Isolation of XAB2 Complex Involved in Pre-mRNA Splicing, Transcription, and Transcription-coupled Repair

Abstract

Nucleotide excision repair is a versatile repair pathway that counteracts the deleterious effects of various DNA lesions. In nucleotide excision repair, there is a transcription-coupled repair (TCR) pathway that focuses on DNA damage that blocks RNA polymerase IIo in transcription elongation. XAB2 (XPAbinding protein 2), containing tetratricopeptide repeats, has been isolated by virtue of its ability to interact with xeroderma pigmentosum group A protein (XPA). Moreover, XAB2 has been shown to interact with Cockayne syndrome group A and B proteins (CSA and CSB) and RNA polymerase II, as well as XPA, and is involved in TCR and transcription. Here we purified XAB2 as a multimeric protein complex consisting of hAquarius, XAB2, hPRP19, CCDC16, hISY1, and PPIE, which are involved in pre-mRNA splicing. Knockdown of XAB2 with small interfering RNA in HeLa cells resulted in a hypersensitivity to killing by UV light and a decreased recovery of RNA synthesis after UV irradiation and regular RNA synthesis. Enhanced interaction of XAB2 with RNA polymerase IIo or XPA was observed in cells treated with DNA-damaging agents, indicating DNA damageresponsive activity of the XAB2 complex. These results indicated that the XAB2 complex is a multifunctional factor involved in pre-mRNA splicing, transcription, and TCR.

Transcription-coupled nucleotide excision repair in mammalian cells: molecular mechanisms and biological effects

“…In addition to XPA, XAB2 interacts with chromatin bound stalled RNAPIIo complex in a UV-and CS-dependent manner [41]. In agreement with these data, Tanaka and coworkers [43] showed in a recent study that XAB2 interacts with RNAPIIo and that this interaction is enhanced after DNA damage. Knock-down of XAB2 resulted in hypersensitivity of cells to cytotoxic effects of UV light and led to reduced RNA synthesis recovery.…”

Mammalian Transcription-Coupled Excision Repair

“…XAB2 is an essential protein involved in pre-mRNA splicing and is indispensable for TC-NER and restoration of damage-inhibited RNA synthesis (Kuraoka et al 2008). Thus, the implication of XAB2 in TC-NER links mRNA splicing to the arrest of transcription elongation, highlighting the multiple levels of complexity that regulate the DDR processes.…”

“…These factors include the CSA and CSB proteins, UVSSA, XAB2, and HMGN1. Cells with either a mutation or down-regulation of these factors show a greater reduction in the rate of repair of lesions in the transcribed versus the nontranscribed strand and/or increased UV sensitivity and a prolonged inhibition of RNA synthesis leading to a strong signal for cellular apoptosis (Troelstra et al 1992b;Henning et al 1995;Nakatsu et al 2000;Spivak et al 2002;Birger et al 2003;Kuraoka et al 2008;Fei and Chen 2012;Nakazawa et al 2012;Schwertman et al 2012;Zhang et al 2012). Thus, although dispensable for the core repair process of NER/ GG-NER, these factors are crucial for TC-NER, underscoring the special requirements of this pathway.…”

Nucleotide excision repair deficient mouse models and neurological disease

“…The exact function of XAB2 in TC-NER is not known. However, immunodepletion of XAB2 or siRNA knock-down causes UV hypersensitivity, impairs RNA synthesis following UV irradiation of cells, as well as inhibiting transcription [71,72]. In the mouse, deletion of Xab2 or even just the C-terminal 162 amino acids of the protein (removing the 15 th tetratricopeptide motif involved in protein:protein interactions) causes early embryonic lethality (E3.5) [73].…”