Pre-mRNA processing factors meet the DNA damage response

Abstract

It is well-known that DNA-damaging agents induce genome instability, but only recently have we begun to appreciate that chromosomes are fragile per se and frequently subject to DNA breakage. DNA replication further magnifies such fragility, because it leads to accumulation of single-stranded DNA. Recent findings suggest that chromosome fragility is similarly increased during transcription. Transcripts produced by RNA polymerase II (RNAPII) are subject to multiple processing steps, including maturation of 5' and 3' ends and splicing, followed by transport to the cytoplasm. RNA maturation starts on nascent transcripts and is mediated by a number of diverse proteins and ribonucleoprotein particles some of which are recruited cotranscriptionally through interactions with the carboxy-terminal domain of RNAPII. This coupling is thought to maximize efficiency of pre-mRNA maturation and directly impacts the choice of alternative splice sites. Mounting evidence suggests that lack of coordination among different RNA maturation steps, by perturbing the interaction of nascent transcripts with the DNA template, has deleterious effects on genome stability. Thus, in the absence of proper surveillance mechanisms, transcription could be a major source of DNA damage in cancer. Recent high-throughput screenings in human cells and budding yeast have identified several factors implicated in RNA metabolism that are targets of DNA damage checkpoint kinases: ATM (ataxia telangiectasia mutated) and ATR (ATM-Rad3 related) (Tel1 and Mec1 in budding yeast, respectively). Moreover, inactivation of various RNA processing factors induces accumulation of γH2AX foci, an early sign of DNA damage. Thus, a complex network is emerging that links DNA repair and RNA metabolism. In this review we provide a comprehensive overview of the role played by pre-mRNA processing factors in the cell response to DNA damage and in the maintenance of genome stability.

Keywords: DNA damage response; RNA binding proteins; checkpoint kinases; pre-mRNA processing; splicing.

FIGURE 1

Phosphorylation controls the subcellular distribution of splicing factor SRSF1. The arginine–serine (RS)-rich domain of the SR protein SRSF1 is phosphorylated by SR protein kinases SRPK and Clk/Sty. The docking motif (DM) restricts phosphorylation of SRSF1 by SRPK1 at the N-terminal portion of the RS domain (RS1), which is required for the interaction with transportin SR and nuclear import. In the nucleus, SRSF1 accumulates in nuclear speckles. Clk/Sty causes release of SRSF1 from speckles by phosphorylating the C-terminal portion of its RS domain (RS2; Ngo et al., 2005).

FIGURE 2

DNA damage affects splicing decisions by modulating the phosphorylation status of RNAPII and the elongation rate of transcription. CPT-induced Top1ccs have immediate and specific effects on RNAPII. CPT triggers a high degree of phosphorylation of the largest subunit (Rpb1) of RNAPII (Baranello et al., 2010). Ultraviolet (UV) irradiation affects cotranscriptional alternative splicing in a p53-independent manner, through hyperphosphorylation of the RNAPII carboxy-terminal domain (CTD) and subsequent inhibition of transcriptional elongation (Muñoz et al., 2009).

FIGURE 3

Protein–protein interactions mediate the cotranscriptional assembly of ribonucleoprotein complexes that are targets of DNA damage-induced signaling pathways. (A) Schematic representation of the communication between the transcriptional and splicing machineries mediated by RPB7, EWS, and YB-1. Camptothecin inhibits the interaction between Ewing’s sarcoma proto-oncoprotein (EWS), an RNAPII-associated factor, and YB-1, a spliceosome-associated factor. This results in the cotranscriptional skipping of several exons of the MDM2 gene (Dutertre et al., 2010). (B) The PRP19 complex functions in transcription and is recruited to the transcription machinery by the C terminus of its component Syf1, the yeast homolog of human XAB2 (adapted from Chanarat et al., 2011). Human XAB2 co-purifies both with factors involved in transcription (RNAPII), splicing (PRP19), and TCR (XPA, CSA and CSB; Kuraoka et al., 2008). The PRP19 complex is required for the recruitment of the THO/TREX complex to nascent transcripts after the switch from the B to the C splicing complex. Thick and thin black lines represent exons and introns, respectively.

FIGURE 4

Upper panel: the translocation of the transcriptional apparatus along the DNA induces positive and negative supercoiling, respectively, in front of and behind RNAPII. The physiological association of RBPs with the pre-mRNA molecule as it emerges from the transcriptional machinery is believed to play a major role in counteracting R-loop formation in negatively super-coiled regions. Middle panel: hybridization of the nascent RNA with the DNA template results in the formation of R-loops and occurs upon down-regulation, inhibition or mutation of several specific RBPs involved in different steps of pre-mRNA synthesis/maturation. The list of RBPs that may influence R-loop formation includes Capping enzymes, splicing factors SRSF1 and SRSF2, the THO/TREX complex, and Sen1/senataxin which is important for transcription termination. Moreover, DNA topoisomerase I (Topo I) by relieving torsional stress and phosphorylating SRSF1 can prevent R-loop formation. Bottom panel: R-loops hamper the movement of the DNA replication fork, which promotes genome instability. Thus, RBPs may be crucial to genome stability programs by inhibiting R-loop formation.