Exon 45: Insights into Dystrophin Gene Splicing

Genetic mutations affecting the dystrophin gene are a primary cause of Duchenne muscular dystrophy (DMD), a severe muscle-wasting disorder. Understanding how specific exons contribute to dystrophin production is crucial for developing targeted therapies.

One key area of research focuses on exon 45, which plays a role in maintaining the structural integrity of the dystrophin protein. Scientists are investigating how this exon is regulated during RNA splicing and how mutations can disrupt its inclusion or exclusion.

Exon 45 In The Dystrophin Gene

Exon 45 is a critical component of the dystrophin gene, which spans over 2.2 million base pairs, making it one of the largest genes in the human genome. It encodes a segment of the dystrophin protein essential for stabilizing the dystrophin-glycoprotein complex, which maintains muscle cell membrane integrity. Proper exon 45 inclusion during mRNA processing ensures dystrophin retains its functional domains, particularly those linking the cytoskeleton to the extracellular matrix. Any disruption in this process can weaken muscle fibers.

Exon 45 lies within a mutational hotspot spanning exons 45–55, a region frequently affected by deletions causing Duchenne and Becker muscular dystrophies. Skipping exon 45 can sometimes yield a partially functional dystrophin protein, making exon-skipping therapies a potential treatment strategy. Modulating exon 45 inclusion has implications for restoring dystrophin function in specific mutations.

Regulatory mechanisms influence exon 45 splicing efficiency. Splicing enhancers and silencers within the exon and its flanking regions determine its retention in the mature transcript. RNA-binding proteins such as serine/arginine-rich splicing factors (SRSFs) and heterogeneous nuclear ribonucleoproteins (hnRNPs) interact with these elements to regulate exon inclusion. Variations in these regulatory sequences can alter splicing patterns, leading to defective dystrophin transcripts that either lack exon 45 or include unintended sequences, worsening disease severity.

Mechanism Of Splicing Control

Exon 45 splicing is governed by cis-regulatory elements and trans-acting factors. Exonic splicing enhancers (ESEs) recruit serine/arginine-rich (SR) proteins to facilitate exon recognition, while exonic splicing silencers (ESSs) interact with hnRNPs to suppress inclusion. The balance between these signals determines whether exon 45 is retained or skipped.

RNA-binding proteins such as SRSF1 and SRSF2 promote exon 45 inclusion, while hnRNP A1 and hnRNP H suppress it. These factors bind specific sequence motifs within exon 45 and its flanking introns. Mutations in these regions can disrupt protein binding, leading to aberrant splicing. Additionally, the secondary structure of pre-mRNA influences exon recognition, as RNA folding can expose or obscure regulatory elements.

Co-transcriptional splicing links transcription kinetics to splice site selection. RNA polymerase II elongation rate affects spliceosome assembly, with slower transcription favoring exon inclusion. Chromatin modifications, such as histone acetylation and methylation, influence transcriptional elongation and splicing by altering nucleosome positioning around exon 45.

Mutations Affecting Exon 45

Genetic alterations in exon 45 can disrupt dystrophin synthesis, leading to varying degrees of functional loss. Deletions involving exon 45 are common in Duchenne and Becker muscular dystrophy. The severity depends on whether the deletion maintains the mRNA reading frame. Out-of-frame deletions typically result in a nonfunctional dystrophin protein, characteristic of Duchenne muscular dystrophy, while in-frame deletions may produce a shorter but partially functional protein, seen in Becker muscular dystrophy.

Point mutations can also impact dystrophin expression by altering splicing efficiency or introducing premature stop codons. Nonsense mutations create premature termination signals, leading to mRNA degradation through nonsense-mediated decay, preventing dystrophin production. Missense mutations may affect protein stability or disrupt interactions within the dystrophin-glycoprotein complex. Splice-site mutations at exon-intron boundaries can lead to exon skipping or the inclusion of cryptic sequences, resulting in dysfunctional proteins.

Experimental Approaches For Examining Exon 45

Studying exon 45 requires molecular, cellular, and computational techniques to analyze its splicing behavior and functional role. RNA sequencing (RNA-seq) allows researchers to identify exon 45 inclusion or skipping in muscle biopsies and patient-derived cell lines. High-throughput sequencing data, combined with bioinformatics tools, helps map splicing regulatory elements and predict mutation effects.

CRISPR/Cas9 genome editing enables precise modifications in patient-derived induced pluripotent stem cells (iPSCs) or myoblast cultures. By introducing specific deletions or mutations, scientists can assess their impact on dystrophin expression and muscle cell function. These models serve as platforms for testing splicing-modulating therapies, such as antisense oligonucleotides (ASOs), designed to promote exon skipping or inclusion.

In vivo approaches include transgenic mouse models carrying humanized dystrophin gene sequences, which help evaluate splicing patterns under physiological conditions. Fluorescent reporter assays linked to exon 45 provide real-time insights into alternative splicing events.