Alexander J. Ritter1,3, Jolene M. Draper2,3, Christopher Vollmers1 and Jeremy R. Sanford2 1Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, California 95064, USA; 2Department of Molecular, Cell, and Developmental Biology and Center for Molecular Biology of RNA, University of California Santa Cruz, Santa Cruz, California 95064, USA

↵3 These authors contributed equally to this work.

Corresponding author: jsanfor2ucsc.edu Abstract

Alternative splicing (AS) alters the cis-regulatory landscape of mRNA isoforms, leading to transcripts with distinct localization, stability, and translational efficiency. To rigorously investigate mRNA isoform-specific ribosome association, we generated subcellular fractionation and sequencing (Frac-seq) libraries using both conventional short reads and long reads from human embryonic stem cells (ESCs) and neural progenitor cells (NPCs) derived from the same ESCs. We performed de novo transcriptome assembly from high-confidence long reads from cytosolic, monosomal, light, and heavy polyribosomal fractions and quantified their abundance using short reads from their respective subcellular fractions. Thousands of transcripts in each cell type exhibited association with particular subcellular fractions relative to the cytosol. Of the multi-isoform genes, 27% and 19% exhibited significant differential isoform sedimentation in ESCs and NPCs, respectively. Alternative promoter usage and internal exon skipping accounted for the majority of differences between isoforms from the same gene. Random forest classifiers implicated coding sequence (CDS) and untranslated region (UTR) lengths as important determinants of isoform-specific sedimentation profiles, and motif analyses reveal potential cell type–specific and subcellular fraction–associated RNA-binding protein signatures. Taken together, our data demonstrate that alternative mRNA processing within the CDS and UTRs impacts the translational control of mRNA isoforms during stem cell differentiation, and highlight the utility of using a novel long-read sequencing–based method to study translational control.

Footnotes

[Supplemental material is available for this article.]

Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.279170.124.

Freely available online through the Genome Research Open Access option.

Received February 20, 2024. Accepted May 21, 2024.