Pathomechanisms and Therapy in PUF60-related disorders

• Applicant: Privatdozent Dr. Hormos Salimi Dafsari
• Subject Area: Human Genetics
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 578221361

Splicing is an essential step in mRNA processing that removes intron regions and generates mature transcripts capable of encoding proteins. This process is catalyzed by the spliceosome, a highly dynamic ribonucleoprotein complex. Mutations in spliceosomal components lead to impaired splicing, aberrant transcripts, dysregulated cellular function, and various developmental and degenerative diseases. PUF60 encodes an essential splicing factor in humans that regulates the splicing process. Pathogenic variants in PUF60 cause a rare genetic disorder ranging from neurodevelopmental disorders to short stature, craniofacial dysmorphia, and skeletal abnormalities. The exact splicing defect leading to PUF60-related disorders remains unclear to this date, especially in regards to its metabolic defect. Here, we will investigate the pathomechanisms of PUF60-related disorders in patient cells, from its splicing defect to metabolic dysregulation, overarchingly aiming to identify a treatment with Vitamin B12 and methionine. We observed a patient with a PUF60-related disorder that was treated with Vitamin B12 for the past 10+ years and shows a significantly ameliorated phenotype. Previous work showed that PUF60 knockdown suppresses mTORC1 activity in HEK293FT cell lines. Our proof-of-principle work indicated that (i) aberrant splicing of NAA35 in patient peripheral blood mononuclear cells (PBMCs) which is part of the NatC complex involved in the N-terminal acetylation of mTOR, (ii) mTOR is downregulated and autophagolysosomal proteins are upregulated in patient PBMCs, (iii) methionine is downregulated in patient plasma, (iv) mTOR activity is downregulated at baseline in three patient fibroblast cell lines, and Vitamin B12 (VB12) and methionine (Met) treatment for 24 hours can rescue mTOR activity in these patient cells. Based on our proof-of-principle work, we now aim to examine these molecular changes in PUF60-related disorders by investigating neuronal cell lines. This will enable us to very clearly investigate for 1) neuronal morphology, 2) NAA35 aberrant splicing, 3) dysregulation in mTOR and autophagolysosomal flux, and 4) methionine depletion at baseline. In addition, we will expand our treatment with Vitamin B12 and methionine to these neurons and examine the rescue of neuronal morphology as well as mTOR activity and autophagolysosomal flux. The results will lay the groundwork for developing a targeted and easily tolerable therapeutic option for PUF60-related disorders. Beyond a singular disease, this knowledge will provide valuable insights into splicing disorders and their metabolic regulation in human disease.

• DFG Programme: Research Grants