Biochemical defects in minor spliceosome function in the developmental disorder MOPD I

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• Biallelic mutations in the RNU4ATAC gene cause MOPD I/TALS, a developmental disorder.
• Nine RNU4ATAC mutations are known in MOPD I patients, all potentially affecting U4atac snRNA function and minor intron splicing.
• The precise molecular mechanisms underlying these effects remain unclear.

Purpose of the Study:

• To elucidate the molecular basis of MOPD I/TALS caused by RNU4ATAC mutations.
• To investigate how specific RNU4ATAC mutations impact U4atac snRNA expression, protein binding, and complex formation.
• To understand the link between these molecular defects and impaired minor intron splicing in patient cells.

Main Methods:

• In vitro and in vivo assays to assess U4atac snRNA expression and protein binding.
• Analysis of MOPD I patient fibroblasts and induced pluripotent stem cells (iPSCs).
• Glycerol gradient sedimentation and immunoprecipitation to study snRNP complex formation.

Main Results:

• One mutation (124G>A) significantly reduced U4atac snRNA expression.
• Four mutations (30G>A, 50G>A, 50G>C, 51G>A) impaired the binding of essential proteins to U4atac snRNA.
• Cells with the common 51G>A mutation showed reduced levels of the U4atac/U6atac.U5 tri-snRNP complex.
• Inefficient splicing of U12-dependent introns in patient cells was linked to defects in minor tri-snRNP formation.

Conclusions:

• RNU4ATAC mutations disrupt multiple aspects of minor snRNA function, including expression, protein association, and complex assembly.
• Defects in minor tri-snRNP formation underlie the inefficient splicing observed in MOPD I patient cells.
• This study establishes a mechanistic basis for MOPD I/TALS, linking RNU4ATAC mutations to specific molecular defects in the minor spliceosome.