Cryptic Exons: A Comprehensive Overview of TDP43’s Role

RNA processing is tightly regulated to ensure proper gene expression, but disruptions can lead to the inclusion of cryptic exons—sequences normally excluded from mature mRNA. These aberrations can alter protein function and contribute to disease, particularly neurodegenerative disorders.

A key regulator of exon exclusion is TDP43, an RNA-binding protein essential for transcript integrity. When its function is impaired, cryptic exons are erroneously incorporated into mRNAs, producing dysfunctional proteins. Understanding TDP43’s role in this process is crucial for deciphering its impact on health and disease.

Mechanisms of Cryptic Exon Generation

Cryptic exons arise from disruptions in pre-mRNA splicing, where noncanonical sequences are mistakenly included in mature transcripts. This misregulation often results from the loss of splicing repressors that normally prevent these sequences from being recognized as exons. RNA-binding proteins, particularly those regulating splicing, ensure only correct sequences are retained. When these mechanisms fail, cryptic exons may be spliced into transcripts, potentially altering protein function.

A major factor in cryptic exon inclusion is the weakening of splicing silencer elements—short RNA sequences that suppress exon recognition. These silencers are bound by proteins such as heterogeneous nuclear ribonucleoproteins (hnRNPs), which prevent the spliceosome from assembling at cryptic splice sites. If these proteins are depleted or dysfunctional, the spliceosome may erroneously recognize and splice in cryptic exons. Mutations in splicing regulator genes can lead to widespread cryptic exon activation, highlighting their role in transcript stability.

Cryptic splice site accessibility also increases due to chromatin structure changes or RNA secondary conformation shifts. Splicing is closely linked to transcription, and modifications like histone acetylation or methylation influence splice site selection by altering RNA polymerase II elongation rates. A slower transcriptional pace can expose weak splice sites longer, increasing cryptic exon inclusion. Similarly, RNA secondary structures can either shield or expose cryptic splice sites. Disruptions in RNA helicases or modifying enzymes can destabilize these structures, inadvertently promoting cryptic exon recognition.

Role of TDP43 in Exon Inclusion

TDP43, a ubiquitously expressed RNA-binding protein, ensures exon recognition fidelity by regulating splicing. It represses cryptic exons by binding UG-rich motifs in pre-mRNA, preventing noncanonical sequences from being included. This repression occurs through interactions with spliceosome components and modulation of RNA secondary structures that influence splice site accessibility. When TDP43 function is lost, cryptic exon inclusion increases, leading to abnormal transcript formation.

TDP43 suppresses cryptic exons by recruiting splicing repressors and influencing RNA polymerase II elongation kinetics. Binding intronic regions near cryptic splice sites, it sterically hinders spliceosome assembly at inappropriate locations. Additionally, it interacts with other RNA-binding proteins, such as hnRNP A1 and hnRNP C, to reinforce exon exclusion. Under normal conditions, this cooperation keeps cryptic exons silent. However, in disease states where TDP43 is depleted or mislocalized, these protective mechanisms fail, allowing noncanonical exons into mature mRNA.

Beyond direct binding, TDP43 regulates exon selection by modulating splicing factors like SRSF2 and PTBP1, which influence splice site recognition. This extends its regulatory reach beyond immediate RNA targets, shaping broader splicing patterns. RNA sequencing and CLIP (crosslinking immunoprecipitation) studies reveal that TDP43 loss alters splicing across many transcripts, incorporating cryptic exons that disrupt protein function.

Cryptic Exons in Neurodegenerative Conditions

Aberrant splicing events leading to cryptic exon inclusion are increasingly linked to neurodegenerative diseases. In conditions like amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), defective RNA processing results in dysfunctional proteins that impair neuronal function. Accumulation of these proteins contributes to cellular stress, synaptic dysfunction, and neurodegeneration. Postmortem analyses of patient brain tissue confirm widespread cryptic exon activation in affected regions, making these splicing errors a defining molecular feature of disease progression.

Cryptic exon inclusion disrupts regulatory networks essential for neuronal survival. Many affected genes encode proteins involved in cytoskeletal maintenance, axonal transport, and synaptic plasticity—functions particularly vulnerable in neurodegeneration. In ALS, cryptic exon incorporation into STMN2, a gene critical for motor neuron stability, leads to a loss of functional stathmin-2 protein, impairing axonal regeneration and accelerating degeneration. In FTD, cryptic exon misregulation in genes like UNC13A affects synaptic vesicle dynamics, further contributing to disease pathology.

Neurodegenerative diseases progress over time, suggesting cryptic exon inclusion is an evolving process. Longitudinal studies using patient-derived neurons show increasing cryptic exon-containing transcripts as disease advances, correlating with greater cellular dysfunction, impaired RNA metabolism, and protein aggregation. The early appearance of cryptic exon misprocessing suggests these splicing abnormalities could serve as biomarkers for early disease detection. Emerging transcriptomic technologies, such as long-read RNA sequencing, now enable high-resolution detection of these aberrant splicing events, offering new diagnostic possibilities.

Laboratory Approaches to Detect Cryptic Exons

Detecting cryptic exons requires precise molecular techniques to identify subtle splicing errors. RNA sequencing (RNA-seq) offers a comprehensive view of transcriptome-wide splicing patterns. While short-read RNA-seq is widely used, its reliance on fragmented reads makes distinguishing full-length isoforms challenging. Long-read sequencing technologies, such as those from Oxford Nanopore and Pacific Biosciences, overcome this limitation by capturing entire transcripts, allowing direct observation of cryptic exon inclusion.

Specialized PCR-based techniques provide targeted detection of cryptic exons in specific genes. Reverse transcription PCR (RT-PCR) followed by gel or capillary electrophoresis quickly identifies aberrantly spliced transcripts. Quantitative PCR (qPCR) measures the relative abundance of cryptic exon-containing mRNA compared to canonical isoforms. Digital droplet PCR (ddPCR) enhances sensitivity, detecting low-frequency cryptic exon events that might be missed by conventional methods. These PCR-based strategies are valuable for validating RNA-seq findings and high-throughput screening of patient-derived samples.