An ultra-deep RNA-seq time-course defines alternative splicing in the cold temperature response of Arabidopsis

Abstract: Alternative splicing (AS) has been implicated in a wide range of developmental and physiological processes. We have shown that AS is important in regulating expression of key circadian clock genes and mediating the response of the clock to changes in temperature. The circadian clock is a cellular mechanism able to organize many physiological processes in anticipation/preparation to daily and seasonal changes. AS may therefore have roles in temperature perception, entrainment, compensation, regulation of downstream physiological responses and acclimation to exposure to low temperature.

We wish to analyse dynamic reprogramming of the cold transcriptome and have a high resolution time-course of Arabidopsis plants grown at 20°C and then transferred to the cold. We are using Salmon to analyse the time-course  RNA-seq data to quantify expression at the transcript level. To use Salmon, we have developed a comprehensive, non-redundant Reference Transcript Dataset (AtRTD2 – bioRxiv May 2016) with ca. 82k unique transcripts. Through extensive validation with high resolution RT-PCR, we established quality control filters to maximise the number of supported transcripts. We also identified inaccurate quantification of transcripts from genes with UTR variation among their transcripts. We modified AtRTD2 to overcome this issue – creating AtRTD2-QUASI – for Quantification of Alternatively Spliced Isoforms.

We have analysed the RNA-seq data using Salmon/AtRTD2-QUASI and are currently analysing differential expression at the gene and transcript level as well as differential alternative splicing. The dynamic time-course data of individual transcripts has allowed the identification of genes regulated only by transcription, only by AS and by both transcription and AS have been identified. Around a third of genes which are differentially expressed in the cold undergo AS. We have identified isoform switches as well as genes whose rhythmic expression changes in the cold. The data will be used to build transcription and splicing networks to identify genes that may regulate alternative splicing of core clock genes at low temperature and the role of AS in reprogramming the cold transcriptome.

Host: Katherine Denby