An integrated DNA and RNA variant detector identifies a highly conserved three base exon in the MAP4K5 kinase locus.

RNA variants that emerge from editing and alternative splicing form important regulatory stages in protein signalling. In this report, we apply an integrated DNA and RNA variant detection workbench to define the range of RNA variants that deviate from the reference genome in a human melanoma cell model. The RNA variants can be grouped into (i) classic ADAR-like or APOBEC-like RNA editing events and (ii) multiple-nucleotide variants (MNVs) including three and six base pair in-frame non-canonical unmapped exons. We focus on validating representative genes of these classes. First, clustered non-synonymous RNA edits (A-I) in the CDK13 gene were validated by Sanger sequencing to confirm the integrity of the RNA variant detection workbench. Second, a highly conserved RNA variant in the MAP4K5 gene was detected that results most likely from the splicing of a non-canonical three-base exon. The two RNA variants produced from the MAP4K5 locus deviate from the genomic reference sequence and produce V569E or V569del isoform variants. Low doses of splicing inhibitors demonstrated that the MAP4K5-V569E variant emerges from an SF3B1-dependent splicing event. Mass spectrometry of the recombinant SBP-tagged MAP4K5V569E and MAP4K5V569del proteins pull-downs in transfected cell systems was used to identify the protein-protein interactions of these two MAP4K5 isoforms and propose possible functions. Together these data highlight the utility of this integrated DNA and RNA variant detection platform to detect RNA variants in cancer cells and support future analysis of RNA variant detection in cancer tissue.

The effects of p53 gene inactivation on mutant proteome expression in a human melanoma cell model.

BACKGROUND: The identification of mutated proteins in human cancer cells-termed proteogenomics, requires several technologically independent research methodologies including DNA variant identification, RNA sequencing, and mass spectrometry. Any one of these methodologies are not optimized for identifying potential mutated proteins and any one output fails to cover completely a specific landscape. METHODS: An isogenic melanoma cell with a p53-null genotype was created by CRISPR/CAS9 system to determine how p53 gene inactivation affects mutant proteome expression. A mutant peptide reference database was developed by comparing two distinct DNA and RNA variant detection platforms using these isogenic cells. Chemically fractionated tryptic peptides from lysates were processed using a TripleTOF 5600+ mass spectrometer and their spectra were identified against this mutant reference database. RESULTS: Approximately 190 mutated peptides were enriched in wt-p53 cells, 187 mutant peptides were enriched in p53-null cells, with an overlap of 147 mutated peptides. STRING analysis highlighted that the wt-p53 cell line was enriched for mutant protein pathways such as CDC5L and POLR1B, whilst the p53-null cell line was enriched for mutated proteins comprising EGF/YES, Ubiquitination, and RPL26/5 nodes. CONCLUSION: Our study produces a well annotated p53-dependent and p53-independent mutant proteome of a common melanoma cell line model. Coupled to the application of an integrated DNA and RNA variant detection platform (CLCbio) and software for identification of proteins (ProteinPilot), this pipeline can be used to detect high confident mutant proteins in cells. GENERAL SIGNIFICANCE: This pipeline forms a blueprint for identifying mutated proteins in diseased cell systems.

Analysis of venom sac constituents from the solitary, aculeate wasp Cerceris rybyensis.

Solitary aculeate wasps are abundant and diverse hymenopteran insects that disable prey using venom. The venom may possess neuromodulation, immunomodulatory, metabolic-modulatory and antimicrobial functions. Venom analysis of transcriptomes and proteomes has been previously performed in social and parasitoid wasp species. We develop methodologies including mass spectrometry-based shotgun proteomics to analyse the protein constituents from venom sacs of the solitary aculeate wasp Cerceris rybyensis. The venom sac constituents of C. rybyensis are discussed with respect to other wasp species.