The response to DNA damage-stalled RNA polymerase II (RNAPIIo) involves the assembly of the transcription-coupled repair (TCR) complex on actively transcribed strands. The function of the TCR proteins CSB, CSA and UVSSA and the manner in which the core DNA repair complex, including transcription factor IIH (TFIIH), is recruited are largely unknown. Here, we define the assembly mechanism of the TCR complex in human isogenic knockout cells. We show that TCR is initiated by RNAPIIo-bound CSB, which recruits CSA through a newly identified CSA-interaction motif (CIM). Once recruited, CSA facilitates the association of UVSSA with stalled RNAPIIo. Importantly, we find that UVSSA is the key factor that recruits the TFIIH complex in a manner that is stimulated by CSB and CSA. Together these findings identify a sequential and highly cooperative assembly mechanism of TCR proteins and reveal the mechanism for TFIIH recruitment to DNA damage-stalled RNAPIIo to initiate repair.

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ranscription of protein-coding and noncoding genes requires RNA polymerase II (RNAPII), which synthesizes RNA transcripts complementary to the DNA template strand. The presence of DNA lesions in the template strand causes stalling of elongating RNAPII (RNAPIIo), which leads to genome-wide transcriptional arrest 1-3 . It is essential that cells overcome this arrest and restore transcription. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions through the proteins CSB, CSA and UVSSA [4][5][6] . Inactivating mutations in CSB and CSA cause Cockayne syndrome (CS), which is characterized by severe neurological dysfunction related to persistent RNAPII arrest at DNA lesions 2,7 .The sequential and cooperative actions of CSB, CSA and UVSSA recruit TFIIH to DNA damage-stalled RNAPII to initiate DNA repair 6 . In addition to protein-protein contacts, efficient transfer of TFIIH onto RNAPII requires ubiquitylation of a single lysine on the largest subunit of RNAPII (RPB1-K1268), which is essential for efficient TCR 2 . This DNA damage-induced modification of RNAPII is dependent on cullin-ring type E3-ligases (CRLs) and is strongly decreased in CSA-deficient cells 2 , which indicates that the CRL4 CSA E3 ligase complex drives RNAPII ubiquitylation.CSB binds to DNA upstream of RNAPII 8 (Extended Data Fig. 1a) and recruits the CRL4 CSA complex through an evolutionarily conserved motif in its carboxy terminus 6 . However, how the activity of CRL4 CSA ubiquitin ligase is specifically directed towards the K1268 site remains to be elucidated. Results A CRISPR screen identifies ELOF1 as a putative TCR gene.To identify unknown TCR genes, we performed a genome-wide CRISPR screen in the presence of the compound illudin S, which induces transcription-blocking DNA lesions that are eliminated by TCR 9 . RPE1-iCas9 cells were transduced with the pLCKO-TKOv3 library, which contains 70,948 single guide RNAs (sgRNAs) targeting open reading frames 10 , and cultured for 12 population doublings, after which sgRNA contents were analysed (Extended Data Fig. 1b).Using a false-discovery rate (FDR) cut-off of 0.01, we found 104 sensitizer hits and 18 hits conferring resistance to illudin S. The strongest resistance was conferred by guide RNAs (gRNAs) targeting PTGR1, which is in line with its known role in bioactivating illudin S 11 (Fig. 1a and Extended Data Fig. 1c). Nine known core TCR genes, including CSB, CSA and UVSSA, but also genes connected to transcription recovery after UV irradiation (HIRA 12 , DOT1L 13 and STK19 (ref. 14 ) (Fig. 1a,b), were required for illudin S tolerance. Consistent with known effects of illudin S on replication 9 , we found the 9-1-1 complex, translesion synthesis and sister-chromatid cohesion components (Fig. 1b). Our screen also identified the ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation Yana van der Weegen 1,10 , Klaas de Lint 2,10 , Diana van den Heuvel

BackgroundClinical trials to test safety and efficacy of drugs for patients with spinal muscular atrophy (SMA) are currently underway. Biomarkers that document treatment-induced effects are needed because disease progression in childhood forms of SMA is slow and clinical outcome measures may lack sensitivity to detect meaningful changes in motor function in the period of 1–2 years of follow-up during randomized clinical trials.ObjectiveTo determine and compare SMN protein and mRNA levels in two cell types (i.e. PBMCs and skin-derived fibroblasts) from patients with SMA types 1–4 and healthy controls in relation to clinical characteristics and SMN2 copy numbers.Materials and methodsWe determined SMN1, SMN2-full length (SMN2-FL), SMN2-delta7 (SMN2-Δ7), GAPDH and 18S mRNA levels and SMN protein levels in blood and fibroblasts from a total of 150 patients with SMA and 293 healthy controls using qPCR and ELISA. We analyzed the association with clinical characteristics including disease severity and duration, and SMN2 copy number.ResultsSMN protein levels in PBMCs and fibroblasts were higher in controls than in patients with SMA (p<0.01). Stratification for SMA type did not show differences in SMN protein (p>0.1) or mRNA levels (p>0.05) in either cell type. SMN2 copy number was associated with SMN protein levels in fibroblasts (p = 0.01), but not in PBMCs (p = 0.06). Protein levels in PBMCs declined with age in patients (p<0.01) and controls (p<0.01)(power 1-beta = 0.7). Ratios of SMN2-Δ7/SMN2-FL showed a broad range, primarily explained by the variation in SMN2-Δ7 levels, even in patients with a comparable SMN2 copy number. Levels of SMN2 mRNA did not correlate with SMN2 copy number, SMA type or age in blood (p = 0.7) or fibroblasts (p = 0.09). Paired analysis between blood and fibroblasts did not show a correlation between the two different tissues with respect to the SMN protein or mRNA levels.ConclusionsSMN protein levels differ considerably between tissues and activity is age dependent in patients and controls. SMN protein levels in fibroblasts correlate with SMN2 copy number and have potential as a biomarker for disease severity.

Transcription-blocking DNA lesions are removed by transcription-coupled nucleotide excision repair (TC-NER) to preserve cell viability. TC-NER is triggered by the stalling of RNA polymerase II at DNA lesions, leading to the recruitment of TC-NER-specific factors such as the CSA–DDB1–CUL4A–RBX1 cullin–RING ubiquitin ligase complex (CRLCSA). Despite its vital role in TC-NER, little is known about the regulation of the CRLCSA complex during TC-NER. Using conventional and cross-linking immunoprecipitations coupled to mass spectrometry, we uncover a stable interaction between CSA and the TRiC chaperonin. TRiC’s binding to CSA ensures its stability and DDB1-dependent assembly into the CRLCSA complex. Consequently, loss of TRiC leads to mislocalization and depletion of CSA, as well as impaired transcription recovery following UV damage, suggesting defects in TC-NER. Furthermore, Cockayne syndrome (CS)-causing mutations in CSA lead to increased TRiC binding and a failure to compose the CRLCSA complex. Thus, we uncover CSA as a TRiC substrate and reveal that TRiC regulates CSA-dependent TC-NER and the development of CS.