RNA dependent DNA damage response

Genetic information stored in DNA is continuously exposed to endogenous or exogenous damaging factors. Efficient DNA damage repair is a fundamental process for every living organism. The accumulation of DNA damage affects cellular viability and leads to a variety of diseases, particularly cancer. Therefore, understanding of the molecular mechanisms necessary for DNA damage repair is of great importance. A myriad of repair factors target double-strand breaks (DSBs) by non-homologous end joining (NHEJ) or homologous recombination (HR) pathways (1, 2). However, the relevance of RNA for the DNA damage response (DDR) is currently not understood and the impact of transcription on genome stability and the role of RNA binding factors as well as function of small RNA (DDRNA) remain enigmatic. Our proposed research aims to elucidate molecular mechanism of RNA dependent DDR. Our results might pave a new way for design of RNA based cancer therapies as well as discovery of new biomarkers.

Transcription at the site of DNA damage

RNA polymerase II (RNAPII) transcription is a highly regulated and essential process. The carboxy-terminal domain (CTD) of human RNAPII consists of 52 repeats of the consensus heptad Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, which undergo dynamic, regulatory phosphorylations. Phosphorylation of CTD S2/S5 is well-characterised hallmark of active transcription of protein-coding genes; CTD Y1 phosphorylation (CTD Y1P) is less understood. CTD Y1P functions as anti-termination signal by blocking recruitment of termination factors to actively transcribing RNAPII in S. cerevisiae. In mammals, CTD Y1P is enriched at promoters and associated with antisense transcription. Transcription is generally regarded as a threat for genome stability and globally impaired by physical blockage, concomitant with heterochromatin formation during DNA repair. However, global analysis of nascent RNA levels identified a subset of damage-induced short, non-coding transcripts, preceding inhibition of RNAPII elongation. Intriguingly, DSBs are repaired faster, when induced in actively transcribed loci.

To investigate RNAPII in response to DNA damage, we employed a U2OS cell line harboring the 4-Hydroxytamoxifen (4OHT)-inducible endonuclease AsiSI-ER (AsiSI-ER U2OS), which allows localised sequence-specific induction of DSBs and new generation sequencing methods.

The role of human nuclear Dicer in DNA damage response

The DNA damage response (DDR) is crucial for the maintenance of genome stability. A number of mechanisms exist to recognize and repair DNA lesions. The homologous recombination and non-homologous end-joining (NHEJ) pathways repair double strand breaks (DSBs). Recently, a new class of small regulatory RNA has been discovered in higher eukaryotes: site-specific DNA-damage RNA (DDRNA). DDRNA originate from both strands of DSBs and function at sequences that are in close proximity to these lesions. Drosha and Dicer processing of dsRNA precursors may mediate the maturation of DDRNA. The activation of major DNA repair factors, such as ATM is diminished upon Drosha or Dicer deletion, which underscores the connection between DDRNA, nuclear RNAi factors and DSB repair. We are currently investigating how Dicer and Drosha function in DNA damage response. Experiments and analysis are performed by Dr. Kaspar Burger.

Human nuclear Dicer is processing misfolded tRNA

Dicer is a type III endoribonuclease and a core part of the RNA interference (RNAi) machinery. RNAi involves processing of double-stranded RNA into small precursors that can have diverse effects but typically involve a repressive effect on a target nucleic acid. We have shown previously (White et al., NSMB, 2014) that Dicer also acts in nucleus to process endogenous double stranded RNA. Transfer RNAs are 75 nt RNAs that constitute 15% of total cellular RNA. There are 500 tRNA genes (tDNAs) in the human genome, of which only 20% are unique. After initial transcription as precursors by RNA polymerase III (pol III), 50 and 30 leader sequences are removed from pre-tRNAs by RNase P and RNAse Z, respectively. A large number of nucleobase modifications are known to occur in tRNAs some of which are thought to aid in the folding into the functional cloverleaf structure. The present study builds on Dicer association with chromatin in human cells, focusing on Dicer occupancy of transfer RNA (tRNA) genes. Our bioinformatics analysis shows that Dicer binds to actively transcribed tRNA, most likely through dsRNA. Knockdown of Dicer does not perturb transcription of selected tDNAs, although a cryptic alternative tRNA structure is observed upon knockdown of Dicer, suggesting a role for Dicer in tRNA quality control.

Classification and functional annotation of endogenous-siRNAs and other small RNAs

Over the course of the last 10 years the role of small RNAs (sRNA) in the regulation of eukaryotic cell biology and gene expression has become well established. However, while microRNAs (miRNAs) are now recognised as a relatively ubiquitous method by which cells control RNA and protein levels, it has recently become clear that endogenous siRNAs (endo-siRNAs) may also play a significant role in the regulation of expression in mammals. In order to better understand this class of molecule and their relationship to other sRNAs it is imperative that tools be developed. Such tools would allow the community to distinguish endo-siRNAs from amongst other sRNA classes in Next generation sequencing data (NGS). The identification of more widespread endo-siRNA expression has coincided with evidence that mammalian endo-siRNAs may play a key role in orchestrating histone modifications to silence transcription and affect alternative splicing. Tools that can predict this process will help guide research by the wider scientific community and significantly improve our knowledge of the regulatory effect of these sRNAs and their contribution to disease states. Experiments and analysis are performed by Dr. Matthew Davis in collaboration with Dr. Anton Enright.