Speaker
Description
The 1 subunits (CACNA1 family) of L-type voltage-gated calcium channels (LTCCs) play significant roles in brain function and neuropsychiatric disorders. Transcription of LTCCs is complex, with each 1 gene producing multiple isoforms. CACNA1S and CACNA1F were reported to have short-length transcripts from their 3’ region in the human brain which were not well characterized. Moreover, whether other CACNA1 genes also produce the 3’ transcripts have not been studied.
This thesis aims to identify and understand the short-length transcripts arising from the 3’ region of LTCC genes in human brain, especially CACNA1S and CACNA1F, using a series of technical approaches and analyses. Firstly, using PCR amplification and long-read nanopore sequencing, multiple 3’ transcripts with exon-skipping possibilities were identified. Secondly, 5’ Rapid Amplification of cDNA Ends (5’ RACE) identified the 3’ transcripts as being complete transcripts, arising from alternative transcription start sites (TSS). These findings were corroborated by exon expression level findings from Genotype-Tissue Expression (GTEx) data. Analyses using PhyloP showed that the 5’-untranslated region (5’-UTR) of the 3’ transcripts are more conserved than the intronic regions. Finally, the translation and localization of the protein encoded by each 3’ transcript was investigated in transfected HEK-293T and SH-SY5Y cell lines. All proteins were detectable by western blot. C-CaV1.3 (encoded by CACNA1D) localized to the nucleus in both cell types; C-CaV1.2 (encoded by CACNA1C) translocated to the nucleus in the excitable (SH-SY5Y) cells but not the non-excitable (HEK-293T) cells. C-CaV1.1 and C-CaV1.4 (encoded by CACNA1S and CACNA1F respectively) were not localized to the nucleus.
This thesis shows that LTCC 1 subunit genes produce 3’ transcripts using alternative TSS in human brain. Their potential functionality is supported by the evolutionary conservation of their 5’-UTRs and by their translation in vitro. However, further studies are required to identify the significance of these transcripts for brain function.
References
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| Keywords | Alternative splicing, LTCC, Nanopore sequencing |
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