Functional and physical interaction between the yeast splicing factors Slu7 and Prp18

Area of Science:

• Molecular Biology
• RNA Splicing

Background:

• Actin pre-mRNA splicing involves multiple protein factors, including Prp18 and Slu7, which are crucial for the second catalytic step.
• The precise roles and interplay of these factors, particularly in relation to substrate sequence, are not fully elucidated.

Purpose of the Study:

• To investigate the specific conditions under which Prp18 is required for actin pre-mRNA splicing.
• To explore the functional relationship and physical interaction between Prp18 and Slu7 during splicing.

Main Methods:

• In vitro splicing assays using actin pre-mRNA with varying distances between the branch point and 3' splice site.
• Two-hybrid assays to detect physical interactions between Prp18 and Slu7.
• Analysis of Slu7 deletion mutants for cellular viability and interaction with Prp18.

Main Results:

• Prp18 is dispensable for splicing when the branch point-3' splice site interval is less than 12 nucleotides.
• Excess Slu7 can functionally replace Prp18 in vitro, suggesting a concerted mechanism.
• A direct physical interaction between Prp18 and Slu7 was confirmed via two-hybrid analysis.
• A specific region (residues 200-224) of Slu7 was identified as essential for Prp18 interaction.

Conclusions:

• The requirement for Prp18 in actin pre-mRNA splicing is context-dependent, influenced by the substrate's structural features.
• Prp18 and Slu7 function collaboratively in the second step of splicing, with Slu7 potentially compensating for Prp18 absence.
• The identified interaction domain provides insights into the molecular mechanism of spliceosome activation and catalysis.