The role of transcriptional stress in Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is caused by reduced levels of the survival motor neuron (SMN) protein, which is essential for snRNP biogenesis and splicing. The snRNA cap hypermethylase TGS1 cooperates with SMN, and its depletion causes neurological phenotypes similar to SMN loss in different animal models. We previously showed that Smn deficiency in Drosophila leads to accumulation of RNA:DNA hybrids (R-loops), DNA damage and activation of the innate immune response. Here we asked whether TGS1 loss triggers a comparable transcriptional stress. In third instar larval brains, Tgs1 RNAi increased both S9.6- and γH2Av-positive cells, as Smn RNAi does. Short- and long-read sequencing (Illumina and Nanopore) revealed splicing defects in both mutants and identified 376 genes misregulated upon Tgs1 depletion. Genes shared with Smn mutants include antimicrobial peptides, the cytokines upd2 and upd3, and replication-dependent histones. Tgs1 depletion reduced histone transcripts and caused accumulation of 3′-extended histone mRNAs. Histone protein levels also fell, with H2A, H2B and H3 reduced by 20–33% in larval brains. We profiled Tgs1 RNAi embryos by CUT&Tag and ATAC-seq in collaboration with the Iovino group (MPI Freiburg). Chromatin compaction did not change globally, but Pol II Ser5P and Ser2P were reduced and promoter-proximal pausing increased, notably at histone gene clusters and at U2 and U5 snRNA loci. Increasing U2/U5 snRNA dosage reduced R-loops and DNA damage in both Tgs1- and Smn-depleted brains, improved eye development and rescued 24% of the splicing events altered by Tgs1 depletion. These results suggest that TGS1, like SMN, protects against transcriptional stress by linking snRNA biogenesis, histone 3′-end processing and genome stability.