PROCESOS AÑO 2012:

5.6.1

Small RNAs and the cell cycle

Our small RNA analysis across the cell cycle was partly limited by technical obstacles during the generation of small RNA libraries, which prevented us from discovering new cell-cycle dependent small RNAs. A very recent development suggests participation of small RNAs in the double-stranded DNA break repair (Francia, Michelini et al. 2012; Michalik, Bottcher et al. 2012; Wei, Ba et al. 2012; Zhang, Chang et al. 2013). Since the genome is continuously challenged by a variety of genotoxic stresses, DNA double-strand breaks (DSBs) represent the most lethal type of damage and require efficient and accurate repair. To deal with DSBs, cells are equipped with non-homologous end-joining (NHEJ) and the homologous recombination (HR) pathway. NHEJ joins two DNA ends irrespective of their sequence thus presenting an efficient but error-prone mode of repair, which takes place throughout the cell cycle. In contrast, HR is dependent on DNA resection of the DSB and a sister chromatid as template to perform error-free repair, which is limited to the late S and G2 phases. Thus cell cycle phases are the major determinant for which repair pathway will be used.

In further experiments, Drosophila S2 cells could be synchronized into different cell cycle stages by the gentle method of centrifugal elutriation and afterwards treated to generate DSBs. The induction of DSBs can be executed either unspecificly via treatment with ultraviolet (UV) radiation or DNA damaging agents or more specifically by light-inducible endonucleases (Schierling and Pingoud 2012). After induction of DNA damage, deep sequencing of small RNAs over a time course will allow to determine the time of occurrence of DSB-induced small RNAs as well as their abundance and change in context of the cell cycle. In parallel, synchronized cells after the induction of DSBs will be monitored for the cell cycle progression by usage of bromodeoxyuridine (BrdU) assay combined with other cell cycle phase specific DNA dyes. DSB-induced small RNAs are speculated to depend on the resection of DSB (Michalik, Bottcher et al. 2012; Wei, Ba et al. 2012), which occurs as part of the HR repair pathway mainly occurring during the S and G2 cell cycle phase. Therefore corresponding small RNAs are suggested to be detected in those cell cycle phases.

In case of identification of specific small RNAs generated at the flanking regions of a DSB, they can further be inhibited by transfecting LNA inhibitors or usage of small tandem target mimics (STTM) to finally monitor

how they will affect the repair of specific DNA damage sites. The cell cycle arrest might be released after re- addition of the specific small RNAs presenting direct association with cell cycle.

The recognition and signaling to the DSB repair machinery are induced by phosphorylation of histone H2AX which occurs independently of small RNAs (Wei, Ba et al. 2012), indicating that they act downstream as guide molecules directing chromatin modifications or the recruitment of protein complexes to DSB sites. Since a lot of changes occur after DNA damage, it is interesting to elucidate if the occurrence of DSB- induced small RNAs correlates with specific chromatin modification patterns. To do so, chromatin immunoprecipitation after specific introduction of DSB will allow capturing the fraction of the genome which carries the histone modification of interest. Afterwards bisulphate sequencing on the immunoprecipitated material can map the DNA methylation pattern. Comparing these results with the chromatin pattern from cells after inhibition DSB-induced small RNAs will define a potential correlation between both pathways.

5.6.2

Somatic piRNA-like RNAs (pilRNAs)

Somatic piRNA-like RNAs (pilRNAs) are supposed to be expressed in specific types of somatic cells. A recent method referred as TU-tagging was described to enable intact cell type-specific RNA isolation e.g. cell types from central nervous system which are hardly attainable by dissection (Miller, Robinson et al. 2009). TU- tagging is based on the cell type specific expression of UPRT which is achieved via mating flies encoding for the transcription factor Gal4 under the control of a tissue-specific promoter with GAL-4-inducible transgenic UAS-UPRT (upstream activating sequence-UPRT) flies. In the offspring, expressed UPRT couples ribose-5-phosphate to the N1 nitrogen of 4-thiouracil supplied in the food. The resulting product is incorporated into RNA, then coupled with thio-biotin in vitro and finally purified via streptavidin. It should be possible to isolate small RNAs via gel purification and generate small RNA libraries for subsequent deep sequencing. In addition, mRNAs from the isolated RNA can be checked for PIWI, AUB and AGO3 expression by qRT-PCR to examine the expression of pilRNA biogenesis factors.

5.6.3

Loqs-PD and R2D2 in endo-siRNA pathway

The redundancy observed between R2D2 and Loqs-PD in endo-siRNA pathway could be further elucidated by focusing on the redundant functional role of different domains encoded in both proteins. To do so, hybrid proteins are generated by exchange of domains between Loqs and R2D2. In our laboratory a lot of work was already performed to generate a variety of such hybrid proteins, which could be introduced into

loqs or r2d2 mutant flies. The recently published cytoplasmic D2 body was suggested to be the cellular location where endo-siRNAs are loaded onto Ago2 (Nishida, Miyoshi et al. 2013). Dcr-2 and R2D2 are required for D2 body formation but function distinctly as Dcr-2 stabilizes R2D2 whereas R2D2 localizes Dcr- 2 to D2 bodies. Furthermore, the dsRNA-binding activity is necessary for R2D2 to localize to D2 bodies. Taken together, analysis of hybrid proteins in context of the D2 body formation will expand our understanding of the redundancy of Loqs-PD and R2D2 in the endo-siRNA pathway.