not been tested in any published studies thus far. On the basis of calculated FACCE patterns, presented to illustrate features by which such comigration of complex(es) and slower-migrating reactant can be identified, the form of the published pattern for a P-lactoglobulin-poly(styrenesulfonate) mixture does not seem to signify the migration behavior required to justify its consideration in such terms. Additional experimental studies are therefore needed to ascertain the validity of FACCE as a means of determining binding data for the characterization of protein-polyelectrolyte interactions. (c) 2005 Elsevier Inc. All rights reserved.