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1.  Computational extraction of a neural molecular network through alternative splicing 
BMC Research Notes  2014;7:934.
Generally, the results of high throughput analyses contain information about gene expressions, and about exon expressions. Approximately 90% of primary protein-coding transcripts undergo alternative splicing in mammals. However, changes induced by alternative exons have not been properly analyzed for their impact on important molecular networks or their biological events. Even when alternative exons are identified, they are usually subjected to bioinformatics analysis in the same way as the gene ignoring the possibility of functionality change because of the alteration of domain caused by alternative exon. Here, we reveal an effective computational approach to explore an important molecular network based on potential changes of functionality induced by alternative exons obtained from our comprehensive analysis of neuronal cell differentiation.
From our previously identified 262 differentially alternatively spliced exons during neuronal cell differentiations, we extracted 241 sets that changed the amino acid sequences between the alternatively spliced sequences. Conserved domain searches indicated that annotated domain(s) were changed in 128 sets. We obtained 49 genes whose terms overlapped between domain description and gene annotation. Thus, these 49 genes have alternatively differentially spliced in exons that affect their main functions. We performed pathway analysis using these 49 genes and identified the EGFR (epidermal growth factor receptor) and mTOR (mammalian target of rapamycin) signaling pathway as being involved frequently. Recent studies reported that the mTOR pathway is associated with neuronal cell differentiation, vindicating that our approach extracted an important molecular network successfully.
Effective informatics approaches for exons should be more complex than those for genes, because changes in alternative exons affect protein functions via alterations of amino acid sequences and functional domains. Our method extracted alterations of functional domains and identified key alternative splicing events. We identified the EGFR and mTOR signaling pathway as the most affected pathway. The mTOR pathway is important for neuronal differentiation, suggesting that this in silico extraction of alternative splicing networks is useful. This preliminary analysis indicated that automated analysis of the effects of alternative splicing would provide a rich source of biologically relevant information.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-0500-7-934) contains supplementary material, which is available to authorized users.
PMCID: PMC4320441  PMID: 25523101
Comprehensive analysis; Neuronal differentiation; Alternative splicing
2.  A View of Pre-mRNA Splicing from RNase R Resistant RNAs 
During pre-mRNA splicing, exons in the primary transcript are precisely connected to generate an mRNA. Intron lariat RNAs are formed as by-products of this process. In addition, some exonic circular RNAs (circRNAs) may also result from exon skipping as by-products. Lariat RNAs and circRNAs are both RNase R resistant RNAs. RNase R is a strong 3' to 5' exoribonuclease, which efficiently degrades linear RNAs, such as mRNAs and rRNAs; therefore, the circular parts of lariat RNAs and the circRNAs can be segregated from eukaryotic total RNAs by their RNase R resistance. Thus, RNase R resistant RNAs could provide unexplored splicing information not available from mRNAs. Analyses of these RNAs identified repeating splicing phenomena, such as re-splicing of mature mRNAs and nested splicing. Moreover, circRNA might function as microRNA sponges. There is an enormous variety of endogenous circRNAs, which are generally synthesized in cells and tissues.
PMCID: PMC4100097  PMID: 24865493
pre-mRNA splicing; RNase R; circular RNA; multiexon skipping
3.  Alternative splicing produces structural and functional changes in CUGBP2 
BMC Biochemistry  2012;13:6.
CELF/Bruno-like proteins play multiple roles, including the regulation of alternative splicing and translation. These RNA-binding proteins contain two RNA recognition motif (RRM) domains at the N-terminus and another RRM at the C-terminus. CUGBP2 is a member of this family of proteins that possesses several alternatively spliced exons.
The present study investigated the expression of exon 14, which is an alternatively spliced exon and encodes the first half of the third RRM of CUGBP2. The ratio of exon 14 skipping product (R3δ) to its inclusion was reduced in neuronal cells induced from P19 cells and in the brain. Although full length CUGBP2 and the CUGBP2 R3δ isoforms showed a similar effect on the inclusion of the smooth muscle (SM) exon of the ACTN1 gene, these isoforms showed an opposite effect on the skipping of exon 11 in the insulin receptor gene. In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3δ isoform was flexible and did not form an RRM structure.
Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms. Alternative splicing of CUGBP2 exon 14 contributes to the regulation of the splicing of the insulin receptor. The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.
PMCID: PMC3368720  PMID: 22433174
4.  Comprehensive Analysis of Alternative Splicing and Functionality in Neuronal Differentiation of P19 Cells 
PLoS ONE  2011;6(2):e16880.
Alternative splicing, which produces multiple mRNAs from a single gene, occurs in most human genes and contributes to protein diversity. Many alternative isoforms are expressed in a spatio-temporal manner, and function in diverse processes, including in the neural system.
Methodology/Principal Findings
The purpose of the present study was to comprehensively investigate neural-splicing using P19 cells. GeneChip Exon Array analysis was performed using total RNAs purified from cells during neuronal cell differentiation. To efficiently and readily extract the alternative exon candidates, 9 filtering conditions were prepared, yielding 262 candidate exons (236 genes). Semiquantitative RT-PCR results in 30 randomly selected candidates suggested that 87% of the candidates were differentially alternatively spliced in neuronal cells compared to undifferentiated cells. Gene ontology and pathway analyses suggested that many of the candidate genes were associated with neural events. Together with 66 genes whose functions in neural cells or organs were reported previously, 47 candidate genes were found to be linked to 189 events in the gene-level profile of neural differentiation. By text-mining for the alternative isoform, distinct functions of the isoforms of 9 candidate genes indicated by the result of Exon Array were confirmed.
Alternative exons were successfully extracted. Results from the informatics analyses suggested that neural events were primarily governed by genes whose expression was increased and whose transcripts were differentially alternatively spliced in the neuronal cells. In addition to known functions in neural cells or organs, the uninvestigated alternative splicing events of 11 genes among 47 candidate genes suggested that cell cycle events are also potentially important. These genes may help researchers to differentiate the roles of alternative splicing in cell differentiation and cell proliferation.
PMCID: PMC3041816  PMID: 21365003
5.  ‘Protected DNA Probes’ capable of strong hybridization without removal of base protecting groups 
Nucleic Acids Research  2008;36(6):1952-1964.
We propose a new strategy called the ‘Protected DNA Probes (PDP) method’ in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac4C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac6az8c7A). It was found that PDP containing ac4C and ac6az8c7A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness.
PMCID: PMC2330233  PMID: 18272535

Results 1-5 (5)