Cellular fractionation reveals transcriptome responses of human fibroblasts to UV-C irradiation

Abstract

Abstract While cells activate a multifaceted DNA damage response to remove transcription-blocking DNA lesions, mechanisms to regulate genome-wide reduction of RNA synthesis and the paradoxical continuous loading of RNAP II at initiation sites are still poorly understood. Uncovering how dramatic changes to the transcriptional program contribute to TC-NER (transcription-coupled nucleotide excision repair) is important in DNA repair research. However, the functional significance of transcriptome dynamics and the mechanisms of chromatin attachment for thousands of unstudied human lncRNAs remain unclear. To address these questions, we examined UV-induced gene expression regulation in human fibroblasts by performing RNA-seq with fractionated chromatin-associated and cytoplasmic transcripts. This approach allowed us to separate the synthesis of nascent transcripts from the accumulation of mature RNAs. In addition to documenting the subcellular locations of coding transcripts, our results also provide a high-resolution view of the transcription activities of noncoding RNAs in response to cellular stress. At the same time, the data showed that vast majority of genes exhibit large changes in chromatin-associated nascent transcripts without corresponding changes in cytoplasmic mRNA levels. Distinct from protein-coding genes that transcripts with shorter length prefer to be recovered first, repression of lncRNA transcription after UV exposure is inactivated first on noncoding transcripts with longer length. This work provides an updated framework for cellular RNA organization in response to stress and may provide useful information in understanding how cells respond to transcription-blocking DNA damage.