Light Regulates Plant Alternative Splicing through the Control of Transcriptional Elongation.

Light makes carbon fixation possible, allowing plant and animal life on Earth. We have previously shown that light regulates alternative splicing in plants. Light initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing of a subset of Arabidopsis thaliana transcripts. Here, we show that light promotes RNA polymerase II (Pol II) elongation in the affected genes, whereas in darkness, elongation is lower. These changes in transcription are consistent with elongation causing the observed changes in alternative splicing, as revealed by different drug treatments and genetic evidence. The light control of splicing and elongation is abolished in an Arabidopsis mutant defective in the transcription factor IIS (TFIIS). We report that the chloroplast control of nuclear alternative splicing in plants responds to the kinetic coupling mechanism found in mammalian cells, providing unique evidence that coupling is important for a whole organism to respond to environmental cues.

Global unleashing of transcription elongation waves in response to genotoxic stress restricts somatic mutation rate.

Complex molecular responses preserve gene expression accuracy and genome integrity in the face of environmental perturbations. Here we report that, in response to UV irradiation, RNA polymerase II (RNAPII) molecules are dynamically and synchronously released from promoter-proximal regions into elongation to promote uniform and accelerated surveillance of the whole transcribed genome. The maximised influx of de novo released RNAPII correlates with increased damage-sensing, as confirmed by RNAPII progressive accumulation at dipyrimidine sites and by the average slow-down of elongation rates in gene bodies. In turn, this transcription elongation 'safe' mode guarantees efficient DNA repair regardless of damage location, gene size and transcription level. Accordingly, we detect low and homogenous rates of mutational signatures associated with UV exposure or cigarette smoke across all active genes. Our study reveals a novel advantage for transcription regulation at the promoter-proximal level and provides unanticipated insights into how active transcription shapes the mutagenic landscape of cancer genomes.

UV Irradiation Induces a Non-coding RNA that Functionally Opposes the Protein Encoded by the Same Gene.

The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ∼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.

Identifying transcription start sites and active enhancer elements using BruUV-seq.

BruUV-seq utilizes UV light to introduce transcription-blocking DNA lesions randomly in the genome prior to bromouridine-labeling and deep sequencing of nascent RNA. By inhibiting transcription elongation, but not initiation, pre-treatment with UV light leads to a redistribution of transcription reads resulting in the enhancement of nascent RNA signal towards the 5'-end of genes promoting the identification of transcription start sites (TSSs). Furthermore, transcripts associated with arrested RNA polymerases are protected from 3'-5' degradation and thus, unstable transcripts such as putative enhancer RNA (eRNA) are dramatically increased. Validation of BruUV-seq against GRO-cap that identifies capped run-on transcripts showed that most BruUV-seq peaks overlapped with GRO-cap signal over both TSSs and enhancer elements. Finally, BruUV-seq identified putative enhancer elements induced by tumor necrosis factor (TNF) treatment concomitant with expression of nearby TNF-induced genes. Taken together, BruUV-seq is a powerful new approach for identifying TSSs and active enhancer elements genome-wide in intact cells.