Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by a deficiency of the Survival Motor Neuron (SMN) protein. SMN, the causative factor of SMA, has a crucial role in snRNPs biogenesis as cells defective for SMN result in aberrantly spliced transcripts. SMN is strickly linked to TGS1, an S-adenosyl-L-methionine dependent methyltransferase responsible for snRNAs cap trimethylation. Our recent work shows a physical and genetic interaction between TGS1 and all the subunits of Drosophila Smn complex. Loss of dTgs1 results in neurodegenerative phenotypes similar to those caused by Smn loss in fly eye imaginal discs, indicating that both dTgs1 and dSmn are required for viability of retinal progenitor cells (1). Loss of TGS1 leads to accumulation of immature snRNA with extended 3' tail both in Drosophila melanogaster and HeLa cells systems. In line with the accumulation of defective snRNAs, sequencing analyses performed on TGS1 and SMN mutant cells reveal a partial overlapping of transcriptome alterations such as unspliced and readthrough transcripts, probably due to incorrect transcription termination. We aim at understanding how 3' extended snRNAs and defective transcripts contribute to neurodegeneration in SMA. Integrating Illumina RNA sequencing with Oxford nanopore technologies, we plan to perform an analysis on Drosophila neuronal tissues like brains and eye imaginal discs as well as HeLa cells in order to gain insight into transcriptome alterations linked to loss of TGS1 and SMN. The power of using long read nanopore sequencing allows the reconstruction of full-length RNA molecules so as to circumvent the ambiguity generated by imprecise attribution of RNA-seq reads to a specific isoform.
Perturbation in RNA metabolism due to defects in various aspects of RNA biogenesis and maturation, is one of the causes leading to neurodegeneration. Accumulation of improperly processed snRNAs precursors upon TOE1 mutations lead to PCH7 disease (11) and loss of TGS1 results in accumulation of unprocessed snRNA precursors that might interfere with spliceosome activity. Our findings provide an insight into the role of TGS1 in preventing neurodegeneration by supporting the involvement of TGS1 in SMA pathogenesis both in human cells and in flies tissues (D. melanogaster).
Illumina deep sequencing analysis on TGS1 and SMN mutant HeLa cells revealed an accumulation of extensive alterations in the human transcriptome, including changes in the efficiency of intron removal and accumulation of transcripts with 3'extensions spanning intergenic regions. This characterization will be improved by Oxford nanopore technologies sequencing, a promising opportunity to improve the efficiency in isoform identification and quantification using long reads for the isoform reconstruction. We hope that, by combining Illumina and Nanopore sequencing approach, we will refine our analyses of aberrant transcripts in human cells. To the best of our knowledge, this is the first time that the Nanopore technology is used to characterize complex transcriptomes and the relationships between splicing and transcription termination. We hope that this powerful technology will enhance the accurancy of the analysis, boosting the investigation of transcript isoforms generated in different mutant backgrounds.
Besides, this kind of analysis performed in Drosophila tissues (eye imaginal discs and brains) will be particularly informative, in order to establish a link between aberrant transcripts and neurodegeneration. We plan to unravel the causative relationships between the accumulation of aberrantly spliced transcript isoforms and neuronal death, by performing transcriptome analyses on larval retinal tissues of the fly model for SMA that we recently developed. These bioinformatic analyses will help to understand how TGS1 and SMN loss affect the expression and splicing of transcripts that are relevant for the development and survival of neuronal cells and crucial aspects in snRNAs biogenesis that are necessary for neuronal stability. Using RNA-seq analysis is undoubtedly helpful to evaluate the transcriptome dynamics in order to compare developmental and physiological changes in normal or mutant tissues or cells, highlighting altered conditions that could induce pathological traits.