SMN, the causative factor in SMA, is conserved from flies to humans. Recent work has shown that the Drosophila model allows identification of proteins that ameliorate the Smn loss-of-function phenotype. We found that RNAi-mediated depletion of Smn in fly neurons results in locomotion and post-eclosion defects that are ameliorated by overexpression of Tgs1, whose loss results in a phenotype similar to that elicited by Smn depletion. TGS1 (Trimethyl Guanosine Synthetase 1) adds a trimethylguanosine cap (TMG cap) to several noncoding RNAs including the snRNAs, the telomerase RNA subunit, and viral RNAs, favouring their compartmentalization in their district of function. TGS1 is enriched at the Cajal body (CB) and its catalytic activity is related to the Survival of Motor Neuron (SMN) complex, whose function is essential to prevent development of Spinal Muscular Atrophy (SMA) in humans. Our preliminary results indicate that Drosophila TGS1 (dTgs1) functions in the same pathways as its human homologue TGS1. One of these pathways involves the SMN complex and appears to be essential for proper locomotory activity. We plan to obtain novel insights into the functional relationships between TGS1 and SMN in the nervous system. We will perform genome-wide RNA seq aimed at the identification of Smn and Tgs1 RNA targets.
SMA is a hereditary neuromuscular disorder and one of the main causes of infant death. No specific therapy to cure SMA has yet been identified. Since most clinical trials for SMA are currently aimed at increasing the amount of the SMN protein by either drug treatments or molecular therapy, the identification of new modifiers of SMN function might provide novel targets for treatments to ameliorate the disease. The identification of such modifiers requires detailed knowledge of the critical steps in the SMN pathway. The Drosophila model of SMA has proven to be a powerful system to assess directly the consequences of mutations in individual components of the SMN pathway in various cell types and at the organismal level, and has helped uncover important aspects of SMA pathogenesis in humans.
The RNA seq experiments we propose are in line with the most recent investigations on SMN mutants in different model systems. A detailed knowledge of the RNA processing changes occurring upon impairment of the SMN pathway is instrumental to understand the molecular mechanisms underlying SMA pathogenesis. RNA seq from larval brain extracts has been previously carried out using a different UAS-Smn RNAi construct and a different pan-neuronal driver (ELAV-GAL4) (28). The authors pointed out that several controls should be carried out to avoid cross contamination from neighboring cells that are not targeted by the expression of the Smn RNAi construct and might therefore impair RNA seq analyses. The same authors concluded that upon performing appropriate controls (that are suggested in detail in their paper) the RNA seq data from brains are both reliable and informative and reveal differences between wild type and RNAi brains. We would like to note that in our RNA seq experiments from brains we will perform all controls suggested by Amaral et al., (2014). Should our first analyses reveal insufficient separation between mutant and control datasets, we will perform ubiquitous expression of the RNAi construct driven by an actin-GAL4 driver rather than pan-neuronal driver. A similar approach has been used to determine the transcriptome profile from pupal heads of TDP43 RNAi animals (29). We would love to be able to analyze RNA expression only in a specific set of cells, rather than in whole brains. For example, it would be very interesting to combine the expression of the UAS-SmnRNAi transgene with a UAS-GFP transgene and use cell sorting (of dissected brain cells) to isolate GFP-positive cells that also express the RNAi construct, and use them for appropriate comparisons and analyses. A cell sorting-based experiment is not included in this grant proposal, as at the moment we do not have the necessary competence for this type of work. However, we hope we will be able to gain it in the next moths, as we plan to perform some pilot experiments following the procedures described in (30), in collaboration with colleagues who have full access to a FACS machine and have expertise in its use.
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