The impact of Hnrnpl deficiency on transcriptional patterns of developing muscle cells

Sep 29, 2026

This research article investigates how Hnrnpl deficiency affects gene expression and splicing during muscle development. The study uses nanopore long-read transcriptome sequencing and qPCR, examining hnRrnpl knockdown in myoblasts and smooth ortholog knockdown in Drosophila. Results show dysregulation of Notch signaling and muscle-related genes, alongside measurable splicing changes in genes such as Lamp2, Fhl1, and Dtna, with opposite trends for Dtna isoforms a-DB1 and a-DB3. A minimum nanopore read depth of 10 helps detect splicing differences above 10% to 20%, supporting further evaluation and potential therapeutic relevance.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages21

Reading Time53 min

AccessFree to download

Introduction

HnRNP L and gene regulation

Links to disease and muscle development

Methods and Experimental Design

Knockdown models in myoblasts and Drosophila

Long-read transcriptome sequencing and qPCR

Results

Dysregulation of Notch and muscle-related genes

Altered splicing patterns and Dtna isoforms

Read-depth requirements for detecting splicing changes

Discussion and Future Directions

Validation across genes, cell types, and conditions

Potential therapeutic implications

RESEARCH ARTICLE The impact of Hnrnpl deﬁciency on transcriptional patterns of developing muscle cells Hannah R. Littel 1, Mekala Gunasekaran 1, Audrey L. Daugherty 1 , Natalya M. Wells 1, Johnnie Turner 1, Christine C. Bruels 1, Christina A. Pacak 1 , Isabelle Draper2 and Peter B. Kang 1,3 1 Greg Marzolf Jr. Muscular Dystrophy Center and Department of Neurology, University of Minnesota Medical School, Minneapolis, MN, USA 2 Molecular Cardiology Research Institute, Tufts Medical Center, Boston, MA, USA 3 Institute for Translational Neuroscience, University of Minnesota, Minneapolis, MN, USA Keywords HnRNP L; nanopore transcriptomics; RNA sequencing; skeletal muscle; splicing regulation Correspondence P. B. Kang, Department of Neurology, University of Minnesota Medical School, 420 Delaware St SE, MMC 295, Minneapolis, MN 55455, USA E-mail: [pkang@umn.edu](pkang@umn.edu) (Received 15 April 2025, revised 13 July 2025, accepted 28 August 2025) doi:10.1002/2211-5463.70117 Heterogeneous nuclear ribonucleoproteins (hnRNPs) bind to RNA, regulating gene expression and splicing. HnRNP L contributes to muscle development and the pathogenesis of myotonic dystrophy. We hypothesized that hnRNP L regulates muscle expression and splicing patterns. Using nanopore long-read transcriptome sequencing and qPCR analyses, we investigated the impact of Hnrnpl knockdown on myoblasts and knockdown of the orthologous gene smooth in Drosophila. Notch signaling genes and muscle-related genes were dysregulated in both models. Several genes had altered splicing patterns, including Lamp2, Fhl1, and Dtna. The a-DB1 isoform of Dtna was downregulated, whereas the a-DB3 isoform was upregulated. Our ﬁndings indicate that hnRNP L regulates both the transcription levels and splicing patterns of genes relevant to skeletal muscle development. We demonstrate the capabilities of long-read transcriptome sequencing to study muscle development. Comparisons between nanopore long-read transcriptome sequencing and data from PCR and qPCR analyses suggest that a minimum read depth of 10 is needed on nanopore sequencing to detect splicing differences greater than 10% to 20% . Future studies could determine whether the minimum read depth that we identiﬁed in our model is valid across a broader range of genes, cell types, and conditions. There are also intriguing hints of therapeutic implications of hnRNPL regulation for muscle diseases that merit further investigation. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a superfamily of RNA binding proteins (RBPs) with diverse functions including alternative splicing, mRNA stabilization, and regulation of transcription and translation [1] . The hnRNPs have been linked to numerous diseases such as cancer [2–7], amyotrophic lateral sclerosis (ALS) with or without frontotemporal dementia (FTD) [8,9], Alzheimer’s disease [10], spinal muscular atrophy (SMA) [11–13], and rare neurodevelopmental disorders [14] . Regulation of mRNA stability and splicing by hnRNPs is important during the stem cell differentiation process, and many hnRNPshave important roles in maintaining the self-renewal capacity of stem cells [15] . Several hnRNPs have been linked to myogenesis and muscle development [16,17] . For example, hnRNP A1 Abbreviations CMA, chaperone-mediated autophagy; DM1, myotonic dystrophy type 1; hnRNP L, heterogeneous nuclear ribonucleoprotein L; hnRNP LL, hnRNP L-like; hnRNPs, heterogeneous nuclear ribonucleoproteins; IGV, integrative genome viewer; LOF, loss of function; LRT, likelihoodratio-test; RBPs, RNA binding proteins; Sm, smooth. 178 FEBS Open Bio 16 (2026) 178–198 ª 2025 The Author(s) . FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly

FAQs

What is the main hypothesis about Hnrnpl in developing muscle cells?

The study hypothesizes that hnRNP L regulates both muscle gene expression levels and specific splicing patterns during muscle development.

Which experimental approaches were used to assess hnRrnpl deficiency?

Nanopore long-read transcriptome sequencing and qPCR analyses were used to study hnRrnpl knockdown in myoblasts and knockdown of the orthologous smooth gene in Drosophila.

What kinds of molecular changes were observed after hnRrnpl knockdown?

Notch signaling genes and muscle-related genes were dysregulated, and several genes showed altered splicing patterns, including Lamp2, Fhl1, and Dtna with isoform-specific up- and down-regulation.

What nanopore sequencing depth is suggested for detecting splicing differences?

The findings suggest a minimum read depth of 10 to detect splicing differences greater than about 10% to 20%.

Sep 29, 2026

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