Both familial ALS and sporadic ALS exhibit dysregulation of RNA metabolism, indicating that homeostasis of the transcriptome is crucial for motor neuron survival. Indeed, almost all ALS eventually converges on TDP-43 pathology and thus, widespread transcriptome alterations. Given that changes in RNA metabolism occur in all forms of ALS, identifying key transcripts altered in the course of disease pathogenesis – particularly those caused by TDP-43 loss of function – could yield novel, broadly applicable therapeutic targets for the treatment of ALS.
We found that alternative polyadenylation (APA), an aspect of RNA metabolism understudied in ALS, occurs broadly upon TDP-43 mutation or depletion. APA affects RNA metabolism by producing distinct transcripts with shortened or lengthened 3′UTRs containing different cis-regulatory elements, such as binding sites for microRNAs (miRNAs) and RNA binding proteins (RBPs), leading to altered RNA stability, protein translation, or subcellular localization of a given transcript. TDP-43 is known to regulate APA by binding target RNAs near polyadenylation signals (PAS); yet, this aspect of TDP-43 biology has not been adequately studied because standard analysis of RNA-sequencing data does not fully capture APA events, which may alter RNA metabolism without affecting steady-state transcript levels.
With our collaborator Dr. Wei Li (UC Irvine), we have applied the dynamic analysis of polyadenylation from RNA-seq (DaPars) tool to ALS RNA-sequencing datasets, finding hundreds of previously unknown APA events in genes that function in pathways implicated in ALS pathogenesis, such as nucleocytoplasmic transport and the oxidative stress response. In subsequent experiments, we functionally validated new disease phenotypes revealed by these APA analyses in neuronal cells.
In addition, we found that APA of MARK3, a tau kinase implicated in Alzheimer’s disease, leads to increased MARK3 protein levels in neuronal cells depleted of TDP-43, reflecting a novel mechanistic link between TDP-43 and tau pathology. Importantly, APA can be directly modulated by antisense oligonucleotides (ASOs); thus, newly identified APA genes may be candidates for rapid therapy development in ALS.