Double-strand break interacting RNAs (diRNAs)

A new role for small RNAs in the repair of DNA double-strand breaks has been reported in Cell. Wei et al. have found diRNAs, derived from the vicinity of DNA double-strand breaks, in both Arabidopsis thaliana and human cells.

DNA double strand breaks (DSBs) are a particularly deleterious form of DNA damage as they can cause chromosomal translocations and induce cell death. To maintain the genome’s integrity, eukaryotic cells employ two different mechanisms of DSB repair. Non-homologous end joining (NHEJ) is an efficient mechanism that rapidly repairs DSBs without requiring an homologous template. However, NHEJ often causes insertions or deletions at the break site. Homologous recombination (HR) is a less error prone mechanism in which a sister chromatid is used as a template for repair. A specialised form of HR, single-strand annealing (SSA) that doesn’t require a sister chromatid can take place at repetitive sequences.

Wei et al. used an assay system that monitors DSB repair by SSA in the model plant Arabidopsis thaliana. A genetic cross causes a single DSB in an inactive reporter gene containing a repeat. SSA mediated repair restores the activity of the reporter gene and allows a quantitative and visible readout of DSB repair events. For instance, when this assay system is introduced (by crossing) into a genetic background mutant for atr (encoding a PI3 kinase known to be involved in DSB response), the researchers observed a large reduction in repair efficiency.

The first clue that suggested that small RNAs may be involved in double strand break repair came when they crossed their DSB repair assay system into lines mutant for Dicer-like proteins (DCL). Dicer and DCLs are responsible for the biogenesis of small RNAs (miRNAs and siRNAs) from double-stranded RNAs. Mutations in three different dcl genes (especially dcl3) all diminished the efficiency of DSB repair. The researchers therefore tried to examine whether small RNAs were produced from sequences adjacent to the DSB site. By probing northern blots with sequence flanking the DSB site, Wei et al detected a population of small RNAs approximately 21nt in length that were only present when DSBs had been induced. Deep sequencing (direct sequencing of RNA populations) revealed that these DSB-induced small RNAs (diRNAs) were specifically produced from the sequences flanking the DSB (approximately 800bp in each direction) and derived from both the sense and antisense strands in equal measure. By using a similar assay that monitored DSB repair by HR, they showed that diRNAs were also produced in this system.

In plants, a well characterised small RNA system mediates heterochromatic silencing of repetitive sequences by DNA methylation. Wei et al. used this pathway as a model to dissect the diRNA system. In the heterochromatic-siRNA system, single stranded RNA transcripts generated by the DNA-dependent RNA polymerase IV (Pol IV)  are converted to dsRNAs by the action of the RNA-dependent RNA polymerase 2 (RDR2). The dsRNAs are then cleaved into hc-siRNAs by Dicer-like proteins. When complexed with the Argonaute protein AGO4, hc-siRNAs direct de novo DNA methylation. By using the DSB assay system in backgrounds mutant for these factors and deep sequencing, Wei et al.  found that diRNA production requires the activity of Pol IV, RDR2 and RDR6 and DCLs, and that this pathway is under the control of DSB responsive kinase ATR. However, diRNA-mediated DSB repair does not involve RNA-directed DNA methylation pathway effector components such as AGO4. Instead, a different Argonaute protein AGO2 was found to complex diRNAs. Both diRNA accumulation and DSB repair were compromised in ago2 mutants.

Wei et al. went on to enquire as to whether diRNAs are involved in DSB repair in animals as well as plants. Using a similar HR mediated DSB repair assay in a human cell line, they showed small RNAs are also produced close to DSBs. Interestingly, whereas in plants the diRNAs were produced from sequences immediately neighbouring the DSB, in human cells they originated from a broader vicinity around the break site and not immediately adjacent. When Dicer or Ago2 were depleted in human cells DSB repair was compromised.

This paper has demonstrated the existence of a new class of small RNAs and their involvement in yet another important biological process. However, the details of how diRNAs act in DSB repair are completely unknown as yet. The authors suggest that diRNAs may guide histone modifications around the DSB site that facilitate DNA repair. Alternatively, diRNA-AGO2 complexes may be directly target DSB repair complexes to break sites. The assay systems used in this study only tested DSB repair by HR and SSA. It would be interesting to know whether diRNAs are also involved in DSB repair by NHEJ.

Wei, W., Ba, Z., Gao, M., Wu, Y., Ma, Y., Amiard, S., White, C., Danielsen, J., Yang, Y., & Qi, Y. (2012). A Role for Small RNAs in DNA Double-Strand Break Repair Cell DOI: 10.1016/j.cell.2012.03.002


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