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.

中文翻译：

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长读长亚细胞分级分离和测序揭示了神经元分化过程中全长 mRNA 亚型的翻译命运


选择性剪接 （AS） 改变了 mRNA 亚型的顺式调节格局，导致转录本具有不同的定位、稳定性和翻译效率。为了严格研究 mRNA 亚型特异性核糖体关联，我们使用来自人胚胎干细胞 （ESC） 和神经祖细胞 （NPC） 的常规短读长生成了亚细胞分级分离和测序 （Frac-seq） 文库。我们从细胞溶质、单体、轻和重多核糖体组分的高置信度长读长中进行了从头转录组组装，并使用来自各自亚细胞组分的短读长定量了它们的丰度。每种细胞类型中的数千个转录本表现出与相对于胞质溶胶的特定亚细胞组分的关联。在多亚型基因中，27% 和 19% 分别在 ESCs 和 NPC 中表现出显著的差异亚型沉降。替代启动子使用和内部外显子跳跃是来自同一基因的亚型之间大部分差异的原因。随机森林分类器表明编码序列 （CDS） 和非翻译区 （UTR） 长度是亚型特异性沉降曲线的重要决定因素，基序分析揭示了潜在的细胞类型特异性和亚细胞组分相关的 RNA 结合蛋白特征。综上所述，我们的数据表明，CDS 和 UTR 中的替代 mRNA 加工会影响干细胞分化过程中 mRNA 亚型的翻译控制，并强调了使用基于长读长测序的新型方法研究翻译控制的实用性。