Cold-Dependent Expression and Alternative Splicing of Arabidopsis Long Non-coding RNAs

Abstract

Plants re-program their gene expression when responding to changing environmental conditions. Besides differential gene expression, extensive alternative splicing (AS) of pre-mRNAs and changes in expression of long non-coding RNAs (lncRNAs) are associated with stress responses. RNA-sequencing of a diel time-series of the initial response of Arabidopsis thaliana rosettes to low temperature showed massive and rapid waves of both transcriptional and AS activity in protein-coding genes. We exploited the high diversity of transcript isoforms in AtRTD2 to examine regulation and post-transcriptional regulation of lncRNA gene expression in response to cold stress. We identified 135 lncRNA genes with cold-dependent differential expression (DE) and/or differential alternative splicing (DAS) of lncRNAs including natural antisense RNAs, sORF lncRNAs, and precursors of microRNAs (miRNAs) and trans-acting small-interfering RNAs (tasiRNAs). The high resolution (HR) of the time-series allowed the dynamics of changes in transcription and AS to be determined and identified early and adaptive transcriptional and AS changes in the cold response. Some lncRNA genes were regulated only at the level of AS and using plants grown at different temperatures and a HR time-course of the first 3 h of temperature reduction, we demonstrated that the AS of some lncRNAs is highly sensitive to small temperature changes suggesting tight regulation of expression. In particular, a splicing event in TAS1a which removed an intron that contained the miR173 processing and phased siRNAs generation sites was differentially alternatively spliced in response to cold. The cold-induced reduction of the spliced form of TAS1a and of the tasiRNAs suggests that splicing may enhance production of the siRNAs. Our results identify candidate lncRNAs that may contribute to the regulation of expression that determines the physiological processes essential for acclimation and freezing tolerance.

A powerful and flexible tool for rapid and accurate differential expression and alternative splicing analysis of RNA-seq data for biologists

“…These results reflect the analysis of the complete time-series (Calixto et al, 2018). Experimental validation of expression, AS and isoform switches by high resolution RT-qPCR and high resolution RT-PCR have been described previously (Calixto et al, 2018(Calixto et al, , 2019.…”

“…The 3D RNA-seq App was applied to selected time-points from time-series RNA-seq data of a study on changes in the Arabidopsis transcriptome in response to cold stress (Supplementary Figure 6) (Calixto et al, 2018(Calixto et al, , 2019. Briefly, 5-week-old Arabidopsis Col-0 plants were grown at 20 o C for 24 hours, then the temperature was reduced to 4 o C. Samples Figure 6A).…”

BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq

“…We used RNAseq data from three biological repeats of five organs/tissues of Morex to quantify transcripts with Salmon and BaRTv1.0-QUASI. Differential expression (DE) at both gene and transcript levels, differential AS (DAS) genes and differential transcript usage (DTU) were analysed using the recently developed 3D RNA-seq App (Calixto et al, 2018(Calixto et al, , 2019Guo et al, personal communication and 2,791 genes showed significant DAS ( Figure 4A&D; Supplementary Table S6). The overlap between DE and DAS genes (those genes regulated by both transcription and AS) was 2,199 such that 592 genes were DAS-only and regulated only at the level of AS with no change in overall gene expression.…”

“…We used RNAseq data from three biological repeats of five organs/tissues of Morex to quantify transcripts with Salmon and BaRTv1.0-QUASI. Differential expression (DE) at both gene and transcript levels, differential AS (DAS) genes and differential transcript usage (DTU) were analysed using the recently developed 3D RNA-seq App (Calixto et al, 2018(Calixto et al, , 2019Guo et al, personal communication log2 fold change of ≥ 1. To identify significant DAS genes, consistency of expression changes (log2 fold change) between the gene and its transcripts was determined along with the change in splice ratio (Δ Percent Spliced -ΔPS).…”

“…with unsupported splice junctions), transcript fragments and redundant transcripts, all of which affected the accuracy of transcript quantification by Salmon/Kallisto (Zhang et al, 2015;Zhang et al, 2017a). AtRTD2 has been used for genome-wide differential expression/differential AS to identify novel regulators of the cold response and splicing factors that regulate AS in innate immunity and root development (Zhang et al, 2017b;Calixto et al, 2018;Bazin et al, 2018;Calixto et al, 2019).…”