The relationship between 3D exon-exon DNA folding and RNA splicing

The National Institute of General Medical Sciences awarded the Board of Regents of the University of Nebraska, through its University of Nebraska Medical Center division, $152,600 under the Biomedical Research and Research Training program (CFDA 93.859) on January 13, 2027, to fund a fellowship project examining the relationship between three-dimensional exon-exon DNA folding and RNA splicing.

The research defines the mechanistic and temporal relationship between chromatin structure and co-transcriptional splicing through two integrated aims. In Aim 1, synchronized transcriptional activation in HCT116 cells will dissect the timing of exon-exon DNA folding, RNA polymerase progression, histone modifications, and spliceosome recruitment using high-resolution HI-C, CUT & RUN, CUT & TAG, and long-read nascent RNA sequencing. In Aim 2, the role of specific histone modifications in modulating internucleosomal interactions will be evaluated using reconstituted chromatin arrays, assessed via atomic force microscopy and in vitro micro-C. The fellowship provides training in genomics, chromatin biochemistry, and single-molecule AFM imaging, with applicant proficiency development in nanopore sequencing, bioinformatics, and chromatin nanoimaging conducted in the Rowley and Lyubchenko Laboratories.

Performance occurs in Omaha, Nebraska. The project runs through July 31, 2029. This is a Project Grant, an assistance type within NIGMS's portfolio of investigator-initiated biomedical research funding.

Abstract

Accurate pre-mrna splicing is essential for generating transcriptomic diversity and maintaining normal cellular function. Dysregulation of RNA splicing contributes to a broad spectrum of human diseases, including cancer and neurological disorders. While splicing has traditionally been viewed at an RNA-centric level governed by CIS-elements and RNA-binding proteins, recent studies suggest that chromatin organization and histone modifications may play an instructive role in splicing decisions. Notably, exon-enriched histone marks such as H3K36ME3 have been associated with splicing factor recruitment, and my preliminary data reveal spatial clustering of DNA encoding exons in three-dimensional nuclear space. However, the temporal relationship between exon-exon chromatin folding and spliceosome assembly remains unknown. This fellowship proposal aims to define the mechanistic and temporal relationship between chromatin structure and CO-transcriptional splicing. In aim 1, synchronized transcriptional activation in HCT116 cells will be used to dissect the timing of exon-exon DNA folding, RNA polymerase progression, histone modifications, and spliceosome recruitment through a combination of high-resolution HI-c, cut & run, cut & tag, and long-read nascent RNA sequencing. In aim 2, the role of specific histone modifications in modulating internucleosomal interactions will be evaluated using reconstituted chromatin arrays and assessed via atomic force microscopy and in vitro micro-c. Training will be conducted in the rowley and lyubchenko laboratories, providing rigorous interdisciplinary mentorship in genomics, chromatin biochemistry, and single-molecule AFM imaging. The applicant will gain proficiency in nanopore sequencing, bioinformatics, and chromatin nanoimaging while participating in structured teaching, mentoring, and writing activities. The integrated training and research plan will provide foundational insights into gene regulation and prepare the applicant for a career as an independent investigator at the intersection of chromatin structure and RNA processing.