Authors:	Wang, Juanjuan
Title:	Characterizing the spatio-temporal cellular response to transcription-blocking lesions by mass spectrometry-based proteomics
Online publication date:	6-Oct-2022
Year of first publication:	2022
Language:	english
Abstract:	Exposure to ultraviolet (UV) radiation induces tumorigenesis of keratinocytes and leads to melanoma. On a molecular level, UV generates transcription-blocking DNA lesions that affect all steps of gene expression, from co-transcriptional splicing on chromatin to translation in the cytoplasm. However, a systematic investigation of protein-protein interactions and posttranslational modifications that regulate UV response remains poorly studied. We used human keratinocytes as a model system and employed tandem mass-tag-based quantitative mass spectrometry (TMT-MS) to quantify the changes in proteome and phosphoproteome with spatial and temporal resolution. Additionally, the localization of proteins on chromatin was investigated. Our data revealed that UV stress activates the main DNA damage response kinases, ATM, ATR, and DNA-PK as well as MAPKs. After a long time of recovery post UV stress (18h), the G2/M checkpoint is activated with the activation of WEE1. The dynamic regulation of phosphorylation states results in changing protein levels and protein recruitment to and exclusion from chromatin, subsequently regulating cellular processes. At an early recovery time (0.5h), when transcription is halted, DNA repair and transcription processing are highly regulated. In comparison, when transcription recovers and restarts (2h and 6h), RNA processing including mRNA and non-coding RNA (ncRNA) processing, and translation initiation are regulated by the changes in the associated protein levels and phosphorylation. Moreover, we observed that UV stress induces R-loop accumulation when transcription restarts (6h). Also, R-loop associated proteins are regulated by phosphorylation upon UV stress. Interestingly, signatures of the inflammatory response are observed after transcription-blocking lesions are obliterated completely. To study the role of the transcription-related kinase JNK1, we combined the chemical inhibition of JNK1 and UV light with quantitative phosphorylation profiling by mass spectrometry to identify JNK kinase substrates. We identified 206 putative JNK substrates in response to UV irradiation, with an overrepresentation of RNA binding proteins. Furthermore, we revealed that the Cleavage factor Im (CFIm) complex component (CPSF6) is phosphorylated by JNK1 on threonine 407 upon exposure to UV. The phosphorylated CPSF6 is excluded from chromatin and re-localizes into paraspeckles together with pre-mRNA upon UV stress. CPSF6 rosettes show liquid-liquid phase separation (LLPS) characteristics. The phosphorylation of CPSF6 regulates alternative polyadenylation after UV exposure and promotes cellular recovery from transcription-blocking lesions.
DDC:	570 Biowissenschaften 570 Life sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 10 Biologie
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-7221
URN:	urn:nbn:de:hebis:77-openscience-bd00a69f-7f0a-4b85-8f38-13e6c011d7351
Version:	Original work
Publication type:	Dissertation
License:	CC BY
Information on rights of use:	https://creativecommons.org/licenses/by/4.0/
Extent:	X, 199 Seiten, Illustrationen, Diagramme
Appears in collections:	JGU-Publikationen