Pain sensitivity varies substantially among humans. A significant part of the human population develops chronic pain conditions that are characterized by heightened pain sensitivity. We identified three genetic variants (haplotypes) of the gene encoding catecholamine-O-methyltransferase (COMT) that we designated as low pain sensitivity (LPS), average pain sensitivity (APS) and high pain sensitivity (HPS). We show that these haplotypes encompass 96% of the human population, and five combinations of these haplotypes are strongly associated (P=0.0004) with variation in the sensitivity to experimental pain. The presence of even a single LPS haplotype diminishes, by as much as 2.3 times, the risk of developing myogenous temporomandibular joint disorder (TMD), a common musculoskeletal pain condition. The LPS haplotype produces much higher levels of COMT enzymatic activity when compared with the APS or HPS haplotypes. Inhibition of COMT in the rat results in a profound increase in pain sensitivity. Thus, COMT activity substantially influences pain sensitivity, and the three major haplotypes determine COMT activity in humans that inversely correlates with pain sensitivity and the risk of developing TMD.

Background: All archaeal and many bacterial genomes contain Clustered Regularly Interspaced Short Palindrome Repeats (CRISPR) and variable arrays of the CRISPR-associated (cas) genes that have been previously implicated in a novel form of DNA repair on the basis of comparative analysis of their protein product sequences. However, the proximity of CRISPR and cas genes strongly suggests that they have related functions which is hard to reconcile with the repair hypothesis.

Catechol-O-methyltransferase (COMT) is a key regulator of pain perception, cognitive function, and affective mood. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous position, code for differences in COMT enzymatic activity and are associated with pain sensitivity. Haplotypes divergent in synonymous changes exhibited the largest difference in COMT enzymatic activity, due to a reduced amount of translated protein. The major COMT haplotypes varied with respect to messenger RNA local stem-loop structures, such that the most stable structure was associated with the lowest protein levels and enzymatic activity. Site-directed mutagenesis that eliminated the stable structure restored the amount of translated protein. These data highlight the functional significance of synonymous variations and suggest the importance of haplotypes over single-nucleotide polymorphisms for analysis of genetic variations.

Small interfering RNAs (siRNAs) and genome-encoded microRNAs (miRNAs) silence genes via complementary interactions with mRNAs. With thousands of miRNA genes identified and genome sequences of diverse eukaryotes available for comparison, the opportunity emerges for insights into origin and evolution of RNA interference (RNAi). The miRNA repertoires of plants and animals appear to have evolved independently. However, conservation of the key proteins involved in RNAi suggests that the last common ancestor of modern eukaryotes possessed siRNAbased mechanisms. Prokaryotes have a RNAi-like defense system that is functionally analogous but not homologous to eukaryotic RNAi. The protein machinery of eukaryotic RNAi seems to have been pieced together from ancestral proteins of archaeal, bacterial and phage origins that are involved in DNA repair and RNA-processing pathways. The miRNA and siRNA machineryRecent transcriptome analyses have shown that most of the eukaryotic genome is transcribed [1,2], and the genomes of all cellular life forms, in addition to protein-coding genes, contain varying numbers of non-protein-coding RNA [3,4]. MicroRNAs (miRNAs) are an abundant class of small (21-22 nucleotides) non-protein-coding RNAs that regulate translation in eukaryotes [5]. MiRNAs are key components of a major, evolutionarily conserved system of gene regulation in plants and animals that typically post-transcriptionally down-regulates gene expression either by inducing degradation of the target mRNAs, or by blocking their translation [6,7]. MiRNA-mediated pathways belong to a vast network of regulatory systems known as RNA interference (RNAi) [4,8]. RNAi consists of three major branches. These branches are small interfering (si)RNA-mediated pathways, miRNA-based pathways that are involved, respectively, in defense against viruses and transposable elements and in regulation of eukaryotic gene expression, and piwi-interacting RNA (piRNA) pathway that appears to be mechanistically distinct from the other two pathways [9].There are two key differences between miRNA and siRNA-mediated systems:i. miRNAs are endogenous non-protein-coding RNA molecules that are encoded by their own, distinct genes; by contrast, there are no dedicated genes for siRNAs. Instead, siRNAs are either generated by degradation of exogenous (e.g., viral) dsRNAs or transcribed from transposable elements integrated in the genome, or from other types of inverted repeats;ii. siRNAs are fully complementary to their targets, whereas miRNAs, at least in animals, show limited complementarity to their recognition sites.Although the structures and mechanisms of animal and plant miRNAs differ substantially, the same or homologous key proteins are involved both in miRNA biogenesis and in the siRNA pathways, suggesting that animal and plant miRNAs derive from the same, ancestral proto-RNAi system, but subsequently evolved along widely different trajectories so that the extant repertoires of miRNAs are unrelated. Prokaryotes have no RNAi systems homologous to the ...

Small, hydrophobic proteins whose synthesis is repressed by small RNAs (sRNAs), denoted type I toxin–antitoxin modules, were first discovered on plasmids where they regulate plasmid stability, but were subsequently found on a few bacterial chromosomes. We used exhaustive PSI-BLAST and TBLASTN searches across 774 bacterial genomes to identify homologs of known type I toxins. These searches substantially expanded the collection of predicted type I toxins, revealed homology of the Ldr and Fst toxins, and suggested that type I toxin–antitoxin loci are not spread by horizontal gene transfer. To discover novel type I toxin–antitoxin systems, we developed a set of search parameters based on characteristics of known loci including the presence of tandem repeats and clusters of charged and bulky amino acids at the C-termini of short proteins containing predicted transmembrane regions. We detected sRNAs for three predicted toxins from enterohemorrhagic Escherichia coli and Bacillus subtilis, and showed that two of the respective proteins indeed are toxic when overexpressed. We also demonstrated that the local free-energy minima of RNA folding can be used to detect the positions of the sRNA genes. Our results suggest that type I toxin–antitoxin modules are much more widely distributed among bacteria than previously appreciated.

In enteric bacteria, the transcription factor s E maintains membrane homeostasis by inducing synthesis of proteins involved in membrane repair and two small regulatory RNAs (sRNAs) that down-regulate synthesis of abundant membrane porins. Here, we describe the discovery of a third s E -dependent sRNA, MicL (mRNA-interfering complementary RNA regulator of Lpp), transcribed from a promoter located within the coding sequence of the cutC gene. MicL is synthesized as a 308-nucleotide (nt) primary transcript that is processed to an 80-nt form. Both forms possess features typical of Hfq-binding sRNAs but surprisingly target only a single mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. We show that the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. We also report that s E activity is sensitive to Lpp abundance and that MicL and Lpp comprise a new s E regulatory loop that opposes membrane stress. Together MicA, RybB, and MicL allow s E to repress the synthesis of all abundant outer membrane proteins in response to stress.

Messenger RNA is a key component of an intricate regulatory network of its own. It accommodates numerous nucleotide signals that overlap protein coding sequences and are responsible for multiple levels of regulation and generation of biological complexity. A wealth of structural and regulatory information, which mRNA carries in addition to the encoded amino acid sequence, raises the question of how these signals and overlapping codes are delineated along non-synonymous and synonymous positions in protein coding regions, especially in eukaryotes. Silent or synonymous codon positions, which do not determine amino acid sequences of the encoded proteins, define mRNA secondary structure and stability and affect the rate of translation, folding and post-translational modifications of nascent polypeptides. The RNA level selection is acting on synonymous sites in both prokaryotes and eukaryotes and is more common than previously thought. Selection pressure on the coding gene regions follows three-nucleotide periodic pattern of nucleotide base-pairing in mRNA, which is imposed by the genetic code. Synonymous positions of the coding regions have a higher level of hybridization potential relative to non-synonymous positions, and are multifunctional in their regulatory and structural roles. Recent experimental evidence and analysis of mRNA structure and interspecies conservation suggest that there is an evolutionary tradeoff between selective pressure acting at the RNA and protein levels. Here we provide a comprehensive overview of the studies that define the role of silent positions in regulating RNA structure and processing that exert downstream effects on proteins and their functions.

The parameters of the spontaneous deleterious mutation process remain poorly known, despite their importance. Here, we report the results of a mutation accumulation experiment performed on panmictic populations of Drosophila melanogaster without any genetic manipulations. Two experimental populations were kept for 30 generations under relaxed natural selection. Each generation, 100 pairs were formed randomly, and every fecund pair contributed a son and a daughter to the next generation. Comparison with two controls, one cryopreserved and the other kept as the experimental populations but with long generation time, showed that the number of surviving offspring per female declined by 0.2% and 2.0% per generation under benign and harsh, competitive conditions, respectively. Thus, the mutational pressure on fitness may be strong and depends critically on the conditions under which fitness is assayed.The intensive spontaneous deleterious mutation process may be crucial for human genetics (1), conservation biology (2), maintenance of genetic variability at the molecular (3) and phenotypic (4) levels, and the evolution of reproduction (5). However, the relevant parameters are controversial, with evidence both for (6-10) and against (11-13) high genomic deleterious mutation rates in multicellular eukaryotes. The necessary data can be obtained by assaying the consequences of accumulation of mutations under relaxed selection. The results of mutation accumulation experiments (except in refs. 13 and 14, in which low mutation rates were reported) recently were questioned (12) because mutations accumulated and the fitness was measured in weak, genetically altered organisms, either deeply inbred or carrying balancer chromosomes, while adaptation in control populations, which would cause overestimation of the mutation rates, could not be ruled out.Here, we report the results of an experiment that is free from these problems. Four fitness-related traits were measured after 10, 20, and 30 generations of relaxed selection in two experimental panmictic populations of Drosophila melanogaster and, simultaneously, in two controls. Without selection, every generation the mean fitness in an outbred population is decremented by the mutational pressure Uh, and the average number of mutant alleles per genome is incremented by U, where U is the diploid genomic deleterious mutation rate and hs is the arithmetic mean of the product of dominance coefficient of a new heterozygous mutation h times coefficient of selection against a new homozygous mutation s. Our main objective was to estimate Uhs, and the rate of decline of the mean fitness is enough for this. In contrast, because the number of mutations in a genome cannot be assayed directly, measuring of U, h, and s separately also must involve a much more difficult estimate of the increase of the variance in fitness, and the unknown variance of deleterious effects among new mutations must be somehow taken into account (6, 12). If Uhs is indeed so low that relaxed selection causes no s...