Identification Of Alternative Splice Variants Research Articles

Identification of Alternative Splice Variants Using Unique Tryptic Peptide Sequences for Database Searches

Alternative splicing is a mechanism in eukaryotes by which different forms of mRNAs are generated from the same gene. Identification of alternative splice variants requires the identification of peptides specific for alternative splice forms. For this purpose, we generated a human database that contains only unique tryptic peptides specific for alternative splice forms from Swiss-Prot entries. Using this database allows an easy access to splice variant-specific peptide sequences that match to MS data. Furthermore, we combined this database without alternative splice variant-1-specific peptides with human Swiss-Prot. This combined database can be used as a general database for searching of LC-MS data. LC-MS data derived from in-solution digests of two different cell lines (LNCaP, HeLa) and phosphoproteomics studies were analyzed using these two databases. Several nonalternative splice variant-1-specific peptides were found in both cell lines, and some of them seemed to be cell-line-specific. Control and apoptotic phosphoproteomes from Jurkat T cells revealed several nonalternative splice variant-1-specific peptides, and some of them showed clear quantitative differences between the two states.

The Evolution of COP9 Signalosome in Unicellular and Multicellular Organisms.

The COP9 signalosome (CSN) is a highly conserved protein complex, recently being crystallized for human. In mammals and plants the COP9 complex consists of nine subunits, CSN 1–8 and CSNAP. The CSN regulates the activity of culling ring E3 ubiquitin and plays central roles in pleiotropy, cell cycle, and defense of pathogens. Despite the interesting and essential functions, a thorough analysis of the CSN subunits in evolutionary comparative perspective is missing. Here we compared 61 eukaryotic genomes including plants, animals, and yeasts genomes and show that the most conserved subunits of eukaryotes among the nine subunits are CSN2 and CSN5. This may indicate a strong evolutionary selection for these two subunits. Despite the strong conservation of the protein sequence, the genomic structures of the intron/exon boundaries indicate no conservation at genomic level. This suggests that the gene structure is exposed to a much less selection compared with the protein sequence. We also show the conservation of important active domains, such as PCI (proteasome lid-CSN-initiation factor) and MPN (MPR1/PAD1 amino-terminal). We identified novel exons and alternative splicing variants for all CSN subunits. This indicates another level of complexity of the CSN. Notably, most COP9-subunits were identified in all multicellular and unicellular eukaryotic organisms analyzed, but not in prokaryotes or archaeas. Thus, genes encoding CSN subunits present in all analyzed eukaryotes indicate the invention of the signalosome at the root of eukaryotes. The identification of alternative splice variants indicates possible “mini-complexes” or COP9 complexes with independent subunits containing potentially novel and not yet identified functions.

Identification of alternative splice variants in Aspergillus flavus through comparison of multiple tandem MS search algorithms

BackgroundDatabase searching is the most frequently used approach for automated peptide assignment and protein inference of tandem mass spectra. The results, however, depend on the sequences in target databases and on search algorithms. Recently by using an alternative splicing database, we identified more proteins than with the annotated proteins in Aspergillus flavus. In this study, we aimed at finding a greater number of eligible splice variants based on newly available transcript sequences and the latest genome annotation. The improved database was then used to compare four search algorithms: Mascot, OMSSA, X! Tandem, and InsPecT.ResultsThe updated alternative splicing database predicted 15833 putative protein variants, 61% more than the previous results. There was transcript evidence for 50% of the updated genes compared to the previous 35% coverage. Database searches were conducted using the same set of spectral data, search parameters, and protein database but with different algorithms. The false discovery rates of the peptide-spectrum matches were estimated < 2%. The numbers of the total identified proteins varied from 765 to 867 between algorithms. Whereas 42% (1651/3891) of peptide assignments were unanimous, the comparison showed that 51% (568/1114) of the RefSeq proteins and 15% (11/72) of the putative splice variants were inferred by all algorithms. 12 plausible isoforms were discovered by focusing on the consensus peptides which were detected by at least three different algorithms. The analysis found different conserved domains in two putative isoforms of UDP-galactose 4-epimerase.ConclusionsWe were able to detect dozens of new peptides using the improved alternative splicing database with the recently updated annotation of the A. flavus genome. Unlike the identifications of the peptides and the RefSeq proteins, large variations existed between the putative splice variants identified by different algorithms. 12 candidates of putative isoforms were reported based on the consensus peptide-spectrum matches. This suggests that applications of multiple search engines effectively reduced the possible false positive results and validated the protein identifications from tandem mass spectra using an alternative splicing database.

Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer

BackgroundMicroarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events.ResultsThe array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer.ConclusionsThis methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies.

Dissecting the role of endothelial SURVIVIN ΔEx3 in angiogenesis

AbstractThe identification of alternative splice variants of Survivin that possess distinct functions from those originally identified for the main Survivin isoform has greatly increased the complexity of our understanding of the role of Survivin in different cells. Previous functional studies of the Survivin splice variants have been performed almost exclusively in cancer cells. However, Survivin has increasingly been implicated in other normal physiologic and pathophysiologic processes, including angiogenesis. In this study, we dissect the involvement of Survivin ΔEx3 in angiogenesis. We show by confocal microscopy that a pool of endothelial Survivin ΔEx3 is localized to membrane ruffles. We also demonstrate that Survivin ΔEx3 is the Survivin splice variant responsible for modulating angiogenesis in vitro, in tube formation assays, and in vivo, in an in vivo angiogenesis assay. Our data indicate that Survivin ΔEx3 may regulate angiogenesis via several mechanisms including cell invasion, migration, and Rac1 activation. Our findings identify a novel pathway regulating angiogenesis through Survivin ΔEx3 and a novel mechanism for Rac1 activation during angiogenesis. In conclusion, our results provide new insights into the regulation of endothelial cell homeostasis and angiogenesis by the Survivin proteins.

Role of Survivin‐ exon3 in angiogenesis

The complexities of the Survivin family of proteins has greatly increased since the identification of alternative splice variants generated from the genomic locus that possess distinct functions from those originally identified for the main Survivin isoform. Previous studies on the functions of Survivin splice variants have been performed almost exclusively within cancer cells, however Survivin has increasingly been implicated in other physiological and pathophysiological processes, including angiogenesis. In this study we dissect the involvement of the Survivin family of proteins in the mediation of angiogenesis. We show by confocal microscopy that a pool of endothelial Survivin-Δexon3 is localized to membrane ruffles. We also demonstrate that Survivin-Δexon3 is the Survivin splice variant responsible for modulation of angiogenesis in vitro in tube formation assays and in vivo by direct in vivo angiogenesis assay. Our data indicate that Survivin-Δexon3 may regulate angiogenesis via several mechanisms including cell invasion, migration and Rac activation. Our findings identify a novel pathway regulating angiogenesis through Survivin-Δexon3 and a novel mechanism for Rac activation during angiogenesis. In conclusion, our results provide new insight into the regulation of endothelial cell homeostasis and angiogenesis by the Survivin family.

Identification of alternative splicing variants of the β subunit of human Ca 2+/calmodulin-dependent protein kinase II with different activities

The β subunit of human Ca 2+/calmodulin-dependent protein kinase II (β CaMKII) was identified by searching through an expressed sequence tag database and rapid amplification of cDNA 5′-ends and was assigned to chromosome 7. Reverse transcription-polymerase chain reaction and sequencing analysis identified at least five alternative splicing variants of β CaMKII (β, β6, βe, β′e, and β7) in brain and two of them (β6 and β7) were first detected in any species. When expressed in HEK 293 cells, the Ca 2+/calmodulin-dependent kinase activity of β7, the shortest variant, was much lower than that of either β (the longest one) or βe (the medium one), suggesting possible regulation of β CaMKII activity by alternative splicing.