Nucleolar R-loops as contributors to Spinal muscular atrophy pathophysiology

Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder characterized by degeneration of lower motor neurons, that leads to muscle weakness, and eventually death. SMA is caused by low levels of survival motor neuron (SMN) protein, due to deletion or mutation of SMN1 gene. SMN is a ubiquitously expressed protein, primarily involved in pre-mRNA splicing through its “canonical” function as chaperone in the assembly of small nuclear Ribonucleoproteins (snRNP); additionally, it is believed that it also plays a role during transcription termination by interacting with RNA pol II and Senataxin, a helicase involved in the resolution of DNA/RNA hybrids (R-loops). However, the mechanism through which depletion of SMN disturbs genome stability and contributes to the SMA phenotype remains unclear.

In this project I investigated the presence of R-loops in human cells to determine if these structures contribute to the physiopathology of SMA. R-loops are three stranded nucleic acid structures, usually formed during transcription when the messenger RNA (mRNA) hybridises with the complementary DNA strand, leaving the other DNA strand exposed. For this experiment, fibroblasts from SMA patients overexpressing an enhanced green fluorescent protein (EGFP), fused with the hybrid binding domain (HB) from RNaseH1, an endonuclease that resolves R-loops was used. The fusion protein acted as a R-loop capture agent for the Chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) approach. Further, I analysed the levels of other markers and proteins associated with DNA damage, to assess the degree of cellular stress present in SMA.