Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56,419 completely sequenced and manually annotated full-length cDNAs.

We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56,419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37,670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants.

Signal sequence and keyword trap in silico for selection of full-length human cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries.

We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5'-end single-pass sequences, cDNAs having the signal sequence were selected by PSORT ('signal sequence trap'). We also applied 'secretion or membrane protein-related keyword trap' based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165(80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by 'keyword trap' preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

Complete sequencing and characterization of 21,243 full-length human cDNAs.

As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

Adaptor protein Shc is an isoform-specific direct activator of the tyrosine kinase c-Src.

The activity of c-Src protein-tyrosine kinase is up-regulated under a number of receptor signaling pathways. However, the activation mechanism of c-Src under physiological conditions has remained unclear. We show here that the Shc adaptor protein is a novel direct activator of c-Src in epidermal growth factor receptor signaling in A431 human epidermoid carcinoma cells. Among the three Shc isoforms, P66 and P52, but not P46, were found to interact with and activate c-Src in vitro and in vivo. Activation of c-Src accompanied autophosphorylation of c-Src in the activation segment, but the carboxyl-terminal dephosphorylation was not observed. We have identified the interaction sites between Shc and c-Src and constructed a point mutant of Shc that abolishes the c-Src activation. Using this mutant, we have confirmed that the Shc-mediated c-Src activation triggers Stat-p21/WAF1/Cip1 pathway that has been implicated in the cell cycle arrest and apoptosis of epidermal growth factor-stimulated A431 cells.