CLN3transcript complexity revealed by long-read RNA sequencing analysis

Abstract

AbstractBackgroundBatten disease is a group of rare inherited neurodegenerative diseases. Juvenile CLN3 disease is the most prevalent type, and the most common mutation shared by most patients is the “1-kb” deletion which removes two internal coding exons (7 and 8) inCLN3. Previously, we identified two transcripts in patient fibroblasts homozygous for the “1-kb” deletion: the “major” and “minor” transcripts. To understand the full variety of disease transcripts and their role in disease pathogenesis, it is necessary to first investigateCLN3transcription in “healthy” samples without juvenile CLN3 disease.MethodsWe leveraged PacBio long-read RNA sequencing datasets from ENCODE to investigate the full range ofCLN3transcripts across various tissues and cell types in human control samples. Then we sought to validate their existence using data from different sources.ResultsWe found that a readthrough gene affects the quantification and annotation ofCLN3.After taking this into account, we detected over 100 novelCLN3transcripts, with no dominantly expressedCLN3transcript. The most abundant transcript has median usage of 42.9%. Surprisingly, the known disease-associated “major” transcripts are detected. Together, they have median usage of 1.51% across 22 samples. Furthermore, we identified 48CLN3ORFs, of which 26 are novel. The predominant ORF that encodes the canonical CLN3 protein isoform has median usage of 66.7%, meaning around one-third ofCLN3transcripts encode protein isoforms with different stretches of amino acids. The same ORFs could be found with alternative UTRs. Moreover, we were able to validate the translational potential of certain transcripts using public mass spectrometry data.ConclusionOverall, these findings provide valuable insights into the complexity ofCLN3transcription, highlighting the importance of studying both canonical and non-canonicalCLN3protein isoforms as well as the regulatory role of UTRs to fully comprehend the regulation and function(s) ofCLN3. This knowledge is essential for investigating the impact of the "1-kb" deletion and rare mutations onCLN3transcription and disease pathogenesis.