Investigation of the structure and function of the spliceosomal 220K/Prp8 protein

Auteurs

• Alexey Rozov

Paramètres

• Manuels
• Manuels universitaires

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Splicing is a crucial phase in eukaryotic pre-mRNA maturation, where introns are removed and exons are joined to form a continuous mRNA template. This process is carried out by the spliceosome, a dynamic RNP machine composed of snRNP particles and various non-snRNP protein factors. The spliceosome assembles on pre-mRNA via conserved signal sequences: the 5' splice site, branch-point, and 3' splice site. Following assembly, significant remodeling occurs in the RNA structure and protein composition, positioning active groups for intron excision and exon ligation without external energy. However, the catalytic process details remain elusive. The 220K protein, a large spliceosomal component, is linked to all three signal sequences, suggesting its critical role in splicing regulation. Initially, knowledge of this protein was limited. This study investigates the 220K protein's domain structure and functions through domain dissection, enabling the creation of a soluble fragment for crystallization and structural analysis. The domain resembles RNase H family enzymes, indicating potential involvement in splicing catalysis. Mutagenesis studies in yeast were conducted to assess the significance of specific residues. Additionally, indications of RNase H-like activity and low-affinity binding of manganese ions to the domain were found, suggesting a more active role for the 220K protein in splicing than previously thought. A protocol for co

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• Année de publication: 2012