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Oxford Nanopore sequencing reveals a pervasive role for the mammalian circadian clock in shaping the tissue transcriptome.

Date
ven 21 oct 2022
Horaires

14 heures

Intervenant(s)

Soutenance de M. CHIKHAOULI Lies, sous la direction de M. PADMANABHAN Kiran

Langue(s) des interventions

Description générale

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.

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