Ribosome profiling at isoform level reveals evolutionary conserved impacts of differential splicing on the proteome

Marina Reixachs-Solé,Jorge Ruiz-Orera,M Mar Albà,Eduardo Eyras

doi:10.1038/s41467-020-15634-w

Abstract

The differential production of transcript isoforms from gene loci is a key cellular mechanism. Yet, its impact in protein production remains an open question. Here, we describe ORQAS (ORF quantification pipeline for alternative splicing), a pipeline for the translation quantification of individual transcript isoforms using ribosome-protected mRNA fragments (ribosome profiling). We find evidence of translation for 40–50% of the expressed isoforms in human and mouse, with 53% of the expressed genes having more than one translated isoform in human, and 33% in mouse. Differential splicing analysis revealed that about 40% of the splicing changes at RNA level are concordant with changes in translation. Furthermore, orthologous cassette exons between human and mouse preserve the directionality of the change, and are enriched in microexons in a comparison between glia and glioma. ORQAS leverages ribosome profiling to uncover a widespread and evolutionarily conserved impact of differential splicing on translation, particularly of microexon-containing isoforms.

Highlights

The differential production of transcript isoforms from gene loci is a key cellular mechanism
After the assignment of Ribo-seq reads to isoform-specific open-reading frames (ORFs), ORQAS only considers ORFs with at least ten Ribo-seq reads after pooling replicates, and with average RNA-seq abundance greater than 0.1 in transcript per million (TPM) units
We have described ORQAS, a method to obtain transcript abundance estimates at isoform level in ribosome space, to identify multiple protein products from a gene, and to investigate differential translation associated with alternative splicing and differential transcript usage between conditions

Summary

Introduction

The differential production of transcript isoforms from gene loci is a key cellular mechanism. Of particular interest are microexons, which can be as short as three nucleotides and carry out conserved neuronal-specific functions, and whose misregulation is linked to autism[22,23,24] Despite their involvement in protein–protein interactions[23,25], the detection of protein variation associated with differential microexon inclusion using proteomics is currently challenging. Sequencing of ribosome-protected RNA fragments, i.e., ribosome profiling, provides information on the messengers being translated in a cell It allows the identification of multiple translated open-reading frames (ORFs) in the same gene and the discovery of novel translated genes[26,27,28,29]. We describe ORQAS (ORF quantification pipeline for alternative splicing), a method to quantify translation abundance at individual transcript level from ribosome profiling by taking into account ribosome signal periodicity and uniformity per isoform. ORQAS provides a powerful strategy to study the impacts of differential RNA processing in translation

Methods

Results

Conclusion