Efficient Detection of the Alternative Spliced Human Proteome Using Translatome Sequencing.

Area of Science:

• Molecular Biology
• Genomics
• Proteomics

Background:

• Alternative splicing (AS) generates diverse proteoforms, increasing genomic complexity.
• Identifying AS proteoforms is challenging due to similar sequences, incomplete databases, and limited mass spectrometry coverage.

Purpose of the Study:

• To develop and validate a full-length ribosome nascent-chain complex-bound mRNA sequencing (RNC-seq) strategy.
• To construct an comprehensive protein database of translating AS isoforms.
• To improve the identification of AS proteoforms using mass spectrometry.

Main Methods:

• Employed a full-length RNC-seq strategy using both short-read and long-read next-generation sequencing.
• Generated a protein database encompassing all translating AS isoforms.
• Utilized the RNC-seq-guided database to analyze mass spectrometry datasets.

Main Results:

• Short-read RNC-seq identified 15,289 genes and 15,906 AS isoforms in a human cell line.
• Long-read RNC-seq identified an additional 4,429 annotated and 4,525 novel AS isoforms.
• Mass spectrometry analysis, guided by the RNC-seq database, identified 6,766 annotated and 50 novel protein isoforms.

Conclusions:

• Full-length RNC-seq is a powerful approach for comprehensive proteome analysis of AS isoforms.
• This method significantly expands the discovery of known and novel protein isoforms.
• RNC-seq enhances the investigation of proteome complexity driven by alternative splicing.