Efficient Detection of the Alternative Spliced Human Proteome Using Translatome Sequencing

Abstract

Alternative splicing (AS) isoforms create numerous proteoforms, expanding the complexity of the genome. Highly similar sequences, incomplete reference databases and the insufficient sequence coverage of mass spectrometry limit the identification of AS proteoforms. Here, we demonstrated full-length translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) sequencing (RNC-seq) strategy to sequence the entire translating mRNA using next-generation sequencing, including short-read and long-read technologies, to construct a protein database containing all translating AS isoforms. Taking the advantage of read length, short-read RNC-seq identified up to 15,289 genes and 15,906 AS isoforms in a single human cell line, much more than the Ribo-seq. The single-molecule long-read RNC-seq supplemented 4,429 annotated AS isoforms that were not identified by short-read datasets, and 4,525 novel AS isoforms that were not included in the public databases. Using such RNC-seq-guided database, we identified 6,766 annotated protein isoforms and 50 novel protein isoforms in mass spectrometry datasets. These results demonstrated the potential of full-length RNC-seq in investigating the proteome of AS isoforms.

Single-gene knockout-coupled omics analysis identifies C9orf85 and CXorf38 as two uncharacterized human proteins associated with ZIP8 malfunction

“…Further research on uncharacterized proteins, especially those encoded by short-ORFs, showed that they were indeed involved in diverse biological processes, including but not limited to cell proliferation ( Galindo et al, 2007 ), DNA damage repair ( Slavoff et al, 2014 ), muscle activities ( Anderson et al, 2015 ; Nelson et al, 2016 ), cell signaling ( Chng et al, 2013 ), mRNA degradation ( D'Lima et al, 2017 ), and phagocytosis ( Pueyo et al, 2016 ). Therefore, uncharacterized proteins have attracted increasing attention in recent years, but systemic research on these proteins is still lacking ( Delcourt et al, 2018 ; Wu et al, 2022 ).…”

Next generation sequencing technologies to address aberrant mRNA translation in cancer

“…Using human cell lines, RNC-seq demonstrated approximately two times higher estimation of protein isoforms compared to ribo-seq for the same number of valid reads ( 49 ). Sequencing technologies enabling longer reads enhance the detection of known and novel isoforms ( 49 ).…”

“…Using human cell lines, RNC-seq demonstrated approximately two times higher estimation of protein isoforms compared to ribo-seq for the same number of valid reads ( 49 ). Sequencing technologies enabling longer reads enhance the detection of known and novel isoforms ( 49 ). RNC-seq holds promise for studying circular RNAs (circRNAs) and their role during translation ( 50 ), with applications in detecting circRNAs bound to ribosomes in glioblastoma.…”