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Investigation of tissue-specific human orthologous alternative splice events in pig.AbstractAlternative splicing of pre-mRNA can contribute to differences between tissues or cells either by regulating gene expression or creating proteins with various functions encoded by one gene. The number of investigated alternative splice events in pig has so far been limited. In this study we have investigated alternative splice events detected in humans, in orthologous pig genes. A total of 17 genes with predicted exon skipping events were selected for further studies. The splice events for the selected genes were experimentally verified using real-time quantitative PCR analysis (qPCR) with splice-specific primers in 19 different tissues. The same splice variants as reported in humans were detected in 15 orthologous pig genes, however, the expression pattern predicted in the in silico analyses was only experimentally verified in a few cases. The results support the findings that splice events resulting in preservation of open reading frame are indicative of a functional significance of the splice variants of the gene.Microarray bioinformatics.AbstractBioinformatics has become an increasingly important tool for molecular biologists, especially for the analysis of microarray data. Microarrays can produce vast amounts of information requiring a series of consecutive analyses to render the data interpretable. The direct output of microarrays cannot be directly interpreted to show differences in settings, conditions of samples, or time points. To make microarray experiments interpretable, it is necessary that a series of algorithms and approaches be applied. After normalization of generated data, which is necessary to make a comparison feasible, significance analysis, clustering of samples and biological compounds of interest and visualization are generally performed. This chapter will focus on providing a basic understanding of the generally approaches and algorithms currently employed in microarray bioinformatics.Expression of conjoined genes: another mechanism for gene regulation in eukaryotes.AbstractFrom the ENCODE project, it is realized that almost every base of the entire human genome is transcribed. One class of transcripts resulting from this arises from the conjoined gene, which is formed by combining the exons of two or more distinct (parent) genes lying on the same strand of a chromosome. Only a very limited number of such genes are known, and the definition and terminologies used for them are highly variable in the public databases. In this work, we have computationally identified and manually curated 751 conjoined genes (CGs) in the human genome that are supported by at least one mRNA or EST sequence available in the NCBI database. 353 representative CGs, of which 291 (82%) could be confirmed, were subjected to experimental validation using RT-PCR and sequencing methods. We speculate that these genes are arising out of novel functional requirements and are not merely artifacts of transcription, since more than 70% of them are conserved in other vertebrate genomes. The unique splicing patterns exhibited by CGs reveal their possible roles in protein evolution or gene regulation. Novel CGs, for which no transcript is available, could be identified in 80% of randomly selected potential CG forming regions, indicating that their formation is a routine process. Formation of CGs is not only limited to human, as we have also identified 270 CGs in mouse and 227 in drosophila using our approach. Additionally, we propose a novel mechanism for the formation of CGs. Finally, we developed a database, ConjoinG, which contains detailed information about all the CGs (800 in total) identified in the human genome. In summary, our findings reveal new insights about the functionality of CGs in terms of another possible mechanism for gene regulation and genomic evolution and the mechanism leading to their formation.MicroRNAs, cancer and cancer stem cells.AbstractMicroRNAs regulate self-renewal, differentiation, and division of cells via post-transcriptional gene silencing. Aberrant microRNA levels, specifically an overall downregulation, are present in many cancers, as compared to their normal tissue counterparts. Therefore, a potential therapeutic use of microRNAs is to correct these aberrant transcript levels involved in the signaling pathways of cancer. This review focuses on the current knowledge of microRNAs and their involvement with cancer cells and cancer stemcells. The methods currently being used to develop miRNA-based cancer therapeutics are examined, and the limitations halting further progress are also discussed.Published by Elsevier Ireland Ltd.Genome-wide identification of human microRNAs located in leukemia-associated genomic alterations.AbstractCytogenetic alterations, such as amplifications, deletions, or translocations, contribute to myeloid malignancies. MicroRNAs (miRNAs) have emerged as critical regulators of hematopoiesis and their aberrant expression has been associated with leukemia. Genomic regions containing sequence alterations and fragile sites in cancers are enriched with miRNAs, however the relevant miRNAs within these regions have not been evaluated on a global basis. Here we investigated miRNAs relevant to acute myeloid leukemia (AML) by; 1) mapping miRNAs within leukemia-associated genomic alterations in human AML cell lines by high-resolution genome arrays, and 2) evaluating absolute expression of these miRNAs by massively parallel small RNA sequencing. Seventy-seven percent (542/706) of miRNAs mapped to leukemia-associated copy-number alterations (CNA) in the cell lines, however, only 18% (99/542) of these miRNAs are expressed above background levels. As evidence that this subset of miRNAs is relevant to leukemia, we show that loss of two miRNAs identified in our analysis, miR-145 and miR-146a, results in leukemia in a mouse model. Small RNA sequencing identified 28 putative novel miRNAs, 18 of which map to leukemia-associated CNA. This detailed genomic and small RNA analysis of human leukemic cell lines points to a subset of miRNAs that may play a role in myeloid malignancies.Post-transcriptional gene-expression regulation by micro RNA (miRNA) network in renal disease.AbstractMicro RNAs (miRNAs) are a recently discovered class of small, non-coding RNAs with the function of post-transcriptional gene expression regulation. MiRNAs may function in networks, forming a complex relationship with diseases. Alterations of specific miRNA levels have significant correlation with diseases of divergent origin, such as diabetic or ischemic organ injury including nephropathy, and malignant diseases including renal tumors. After identification of disease-associated miRNAs, there are two options of influencing their tissue expression. The function of miRNAs can be inhibited by antisenseoligonucleotides (ASOs), which have been shown to silence specific miRNAs in vivo. Moreover, miRNA activity can be also mimicked or enhanced by delivering chemically synthesized miRNAs. Thus, modifying the expression of miRNAs is a potential future gene-therapeutic tool to influence posttranscriptional regulation of multiple genes in a single therapy. In this review we focus on key renal miRNAs with the aim of revealing the pathomechanisms of renal diseases. Nucleic acid therapy with oligonucleotides and short interfering RNA (siRNA) are under clinical evaluation presently. Similar therapeutic strategies, to influence miRNA function is also already under clinical investigation in RNA interference trials. We summarize here studies specifically aimed at the modification of miRNA expression. Research on the post-transcriptional regulation of gene expression by miRNA may reshape our understanding of renal pathophysiology and consequently may bring new diagnostic markers and therapeutic agents.Target-align: a tool for plant microRNA target identification.AbstractMOTIVATION: MicroRNAs (miRNAs) are important regulatory molecules. A critical step in elucidating miRNA function is identifying potential miRNA targets. However, few reliable tools have been developed for identifying miRNA targets in plants.RESULTS: Here, we developed a Smith-Waterman-like alignment tool in order to accurately predict miRNA targets. Dynamic programming was used to build a score matrix based on the complementarity of nucleotides in order to trace the optimal local alignments. Important parameters, such as maximum mismatches and maximum consecutive mismatches between miRNAs and their targets, were also used for filtering the optimal local alignments. Almost all of the parameters in this alignment tool can be adjusted by users. Compared to other target prediction tools, Target-align exhibits strong sensitivity and accuracy for identifying miRNA targets. More importantly, Target-align can identify multi-target sites as well potential for non-cleaved targets sites by change the default settings. Windows, web, and command-line versions were developed to better serve different users.。
