# Stage-Specific Alternative Polyadenylation During Human Neural Differentiation Revealed by Integrated Long- and Short-Read Sequencing

## TL;DR

This study reveals how alternative polyadenylation changes during human neural cell development, using advanced sequencing techniques to uncover new regulatory patterns.

## Contribution

The integration of long- and short-read sequencing provides a high-resolution map of APA events during neural differentiation, revealing novel transcripts and polyadenylation sites.

## Key findings

- Integration of ONT and Illumina sequencing identified 20,823 novel transcripts and 8,241 new poly(A) sites during neural differentiation.
- Stage-specific intronic APA events were observed in neural precursor cells, with SLC1A3 as an example.
- SOX11 was identified as a key APA-regulated transcript influencing neural differentiation.

## Abstract

The differentiation of pluripotent embryonic stem cells into specialized neural cells is a highly complex and precise process. Alternative polyadenylation (APA), a crucial post-transcriptional regulatory mechanism, plays a significant role in shaping gene expression. In this study, we integrated long- and short-read sequencing to profile the dynamic landscape of APA during early neural differentiation. We identified four distinct dynamic patterns of mRNA 3′ untranslated region (3′ UTR) length variation and uncovered widespread intronic polyadenylation events, particularly during the neural precursor cell (NPC) stage. Among these, SOX11 and SLC1A3 were highlighted as novel APA-regulated transcripts. By providing a high-resolution atlas of APA regulation, this work offers new insights into the mechanisms of neural lineage differentiation and supplies valuable molecular clues for exploring the etiology and potential therapeutic avenues of nervous system disorders.

Human embryonic stem cell (hESC) neural differentiation involves extensive APA; however, reliance on short-read sequencing in prior studies has offered only a limited view of its complexity and dynamic regulation. Here, we integrated Oxford Nanopore (ONT) long-read sequencing with Illumina short-read sequencing to systematically map the APA landscape during early hESC neural differentiation. Our hybrid approach uncovered remarkable transcriptomic complexity, identifying 20,823 novel transcripts and 8241 previously unannotated poly(A) sites (PASs). We characterized distinct dynamic patterns of 3′ UTR-APA across differentiation and pinpointed SOX11 as a key APA-regulated target. Furthermore, we observed stage-specific enrichment of intronic APA in NPCs, as exemplified by SLC1A3, and performed a comprehensive, large-scale identification of high-confidence exon APA events. These results substantially expand the catalog of PASs during human neural differentiation and provide new insights into how APA-mediated post-transcriptional regulation contributes to cell-fate decisions.