Long-read subcellular fractionation and sequencing reveals the translational fate of full-length mRNA isoforms during neuronal differentiation.

Autor:	Ritter AJ; Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, California 95064, USA., Draper JM; Department of Molecular, Cell, and Developmental Biology and Center for Molecular Biology of RNA, University of California Santa Cruz, Santa Cruz, California 95064, USA., Vollmers C; Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, California 95064, USA., Sanford JR; Department of Molecular, Cell, and Developmental Biology and Center for Molecular Biology of RNA, University of California Santa Cruz, Santa Cruz, California 95064, USA jsanfor2@ucsc.edu.
Jazyk:	angličtina
Zdroj:	Genome research [Genome Res] 2024 Nov 20; Vol. 34 (11), pp. 2000-2011. Date of Electronic Publication: 2024 Nov 20.
DOI:	10.1101/gr.279170.124
Abstrakt:	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. (© 2024 Ritter et al.; Published by Cold Spring Harbor Laboratory Press.)
Databáze:	MEDLINE
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