Nanopore full-length sequencing reveals Nudt21 knockdown drives genome-wide 3'UTR shortening and transcriptome...

Alternative polyadenylation (APA) fine-tunes gene expression at the post-transcriptional level by generating transcript isoforms with distinct 3' untranslated region (3'UTR) lengths. Nudix hydrolase 21 (Nudt21), a core subunit of the cleavage factor Im (CFIm) complex, is a key regulator of APA; however, its function in normal hepatocytes remains poorly characterized. This study aims to elucidate the regulatory role of Nudt21 in shaping APA landscapes and gene expression in normal hepatocytes.

Knockdown of Nudt21 in the normal mouse hepatocyte cell line AML12 was performed, followed by full-length transcriptome sequencing using Nanopore Tail ISO-seq technology, which systematically mapped poly(A) site selection and tail length dynamics at single-molecule resolution. Integrative analysis revealed that Nudt21 knockdown led to differential expression of 1,386 genes and triggered genome-wide remodeling of APA patterns: 2,864 genes exhibited significant APA changes, among which 2,682 (93.6%) showed 3'UTR shortening and only 182 (6.4%) showed lengthening. Genes with 3'UTR shortening were significantly enriched in pathways related to cell cycle, protein transport and degradation, and RNA processing. Among the genes that exhibited both 3'UTR shortening and differential expression, the numbers of upregulated and downregulated genes were similar, suggesting that 3'UTR shortening exerts bidirectional regulatory effects on gene expression and also indicating that APA remodeling is an important mechanism driving transcriptomic reprogramming.

This study provides the first systematic dissection of Nudt21-mediated maintenance of 3'UTR homeostasis in normal hepatocytes, demonstrating its role in modulating gene expression through APA regulation. These findings offer a valuable resource and novel insights into the contribution of APA dysregulation to liver physiology and pathology.