SERpredict: Detection of tissue- or tumor-specific isoforms generated through exonization of transposable elements

Background: Transposed elements (TEs) are known to affect transcriptomes, because either new exons are generated from intronic transposed elements (this is called exonization), or the element inserts into the exon, leading to a new transcript. Several examples in the literature show that isoforms generated by an exonization are specific to a certain tissue (for example the heart muscle) or inflict a disease. Thus, exonizations can have negative effects for the transcriptome of an organism. Results: As we aimed at detecting other tissue- or tumor-specific isoforms in human and mouse genomes which were generated through exonization of a transposed element, we designed the automated analysis pipeline SERpredict (SER = Specific Exonized Retroelement) making use of Bayesian Statistics. With this pipeline, we found several genes in which a transposed element formed a tissue- or tumor-specific isoform. Conclusion: Our results show that SERpredict produces relevant results, demonstrating the importance of transposed elements in shaping both the human and the mouse transcriptomes. The effect of transposed elements on the human transcriptome is several times higher than the effect on the mouse transcriptome, due to the contribution of the primate-specific Alu element

Automatic detection of exonic splicing enhancers (ESEs) using SVMs

<p>Abstract</p> <p>Background</p> <p>Exonic splicing enhancers (ESEs) activate nearby splice sites and promote the inclusion (vs. exclusion) of exons in which they reside, while being a binding site for SR proteins. To study the impact of ESEs on alternative splicing it would be useful to have a possibility to detect them in exons. Identifying SR protein-binding sites in human DNA sequences by machine learning techniques is a formidable task, since the exon sequences are also constrained by their functional role in coding for proteins.</p> <p>Results</p> <p>The choice of training examples needed for machine learning approaches is difficult since there are only few exact locations of human ESEs described in the literature which could be considered as positive examples. Additionally, it is unclear which sequences are suitable as negative examples. Therefore, we developed a motif-oriented data-extraction method that extracts exon sequences around experimentally or theoretically determined ESE patterns. Positive examples are restricted by heuristics based on known properties of ESEs, e.g. location in the vicinity of a splice site, whereas negative examples are taken in the same way from the middle of long exons. We show that a suitably chosen SVM using optimized sequence kernels (e.g., combined oligo kernel) can extract meaningful properties from these training examples. Once the classifier is trained, every potential ESE sequence can be passed to the SVM for verification. Using SVMs with the combined oligo kernel yields a high accuracy of about 90 percent and well interpretable parameters.</p> <p>Conclusion</p> <p>The motif-oriented data-extraction method seems to produce consistent training and test data leading to good classification rates and thus allows verification of potential ESE motifs. The best results were obtained using an SVM with the combined oligo kernel, while oligo kernels with oligomers of a certain length could be used to extract relevant features.</p

Comparative analysis of transposed element insertion within human and mouse genomes reveals Alu's unique role in shaping the human transcriptome

Background: Transposed elements (TEs) have a substantial impact on mammalian evolution and are involved in numerous genetic diseases. We compared the impact of TEs on the human transcriptome and the mouse transcriptome. Results: We compiled a dataset of all TEs in the human and mouse genomes, identifying 3,932,058 and 3,122,416 TEs, respectively. We than extracted TEs located within human and mouse genes and, surprisingly, we found that 60% of TEs in both human and mouse are located in intronic sequences, even though introns comprise only 24% of the human genome. All TE families in both human and mouse can exonize. TE families that are shared between human and mouse exhibit the same percentage of TE exonization in the two species, but the exonization level of Alu, a primatespecific retroelement, is significantly greater than that of other TEs within the human genome, leading to a higher level of TE exonization in human than in mouse (1,824 exons compared with 506 exons, respectively). We detected a primate-specific mechanism for intron gain, in which Alu insertion into an exon creates a new intron located in the 3' untranslated region (termed 'intronization'). Finally, the insertion of TEs into the first and last exons of a gene is more frequent in human than in mouse, leading to longer exons in human. Conclusion: Our findings reveal many effects of TEs on these two transcriptomes. These effects are substantially greater in human than in mouse, which is due to the presence of Alu elements in human

New miRNAs cloned from neuroblastoma

Research articl

Off-target effects of siRNA specific for GFP

<p>Abstract</p> <p>Background</p> <p>Gene knock down by RNAi is a highly effective approach to silence gene expression in experimental as well as therapeutic settings. However, this widely used methodology entails serious pitfalls, especially concerning specificity of the RNAi molecules.</p> <p>Results</p> <p>We tested the most widely used control siRNA directed against <it>GFP </it>for off-target effects and found that it deregulates in addition to <it>GFP </it>a set of endogenous target genes. The off-target effects were dependent on the amount of <it>GFP </it>siRNA transfected and were detected in a variety of cell lines. Since the respective siRNA molecule specific for <it>GFP </it>is widely used as negative control for RNAi experiments, we studied the complete set of off-target genes of this molecule by genome-wide expression profiling. The detected modulated mRNAs had target sequences homologous to the siRNA as small as 8 basepairs in size. However, we found no restriction of sequence homology to 3'UTR of target genes.</p> <p>Conclusion</p> <p>We can show that even siRNAs without a physiological target have sequence-specific off-target effects in mammalian cells. Furthermore, our analysis defines the off-target genes affected by the siRNA that is commonly used as negative control and directed against <it>GFP</it>. Since off-target effects can hardly be avoided, the best strategy is to identify false positives and exclude them from the results. To this end, we provide the set of false positive genes deregulated by the commonly used <it>GFP </it>siRNA as a reference resource for future siRNA experiments.</p

De novo annotation of the transcriptome of the Northern Wheatear (Oenanthe oenanthe)

We have sequenced a partial transcriptome of the Northern Wheatear (Oenanthe oenanthe), a species with one of the longest migrations on Earth. The transcriptome was constructed de novo using RNA-Seq sequence data from the pooled mRNA of six different tissues: brain, muscle, intestine, liver, adipose tissue and skin. The samples came from nine captive-bred wheatears collected at three different stages of the endogenous autumn migratory period: (1) lean birds prior the onset of migration, (2) during the fattening stage and (3) individuals at their migratory body mass plateau, when they have almost doubled their lean body mass. The sample structure used to build up the transcriptome of the Northern Wheatears concerning tissue composition and time guarantees the future survey of the regulatory genes involved in the development of the migratory phenotype. Through the pre-migratory period, birds accomplish outstanding physical and behavioural changes that involve all organ systems. Nevertheless, the molecular mechanisms through which birds synchronize and control hyperphagia, fattening, restlessness increase, immunity boosting and tuning the muscles for such endurance flight are still largely unknown. The use of RNA-Seq has emerged as a powerful tool to analyse complex traits on a broad scale, and we believe it can help to characterize the migratory phenotype of wheatears at an unprecedented level. The primary challenge to conduct quantitative transcriptomic studies in non-model species is the availability of a reference transcriptome, which we have constructed and described in this paper. The cDNA was sequenced by pyrosequencing using the Genome Sequencer Roche GS FLX System; with single paired-end reads of about 400 bp. We estimate the total number of genes at 15,640, of which  67% could be annotated using Turkey and Zebra Finch genomes, or protein sequence information from SwissProt and NCBI databases. With our study, we have made a first step towards understanding the migratory phenotype regarding gene expression of a species that has become a model to study birds long-distance migrations

Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

BACKGROUND: Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. PRINCIPAL FINDINGS: Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. CONCLUSIONS: The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades

Functions, structure, and read-through alternative splicing of feline APOBEC3 genes

APOBEC3 (A3, Apolipoprotein B mRNA-editing catalytic polypeptide 3) genes in the genome of domestic cat (Felis catus) were identified and characterize