Small Cajal body-associated RNA 2 (scaRNA2) regulates DNA repair pathway choice by inhibiting DNA-PK.

Evidence that long non-coding RNAs (lncRNAs) participate in DNA repair is accumulating, however, whether they can control DNA repair pathway choice is unknown. Here we show that the small Cajal body-specific RNA 2 (scaRNA2) can promote HR by inhibiting DNA-dependent protein kinase (DNA-PK) and, thereby, NHEJ. By binding to the catalytic subunit of DNA-PK (DNA-PKcs), scaRNA2 weakens its interaction with the Ku70/80 subunits, as well as with the LINP1 lncRNA, thereby preventing catalytic activation of the enzyme. Inhibition of DNA-PK by scaRNA2 stimulates DNA end resection by the MRN/CtIP complex, activation of ATM at DNA lesions and subsequent repair by HR. ScaRNA2 is regulated in turn by WRAP53ß, which binds this RNA, sequestering it away from DNA-PKcs and allowing NHEJ to proceed. These findings reveal that RNA-dependent control of DNA-PK catalytic activity is involved in regulating whether the cell utilizes NHEJ or HR.

Biallelic mutations in WRAP53 result in dysfunctional telomeres, Cajal bodies and DNA repair, thereby causing Hoyeraal-Hreidarsson syndrome.

Approximately half of all cases of Hoyeraal-Hreidarsson syndrome (HHS), a multisystem disorder characterized by bone marrow failure, developmental defects and very short telomeres, are caused by germline mutations in genes related to telomere biology. However, the varying symptoms and severity of the disease indicate that additional mechanisms are involved. Here, a 3-year-old boy with HHS was found to carry biallelic germline mutations in WRAP53 (WD40 encoding RNA antisense to p53), that altered two highly conserved amino acids (L283F and R398W) in the WD40 scaffold domain of the protein encoded. WRAP53ß (also known as TCAB1 or WDR79) is involved in intracellular trafficking of telomerase, Cajal body functions and DNA repair. We found that both mutations cause destabilization, mislocalization and faulty interactions of WRAP53ß, defects linked to misfolding by the TRiC chaperonin complex. Consequently, WRAP53ß HHS mutants cannot elongate telomeres, maintain Cajal bodies or repair DNA double-strand breaks. These findings provide a molecular explanation for the pathogenesis underlying WRAP53ß-associated HHS and highlight the potential contribution of DNA damage and/or defects in Cajal bodies to the early onset and/or severity of this disease.

The Cajal Body Protein WRAP53ß Prepares the Scene for Repair of DNA Double-Strand Breaks by Regulating Local Ubiquitination.

Proper repair of DNA double-strand breaks is critical for maintaining genome integrity and avoiding disease. Modification of damaged chromatin has profound consequences for the initial signaling and regulation of repair. One such modification involves ubiquitination by E3 ligases RNF8 and RNF168 within minutes after DNA double-strand break formation, altering chromatin structure and recruiting factors such as 53BP1 and BRCA1 for repair via non-homologous end-joining (NHEJ) and homologous recombination (HR), respectively. The WD40 protein WRAP53ß plays an essential role in localizing RNF8 to DNA breaks by scaffolding its interaction with the upstream factor MDC1. Loss of WRAP53ß impairs ubiquitination at DNA lesions and reduces downstream repair by both NHEJ and HR. Intriguingly, WRAP53ß depletion attenuates repair of DNA double-strand breaks more than depletion of RNF8, indicating functions other than RNF8-mediated ubiquitination. WRAP53ß plays key roles with respect to the nuclear organelles Cajal bodies, including organizing the genome to promote associated transcription and collecting factors involved in maturation of the spliceosome and telomere elongation within these organelles. It is possible that similar functions may aid also in DNA repair. Here we describe the involvement of WRAP53ß in Cajal bodies and DNA double-strand break repair in detail and explore whether and how these processes may be linked. We also discuss the possibility that the overexpression of WRAP53ß detected in several cancer types may reflect its normal participation in the DNA damage response rather than oncogenic properties.

Cyclosporin promotes neurorestoration and cell replacement therapy in pre-clinical models of Parkinson's disease.

BACKGROUND: The early clinical trials using fetal ventral mesencephalic (VM) allografts in Parkinson's disease (PD) patients have shown efficacy (albeit not in all cases) and have paved the way for further development of cell replacement therapy strategies in PD. The preclinical work that led to these clinical trials used allografts of fetal VM tissue placed into 6-OHDA lesioned rats, while the patients received similar allografts under cover of immunosuppression in an α-synuclein disease state. Thus developing models that more faithfully replicate the clinical scenario would be a useful tool for the translation of such cell-based therapies to the clinic. RESULTS: Here, we show that while providing functional recovery, transplantation of fetal dopamine neurons into the AAV-α-synuclein rat model of PD resulted in smaller-sized grafts as compared to similar grafts placed into the 6-OHDA-lesioned striatum. Additionally, we found that cyclosporin treatment was able to promote the survival of the transplanted cells in this allografted state and surprisingly also provided therapeutic benefit in sham-operated animals. We demonstrated that delayed cyclosporin treatment afforded neurorestoration in three complementary models of PD including the Thy1-α-synuclein transgenic mouse, a novel AAV-α-synuclein mouse model, and the MPTP mouse model. We then explored the mechanisms for this benefit of cyclosporin and found it was mediated by both cell-autonomous mechanisms and non-cell autonomous mechanisms. CONCLUSION: This study provides compelling evidence in favor for the use of immunosuppression in all grafted PD patients receiving cell replacement therapy, regardless of the immunological mismatch between donor and host cells, and also suggests that cyclosporine treatment itself may act as a disease-modifying therapy in all PD patients.