Insights into the role of ribonuclease 4 polymorphisms in amyotrophic lateral sclerosis

Abstract

Mutations in certain genes of the Ribonuclease (RNASE) superfamily can cause amyotrophic lateral sclerosis (ALS) through altered RNA processing mechanisms. About 30 of these missense mutations in RNASE5/ANG gene have already been reported in ALS patients. In another gene of the ribonuclease superfamily, ribonuclease 4 (RNASE4), missense mutations and single nucleotide polymorphisms have been identified in patients suffering from ALS. However, their plausible molecular mechanisms of association with ALS are not known. Here, we present the molecular mechanisms of RNASE4 polymorphisms with ALS using all-atom molecular dynamics (MD) simulations followed by functional assay experiments. As most ALS causing mutations in RNASE superfamily proteins affect either the ribonucleolytic or nuclear translocation activity, we examined these functional properties of wild-type and known RNASE4 variants, R10W, A98V, E48D and V75I, using MD simulations. Our simulation predicted that these variants would retain nuclear translocation activity and that E48D would exhibit loss of ribonucleolytic activity, which was subsequently validated by ribonucleolytic assay. Our results give a mechanistic insight into the association of RNASE4 polymorphisms with ALS and show that E48D-RNASE4 would probably be deleterious and cause ALS in individuals harbouring this polymorphism.

Keywords:

• amyotrophic lateral sclerosis
• loss-of-functions
• molecular dynamics
• polymorphism
• RNA processing pathway
• ribonuclease 4

Abbreviations:

• ALS: amyotrophic lateral sclerosis
• CD: circular dichroism
• DAPI: 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride
• FBS: foetal bovine serum
• GST: glutathione S-transferase
• HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
• IPTG: isopropyl β-D-1-thiogalactopyranoside
• MD: molecular dynamics
• Ni-NTA: Ni-nitrilotriacetic acid
• OD: optical density
• PBS: phosphate buffered saline
• RNASE4: ribonuclease 4
• RMSD: root mean square deviation
• SDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis
• TIP3P: three-point transferable intermolecular potential
• tRNA: transfer RNA
• VMD: visual molecular dynamics

Acknowledgements

Aditya K. Padhi acknowledges Council of Scientific and Industrial Research (CSIR), Government of India, for research fellowship. Priyam Narain and Upma Dave are supported by research fellowship from IIT Delhi. The authors acknowledge Prof B. Jayaram (Department of Chemistry and Kusuma School of Biological Sciences, IIT Delhi) and Prof Manidipa Banerjee (Kusuma School of Biological Sciences, IIT Delhi) for their valuable suggestions. The authors also thank Supercomputing Facility for Bioinformatics & Computational Biology (SCFBio), IIT Delhi for providing the computational facility to carry out molecular dynamics simulations.