The yeast PRP8 protein interacts directly with pre-mRNA

Area of Science:

• Molecular Biology
• RNA Splicing Mechanisms

Background:

• The PRP8 protein is essential for nuclear pre-mRNA splicing in Saccharomyces cerevisiae.
• Immunological studies identified PRP8 as a component of the U5 small nuclear ribonucleoprotein particle (U5 snRNP).
• PRP8 protein stably associates with the spliceosome throughout the splicing reaction's two steps.

Purpose of the Study:

• To investigate the interaction between PRP8 protein and pre-mRNA using a combination of immunological analysis and UV-crosslinking.
• To determine if PRP8 protein directly contacts splicing substrate RNA during the splicing process.

Main Methods:

• Immunological analysis
• UV-crosslinking assay
• In vitro splicing reactions using wild-type and mutant RNA substrates
• Analysis with truncated RNA substrates

Main Results:

• PRP8 protein directly interacts with splicing substrate RNA during in vitro splicing.
• This interaction is splicing-specific, requiring ATP and functional 5' splice sites and branchpoints.
• PRP8 protein UV-crosslinks to RNA only in substrates capable of undergoing the first step of splicing.

Conclusions:

• PRP8 protein plays a direct role in interacting with pre-mRNA during splicing.
• The interaction is dependent on the splicing reaction's progression, particularly step 1.
• PRP8 protein likely has a significant structural and/or regulatory function within the spliceosome.