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

Jiena Liu,Zhenzhen Wu,Yuming Wang,Jin He

doi:10.1038/s41419-022-04634-x

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.

Highlights

The response to DNA damage intersects with many other physiological processes in the cell, including initiation of DNA repair, chromatin remodeling, regulation of transcription and translation, and the cell cycle to contend with the challenge
The model proposes that CSB recruits CSA to TSS sites, mediating the ubiquitination and degradation of the ATF3 repressor on chromatin to elicit the restart of RNA synthesis after genotoxic stress [10]
The experimental strategy described in this study is critical in its ability to simultaneously provide information about the action of transcripts that remain associated with chromatin and the changes the transcripts proceed to the cytoplasm

Summary

INTRODUCTION

The response to DNA damage intersects with many other physiological processes in the cell, including initiation of DNA repair, chromatin remodeling, regulation of transcription and translation, and the cell cycle to contend with the challenge. The level of p-H3S10 genes in the early phase of DNA repair process were involved in proteins was not significantly altered at 24 h after UV treatment, DNA damage response (clusters 2 and 3, Table S3), suggesting that the accumulation of cells at G2/M phase at 24 h genes activated in the late response time, by contrast, encoded post irradiation results from a G2/M checkpoint arrest instead of proteins that are enriched in the translational regulation process mitotic catastrophe. Gene Ontology analysis indicated that the biological functions of mRNAs co-expressed with UV-induced annotated lncRNAs are associated with cell cycle regulation and cellular response to DNA damage (Fig. 5E showing the representative GO terms for lncRNAs in the clusters 2, 4, and 5, Table S10). These results imply that lncRNAs and mRNAs may have different mechanisms regulating their biogenesis, stability, and spatialtemporal expression patterns during DNA damage repair

DISCUSSION

Findings

MATERIALS AND METHODS