Comparative analysis of sequence features involved in the recognition of tandem splice sites

<p>Abstract</p> <p>Background</p> <p>The splicing of pre-mRNAs is conspicuously often variable and produces multiple alternatively spliced (AS) isoforms that encode different messages from one gene locus. Computational studies uncovered a class of highly similar isoforms, which were related to tandem 5'-splice sites (5'ss) and 3'-splice sites (3'ss), yet with very sparse anecdotal evidence in experimental studies. To compare the types and levels of alternative tandem splice site exons occurring in different human organ systems and cell types, and to study known sequence features involved in the recognition and distinction of neighboring splice sites, we performed large-scale, stringent alignments of cDNA sequences and ESTs to the human and mouse genomes, followed by experimental validation.</p> <p>Results</p> <p>We analyzed alternative 5'ss exons (A5Es) and alternative 3'ss exons (A3Es), derived from transcript sequences that were aligned to assembled genome sequences to infer patterns of AS occurring in several thousands of genes. Comparing the levels of overlapping (tandem) and non-overlapping (competitive) A5Es and A3Es, a clear preference of isoforms was seen for tandem acceptors and donors, with four nucleotides and three to six nucleotides long exon extensions, respectively. A subset of inferred A5E tandem exons was selected and experimentally validated. With the focus on A5Es, we investigated their transcript coverage, sequence conservation and base-paring to U1 snRNA, proximal and distal splice site classification, candidate motifs for <it>cis</it>-regulatory activity, and compared A5Es with A3Es, constitutive and pseudo-exons, in <it>H. sapiens </it>and <it>M. musculus</it>. The results reveal a small but authentic enriched set of tandem splice site preference, with specific distances between proximal and distal 5'ss (3'ss), which showed a marked dichotomy between the levels of in- and out-of-frame splicing for A5Es and A3Es, respectively, identified a number of candidate NMD targets, and allowed a rough estimation of a number of undetected tandem donors based on splice site information.</p> <p>Conclusion</p> <p>This comparative study distinguishes tandem 5'ss and 3'ss, with three to six nucleotides long extensions, as having unusually high proportions of AS, experimentally validates tandem donors in a panel of different human tissues, highlights the dichotomy in the types of AS occurring at tandem splice sites, and elucidates that human alternative exons spliced at overlapping 5'ss posses features of typical splice variants that could well be beneficial for the cell.</p

Violating the splicing rules: TG dinucleotides function as alternative 3' splice sites in U2-dependent introns

TG dinucleotides functioning as alternative 3' splice sites were identified and experimentally verified in 36 human genes

The Mating Pattern of Captive Naked Mole-Rats Is Best Described by a Monogamy Model

Naked mole-rats form colonies with a single reproductively active female surrounded by subordinate workers. Workers perform offspring care, construction and defense of the burrow system, and food supply. Such division of labor, called “cooperative breeding,” is strongly associated with the evolution of monogamous mating behavior, as seen in several mammalian lineages. This association is explained by the evolutionary theory of kin selection, according to which a subordinate adult may help to raise other’s offspring if they are in full sibling relationship. In conflict with this theory, the naked mole-rat is widely considered to be polyandrous, based on reports on multiple males contributing to a colony’s progeny. In order to resolve this contrast, we undertook an in-depth microsatellite-based kinship analysis on captive colonies. Four independent colonies comprising a total of 265 animals were genotyped using a panel of 73 newly established microsatellite markers. Our results show that each mole-rat colony contains a single monogamous breeder pair, which translates to a reproductive skew of 100% for both sexes. This finding, also in conjunction with previously published parental data, favors monogamy as the best-fitting model to describe naked mole-rat reproduction patterns. Polyandry or other polygamous reproduction models are disfavored and should be considered as exceptional. Overall, the empirical genetic data are in agreement with the kin selection theory.Peer Reviewe

The Dictyostelium genome encodes numerous RasGEFs with multiple biological roles

BACKGROUND: Dictyostelium discoideum is a eukaryote with a simple lifestyle and a relatively small genome whose sequence has been fully determined. It is widely used for studies on cell signaling, movement and multicellular development. Ras guanine-nucleotide exchange factors (RasGEFs) are the proteins that activate Ras and thus lie near the top of many signaling pathways. They are particularly important for signaling in development and chemotaxis in many organisms, including Dictyostelium. RESULTS: We have searched the genome for sequences encoding RasGEFs. Despite its relative simplicity, we find that the Dictyostelium genome encodes at least 25 RasGEFs, with a few other genes encoding only parts of the RasGEF consensus domains. All appear to be expressed at some point in development. The 25 genes include a wide variety of domain structures, most of which have not been seen in other organisms. The LisH domain, which is associated with microtubule binding, is seen particularly frequently; other domains that confer interactions with the cytoskeleton are also common. Disruption of a sample of the novel genes reveals that many have clear phenotypes, including altered morphology and defects in chemotaxis, slug phototaxis and thermotaxis. CONCLUSION: These results suggest that the unexpectedly large number of RasGEF genes reflects an evolutionary expansion of the range of Ras signaling rather than functional redundancy or the presence of multiple pseudogenes

Polymorphic segmental duplications at 8p23.1 challenge the determination of individual defensin gene repertoires and the assembly of a contiguous human reference sequence

BACKGROUND: Defensins are important components of innate immunity to combat bacterial and viral infections, and can even elicit antitumor responses. Clusters of defensin (DEF) genes are located in a 2 Mb range of the human chromosome 8p23.1. This DEF locus, however, represents one of the regions in the euchromatic part of the final human genome sequence which contains segmental duplications, and recalcitrant gaps indicating high structural dynamics. RESULTS: We find that inter- and intraindividual genetic variations within this locus prevent a correct automatic assembly of the human reference genome (NCBI Build 34) which currently even contains misassemblies. Manual clone-by-clone alignment and gene annotation as well as repeat and SNP/haplotype analyses result in an alternative alignment significantly improving the DEF locus representation. Our assembly better reflects the experimentally verified variability of DEF gene and DEF cluster copy numbers. It contains an additional DEF cluster which we propose to reside between two already known clusters. Furthermore, manual annotation revealed a novel DEF gene and several pseudogenes expanding the hitherto known DEF repertoire. Analyses of BAC and working draft sequences of the chimpanzee indicates that its DEF region is also complex as in humans and DEF genes and a cluster are multiplied. Comparative analysis of human and chimpanzee DEF genes identified differences affecting the protein structure. Whether this might contribute to differences in disease susceptibility between man and ape remains to be solved. For the determination of individual DEF gene repertoires we provide a molecular approach based on DEF haplotypes. CONCLUSIONS: Complexity and variability seem to be essential genomic features of the human DEF locus at 8p23.1 and provides an ongoing challenge for the best possible representation in the human reference sequence. Dissection of paralogous sequence variations, duplicon SNPs ans multisite variations as well as haplotypes by sequencing based methods is the way for future studies of interindividual DEF locus variability and its disease association

RNA Sequencing Reveals Differential Expression of Mitochondrial and Oxidation Reduction Genes in the Long-Lived Naked Mole-Rat When Compared to Mice

The naked mole-rat (Heterocephalus glaber) is a long-lived, cancer resistant rodent and there is a great interest in identifying the adaptations responsible for these and other of its unique traits. We employed RNA sequencing to compare liver gene expression profiles between naked mole-rats and wild-derived mice. Our results indicate that genes associated with oxidoreduction and mitochondria were expressed at higher relative levels in naked mole-rats. The largest effect is nearly 300-fold higher expression of epithelial cell adhesion molecule (Epcam), a tumour-associated protein. Also of interest are the protease inhibitor, alpha2-macroglobulin (A2m), and the mitochondrial complex II subunit Sdhc, both ageing-related genes found strongly over-expressed in the naked mole-rat. These results hint at possible candidates for specifying species differences in ageing and cancer, and in particular suggest complex alterations in mitochondrial and oxidation reduction pathways in the naked mole-rat. Our differential gene expression analysis obviated the need for a reference naked mole-rat genome by employing a combination of Illumina/Solexa and 454 platforms for transcriptome sequencing and assembling transcriptome contigs of the non-sequenced species. Overall, our work provides new research foci and methods for studying the naked mole-rat's fascinating characteristics

Tnfaip2/exoc3 ‐driven lipid metabolism is essential for stem cell differentiation and organ homeostasis

Abstract Lipid metabolism influences stem cell maintenance and differentiation but genetic factors that control these processes remain to be delineated. Here, we identify Tnfaip2 as an inhibitor of reprogramming of mouse fibroblasts into induced pluripotent stem cells. Tnfaip2 knockout impairs differentiation of embryonic stem cells (ESCs), and knockdown of the planarian para‐ortholog, Smed‐exoc3 , abrogates in vivo tissue homeostasis and regeneration—processes that are driven by somatic stem cells. When stimulated to differentiate, Tnfaip2 ‐deficient ESCs fail to induce synthesis of cellular triacylglycerol (TAG) and lipid droplets (LD) coinciding with reduced expression of vimentin ( Vim )—a known inducer of LD formation. Smed‐exoc3 depletion also causes a strong reduction of TAGs in planarians. The study shows that Tnfaip2 acts epistatically with and upstream of Vim in impairing cellular reprogramming. Supplementing palmitic acid (PA) and palmitoyl‐L‐carnitine (the mobilized form of PA) restores the differentiation capacity of Tnfaip2 ‐deficient ESCs and organ maintenance in Smed‐exoc3 ‐depleted planarians. Together, these results identify a novel role of Tnfaip2 and exoc3 in controlling lipid metabolism, which is essential for ESC differentiation and planarian organ maintenance