In mammals, robust circadian clocks determine the rhythmicity of many biological phenomena and coordinate gene expression programs in a tissue-specific manner. Transcriptome analyses using conventional technologies, such as microarray and Illumina-seq, have revealed rhythms in approximately 10-15% of the genes expressed in the liver. Direct RNA sequencing with Oxford Nanopore technology allows the detection of different gene isoforms and thus the study of alternative splicing/promoters, the precise
measurement of poly(A) tail length, and the identification and quantification of epitranscriptomic modifications, thus generating a comprehensive view of the transcriptome. We performed long-read sequencing with Promethion at an unprecedented level, generating over 100 million direct RNA reads from livers of wild-type mice over 24 hours as well as from Per1-/-;Per2-/- mice lacking circadian rhythms.
Results of the analysis reveal new cyclic transcripts, as well as isoforms with different dynamics (or even antiphase) generated from the same gene, suggesting a circadian activity of alternative splicing. Furthermore, we identified about 2000 deregulated alternative splicing events in PerKO mice and observed isoform switches that could impact metabolism. A Proteomics analysis allowed us to identify potential players in this altered splicing program. In addition, we found time-dependent polyA tail length dynamics as well as a global change in tail length in PerKO mice.
Finally, preliminary results from the study of the epi-transcriptome at nucleotide resolution enabled by this technology are ongoing to determine whether there are circadian dynamics to these modifications.
Gratuit
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