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Identification of rare alternative splicing events in MS/MS data reveals a significant fraction of alternative translation initiation sites.

Kroll JE, de Souza SJ, de Souza GA - PeerJ (2014)

Bottom Line: Although the types of splicing variants (exon skipping, alternative splice sites and intron retention) were found at the same frequency as in the transcriptome, we observed a large variety of modifications at the protein level induced by alternative splicing events.Surprisingly, we found that 40% of all protein modifications induced by alternative splicing led to the use of alternative translation initiation sites.Other modifications include frameshifts in the open reading frame and inclusion or deletion of peptide sequences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Bioinformatics and Biotechnology , Natal , Brazil ; Brain Institute, UFRN , Natal , Brazil.

ABSTRACT
Integration of transcriptome data is a crucial step for the identification of rare protein variants in mass-spectrometry (MS) data with important consequences for all branches of biotechnology research. Here, we used Splooce, a database of splicing variants recently developed by us, to search MS data derived from a variety of human tumor cell lines. More than 800 new protein variants were identified whose corresponding MS spectra were specific to protein entries from Splooce. Although the types of splicing variants (exon skipping, alternative splice sites and intron retention) were found at the same frequency as in the transcriptome, we observed a large variety of modifications at the protein level induced by alternative splicing events. Surprisingly, we found that 40% of all protein modifications induced by alternative splicing led to the use of alternative translation initiation sites. Other modifications include frameshifts in the open reading frame and inclusion or deletion of peptide sequences. To make the dataset generated here available to the community in a more effective form, the Splooce portal (http://www.bioinformatics-brazil.org/splooce) was modified to report the alternative splicing events supported by MS data.

No MeSH data available.


Related in: MedlinePlus

Alignments between normal (Uniprot/RefSeq) and alternative (Splooce) proteins, showing different categories of alternative TIS observed for our data.Sequences highlighted in orange represent MS peptides found for the Uniprot/RefSeq proteins, and sequences highlighted in yellow represent peptides found exclusively in the alternative sequences from Splooce. Peptides that align specifically to a sequence from Splooce are supposed to characterize ASEs. (A) Alternative TIS is downstream the original one; (B) Same as A, although the beginning of the protein sequence is directly affected by the ASE. (C) Alternative TIS is upstream the original one.
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fig-3: Alignments between normal (Uniprot/RefSeq) and alternative (Splooce) proteins, showing different categories of alternative TIS observed for our data.Sequences highlighted in orange represent MS peptides found for the Uniprot/RefSeq proteins, and sequences highlighted in yellow represent peptides found exclusively in the alternative sequences from Splooce. Peptides that align specifically to a sequence from Splooce are supposed to characterize ASEs. (A) Alternative TIS is downstream the original one; (B) Same as A, although the beginning of the protein sequence is directly affected by the ASE. (C) Alternative TIS is upstream the original one.

Mentions: We further explored what types of events were observed in the identified peptides. Interestingly, 355 ASEs, out of the 892 (40%), showed a pattern consistent with the use of an alternative TIS due to an ASE (Fig. 3, File S2). The remaining 537 proteins showed different types of variations along their protein sequences (File S3). Files S2 and S3 not only contain a resumed version of the results described in this section, but also report protein sequence alignments for Uniprot and Splooce sequences of all proteins identified with a rare ASE. Peptides shared between both databases, in addition to the Splooce-specific peptide(s), are highlighted in the alignment. Most importantly, each alignment contains a link to the Splooce website where information and statistics for that rare ASE can be collected.


Identification of rare alternative splicing events in MS/MS data reveals a significant fraction of alternative translation initiation sites.

Kroll JE, de Souza SJ, de Souza GA - PeerJ (2014)

Alignments between normal (Uniprot/RefSeq) and alternative (Splooce) proteins, showing different categories of alternative TIS observed for our data.Sequences highlighted in orange represent MS peptides found for the Uniprot/RefSeq proteins, and sequences highlighted in yellow represent peptides found exclusively in the alternative sequences from Splooce. Peptides that align specifically to a sequence from Splooce are supposed to characterize ASEs. (A) Alternative TIS is downstream the original one; (B) Same as A, although the beginning of the protein sequence is directly affected by the ASE. (C) Alternative TIS is upstream the original one.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232841&req=5

fig-3: Alignments between normal (Uniprot/RefSeq) and alternative (Splooce) proteins, showing different categories of alternative TIS observed for our data.Sequences highlighted in orange represent MS peptides found for the Uniprot/RefSeq proteins, and sequences highlighted in yellow represent peptides found exclusively in the alternative sequences from Splooce. Peptides that align specifically to a sequence from Splooce are supposed to characterize ASEs. (A) Alternative TIS is downstream the original one; (B) Same as A, although the beginning of the protein sequence is directly affected by the ASE. (C) Alternative TIS is upstream the original one.
Mentions: We further explored what types of events were observed in the identified peptides. Interestingly, 355 ASEs, out of the 892 (40%), showed a pattern consistent with the use of an alternative TIS due to an ASE (Fig. 3, File S2). The remaining 537 proteins showed different types of variations along their protein sequences (File S3). Files S2 and S3 not only contain a resumed version of the results described in this section, but also report protein sequence alignments for Uniprot and Splooce sequences of all proteins identified with a rare ASE. Peptides shared between both databases, in addition to the Splooce-specific peptide(s), are highlighted in the alignment. Most importantly, each alignment contains a link to the Splooce website where information and statistics for that rare ASE can be collected.

Bottom Line: Although the types of splicing variants (exon skipping, alternative splice sites and intron retention) were found at the same frequency as in the transcriptome, we observed a large variety of modifications at the protein level induced by alternative splicing events.Surprisingly, we found that 40% of all protein modifications induced by alternative splicing led to the use of alternative translation initiation sites.Other modifications include frameshifts in the open reading frame and inclusion or deletion of peptide sequences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Bioinformatics and Biotechnology , Natal , Brazil ; Brain Institute, UFRN , Natal , Brazil.

ABSTRACT
Integration of transcriptome data is a crucial step for the identification of rare protein variants in mass-spectrometry (MS) data with important consequences for all branches of biotechnology research. Here, we used Splooce, a database of splicing variants recently developed by us, to search MS data derived from a variety of human tumor cell lines. More than 800 new protein variants were identified whose corresponding MS spectra were specific to protein entries from Splooce. Although the types of splicing variants (exon skipping, alternative splice sites and intron retention) were found at the same frequency as in the transcriptome, we observed a large variety of modifications at the protein level induced by alternative splicing events. Surprisingly, we found that 40% of all protein modifications induced by alternative splicing led to the use of alternative translation initiation sites. Other modifications include frameshifts in the open reading frame and inclusion or deletion of peptide sequences. To make the dataset generated here available to the community in a more effective form, the Splooce portal (http://www.bioinformatics-brazil.org/splooce) was modified to report the alternative splicing events supported by MS data.

No MeSH data available.


Related in: MedlinePlus