Identifying and Characterizing a Novel Protein Kinase STK35L1 and Deciphering Its Orthologs and Close-Homologs in Vertebrates

The human kinome containing 478 eukaryotic protein kinases has over 100 uncharacterized kinases with unknown substrates and biological functions. The Ser/Thr kinase 35 (STK35, Clik1) is a member of the NKF 4 (New Kinase Family 4) in the kinome with unknown substrates and biological functions. Various high throughput studies indicate that STK35 could be involved in various human diseases such as colorectal cancer and malaria. In this study, we found that the previously published coding sequence of the STK35 gene is incomplete. The newly identified sequence of the STK35 gene codes for a protein of 534 amino acids with a N-terminal elongation of 133 amino acids. It has been designated as STK35L (STK35 long). Since it is the first of further homologous kinases we termed it as STK35L1. The STK35L1 protein (58 kDa on SDS-PAGE), but not STK35 (44 kDa), was found to be expressed in all human cells studied (endothelial cells, HeLa, and HEK cells) and was down-regulated after silencing with specific siRNA. EGFP-STK35L1 was localized in the nucleus and the nucleolus. By combining syntenic and gene structure pattern data and homology searches, two further STK35L1 homologs, STK35L2 (previously known as PDIK1L) and STK35L3, were found. All these protein kinase homologs were conserved throughout the vertebrates. The STK35L3 gene was specifically lost during placental mammalian evolution. Using comparative genomics, we have identified orthologous sets of these three protein kinases genes and their possible ancestor gene in two sea squirt genomes. We found the full-length coding sequence of the STK35 gene and termed it as STK35L1. We identified a new third STK35-like gene, STK35L3, in vertebrates and a possible ancestor gene in sea squirt genome. This study will provide a comprehensive platform to explore the role of STK35L kinases in cell functions and human diseases

Candidates in Astroviruses, Seadornaviruses, Cytorhabdoviruses and Coronaviruses for +1 frame overlapping genes accessed by leaky scanning

<p>Abstract</p> <p>Background</p> <p>Overlapping genes are common in RNA viruses where they serve as a mechanism to optimize the coding potential of compact genomes. However, annotation of overlapping genes can be difficult using conventional gene-finding software. Recently we have been using a number of complementary approaches to systematically identify previously undetected overlapping genes in RNA virus genomes. In this article we gather together a number of promising candidate new overlapping genes that may be of interest to the community.</p> <p>Results</p> <p>Overlapping gene predictions are presented for the astroviruses, seadornaviruses, cytorhabdoviruses and coronaviruses (families <it>Astroviridae</it>, <it>Reoviridae</it>, <it>Rhabdoviridae </it>and <it>Coronaviridae</it>, respectively).</p

Identification of novel conserved peptide uORF homology groups in Arabidopsis and rice reveals ancient eukaryotic origin of select groups and preferential association with transcription factor-encoding genes

Abstract Background Upstream open reading frames (uORFs) can mediate translational control over the largest, or major ORF (mORF) in response to starvation, polyamine concentrations, and sucrose concentrations. One plant uORF with conserved peptide sequences has been shown to exert this control in an amino acid sequence-dependent manner but generally it is not clear what kinds of genes are regulated, or how extensively this mechanism is invoked in a given genome. Results By comparing full-length cDNA sequences from Arabidopsis and rice we identified 26 distinct homology groups of conserved peptide uORFs, only three of which have been reported previously. Pairwise Ka/Ks analysis showed that purifying selection had acted on nearly all conserved peptide uORFs and their associated mORFs. Functions of predicted mORF proteins could be inferred for 16 homology groups and many of these proteins appear to have a regulatory function, including 6 transcription factors, 5 signal transduction factors, 3 developmental signal molecules, a homolog of translation initiation factor eIF5, and a RING finger protein. Transcription factors are clearly overrepresented in this data set when compared to the frequency calculated for the entire genome (p = 1.2 × 10-7). Duplicate gene pairs arising from a whole genome duplication (ohnologs) with a conserved uORF are much more likely to have been retained in Arabidopsis (Arabidopsis thaliana) than are ohnologs of other genes (39% vs 14% of ancestral genes, p = 5 × 10-3). Two uORF groups were found in animals, indicating an ancient origin of these putative regulatory elements. Conclusion Conservation of uORF amino acid sequence, association with homologous mORFs over long evolutionary time periods, preferential retention after whole genome duplications, and preferential association with mORFs coding for transcription factors suggest that the conserved peptide uORFs identified in this study are strong candidates for translational controllers of regulatory genes.</p

Expression Patterns of Protein Kinases Correlate with Gene Architecture and Evolutionary Rates

Protein kinase (PK) genes comprise the third largest superfamily that occupy ∼2% of the human genome. They encode regulatory enzymes that control a vast variety of cellular processes through phosphorylation of their protein substrates. Expression of PK genes is subject to complex transcriptional regulation which is not fully understood.Our comparative analysis demonstrates that genomic organization of regulatory PK genes differs from organization of other protein coding genes. PK genes occupy larger genomic loci, have longer introns, spacer regions, and encode larger proteins. The primary transcript length of PK genes, similar to other protein coding genes, inversely correlates with gene expression level and expression breadth, which is likely due to the necessity to reduce metabolic costs of transcription for abundant messages. On average, PK genes evolve slower than other protein coding genes. Breadth of PK expression negatively correlates with rate of non-synonymous substitutions in protein coding regions. This rate is lower for high expression and ubiquitous PKs, relative to low expression PKs, and correlates with divergence in untranslated regions. Conversely, rate of silent mutations is uniform in different PK groups, indicating that differing rates of non-synonymous substitutions reflect variations in selective pressure. Brain and testis employ a considerable number of tissue-specific PKs, indicating high complexity of phosphorylation-dependent regulatory network in these organs. There are considerable differences in genomic organization between PKs up-regulated in the testis and brain. PK genes up-regulated in the highly proliferative testicular tissue are fast evolving and small, with short introns and transcribed regions. In contrast, genes up-regulated in the minimally proliferative nervous tissue carry long introns, extended transcribed regions, and evolve slowly.PK genomic architecture, the size of gene functional domains and evolutionary rates correlate with the pattern of gene expression. Structure and evolutionary divergence of tissue-specific PK genes is related to the proliferative activity of the tissue where these genes are predominantly expressed. Our data provide evidence that physiological requirements for transcription intensity, ubiquitous expression, and tissue-specific regulation shape gene structure and affect rates of evolution

Haemonchus contortus Acetylcholine Receptors of the DEG-3 Subfamily and Their Role in Sensitivity to Monepantel

Gastro-intestinal nematodes in ruminants, especially Haemonchus contortus, are a global threat to sheep and cattle farming. The emergence of drug resistance, and even multi-drug resistance to the currently available classes of broad spectrum anthelmintics, further stresses the need for new drugs active against gastro-intestinal nematodes. A novel chemical class of synthetic anthelmintics, the Amino-Acetonitrile Derivatives (AADs), was recently discovered and the drug candidate AAD-1566 (monepantel) was chosen for further development. Studies with Caenorhabditis elegans suggested that the AADs act via nicotinic acetylcholine receptors (nAChR) of the nematode-specific DEG-3 subfamily. Here we identify nAChR genes of the DEG-3 subfamily from H. contortus and investigate their role in AAD sensitivity. Using a novel in vitro selection procedure, mutant H. contortus populations of reduced sensitivity to AAD-1566 were obtained. Sequencing of full-length nAChR coding sequences from AAD-susceptible H. contortus and their AAD-1566-mutant progeny revealed 2 genes to be affected. In the gene monepantel-1 (Hco-mptl-1, formerly named Hc-acr-23H), a panel of mutations was observed exclusively in the AAD-mutant nematodes, including deletions at intron-exon boundaries that result in mis-spliced transcripts and premature stop codons. In the gene Hco-des-2H, the same 135 bp insertion in the 5′ UTR created additional, out of frame start codons in 2 independent H. contortus AAD-mutants. Furthermore, the AAD mutants exhibited altered expression levels of the DEG-3 subfamily nAChR genes Hco-mptl-1, Hco-des-2H and Hco-deg-3H as quantified by real-time PCR. These results indicate that Hco-MPTL-1 and other nAChR subunits of the DEG-3 subfamily constitute a target for AAD action against H. contortus and that loss-of-function mutations in the corresponding genes may reduce the sensitivity to AADs

Insight into cross-talk between intra-amoebal pathogens

Abstract: Background: Amoebae are phagocytic protists where genetic exchanges might take place between amoeba-resistant bacteria. These amoebal pathogens are able to escape the phagocytic behaviour of their host. They belong to different bacterial phyla and often show a larger genome size than human-infecting pathogens. This characteristic is proposed to be the result of frequent gene exchanges with other bacteria that share a sympatric lifestyle and contrasts with the genome reduction observed among strict human pathogens.Results: We sequenced the genome of a new amoebal pathogen, Legionella drancourtii, and compared its gene content to that of a Chlamydia-related bacterium, Parachlamydia acanthamoebae. Phylogenetic reconstructions identified seven potential horizontal gene transfers (HGTs) between the two amoeba-resistant bacteria, including a complete operon of four genes that encodes an ABC-type transporter. These comparisons pinpointed potential cases of gene exchange between P. acanthamoebae and Legionella pneumophila, as well as gene exchanges between other members of the Legionellales and Chlamydiales orders. Moreover, nine cases represent possible HGTs between representatives from the Legionellales or Chlamydiales and members of the Rickettsiales order.Conclusions: This study identifies numerous gene exchanges between intracellular Legionellales and Chlamydiales bacteria, which could preferentially occur within common inclusions in their amoebal hosts. Therefore it contributes to improve our knowledge on the intra-amoebal gene properties associated to their specific lifestyle

Mutation of a Single Residue Renders Human Tetherin Resistant to HIV-1 Vpu-Mediated Depletion

The recently identified restriction factor tetherin/BST-2/CD317 is an interferon-inducible trans-membrane protein that restricts HIV-1 particle release in the absence of the HIV-1 countermeasure viral protein U (Vpu). It is known that Tantalus monkey CV1 cells can be rendered non-permissive to HIV-1 release upon stimulation with type 1 interferon, despite the presence of Vpu, suggesting species-specific sensitivity of tetherin proteins to viral countermeasures such as Vpu. Here we demonstrate that Tantalus monkey tetherin restricts HIV-1 by nearly two orders of magnitude, but in contrast to human tetherin the Tantalus protein is insensitive to HIV-1 Vpu. We have investigated tetherin's sensitivity to Vpu using positive selection analyses, seeking evidence for evolutionary conflict between tetherin and viral countermeasures. We provide evidence that tetherin has undergone positive selection during primate evolution. Mutation of a single amino acid (showing evidence of positive selection) in the trans-membrane cap of human tetherin to that in Tantalus monkey (T45I) substantially impacts on sensitivity to HIV-1 Vpu, but not on antiviral activity. Finally, we provide evidence that cellular steady state levels of tetherin are substantially reduced by Vpu, and that the T45I mutation abrogates this effect. This study provides evidence that tetherin is important in protecting mammals against viral infection, and that the HIV-1 Vpu–mediated countermeasure is specifically adapted to act against human tetherin. It also emphasizes the power of selection analyses to illuminate the molecular details of host–virus interactions. This work suggests that tetherin binding agents might protect it from viral encoded countermeasures and thus make powerful antivirals

Conserved Secondary Structures in Aspergillus

Background: Recent evidence suggests that the number and variety of functional RNAs (ncRNAs as well as cis-acting RNA elements within mRNAs) is much higher than previously thought; thus, the ability to computationally predict and analyze RNAs has taken on new importance. We have computationally studied the secondary structures in an alignment of six Aspergillus genomes. Little is known about the RNAs present in this set of fungi, and this diverse set of genomes has an optimal level of sequence conservation for observing the correlated evolution of base-pairs seen in RNAs. Methodology/Principal Findings: We report the results of a whole-genome search for evolutionarily conserved secondary structures, as well as the results of clustering these predicted secondary structures by structural similarity. We find a total of 7450 predicted secondary structures, including a new predicted,60 bp long hairpin motif found primarily inside introns. We find no evidence for microRNAs. Different types of genomic regions are over-represented in different classes of predicted secondary structures. Exons contain the longest motifs (primarily long, branched hairpins), 59 UTRs primarily contain groupings of short hairpins located near the start codon, and 39 UTRs contain very little secondary structure compared to other regions. There is a large concentration of short hairpins just inside the boundaries of exons. The density of predicted intronic RNAs increases with the length of introns, and the density of predicted secondary structures within mRNA coding regions increases with the number of introns in a gene

Cloning of a gene (SR-A1), encoding for a new member of the human Ser/Arg-rich family of pre-mRNA splicing factors: overexpression in aggressive ovarian cancer

By using the positional cloning gene approach, we were able to identify a novel gene encoding for a serine/arginine-rich protein, which appears to be the human homologue of the rat A1 gene. We named this new gene SR-A1. Members of the SR family of proteins have been shown to interact with the C-terminal domain (CTD) of the large subunit of RNA polymerase II and participate in pre-mRNA splicing. We have localized the SR-A1 gene between the known genes IRF3 and RRAS on chromosome 19q13.3. The novel gene spans 16.7 kb of genomic sequence and it is formed of 11 exons and 10 intervening introns. The SR-A1 protein is composed of 1312 amino acids, with a molecular mass of 139.3 kDa and a theoretical isoelectric point of 9.31. The SR-A1 protein contains an SR-rich domain as well as a CTD-binding domain present only in a subset of SR-proteins. Through interactions with the pre-mRNA and the CTD domain of the Polymerase II, SR proteins have been shown to regulate alternative splicing. The SR-A1 gene is expressed in all tissues tested, with highest levels found in fetal brain and fetal liver. Our data suggest that this gene is overexpressed in a subset of ovarian cancers which are clinically more aggressive. Studies with the steroid hormone receptor-positive breast and prostate carcinoma cell lines ZR-75-1, BT-474 and LNCaP, respectively, suggest that SR-A1 is constitutively expressed. Furthermore, the mRNA of the SR-A1 gene in these cell lines appears to increase by estrogens, androgens and glucocorticoids, and to a lesser extend by progestins. © 2001 Cancer Research Campaign http://www.bjcancer.co