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The yeast PRP8 protein interacts directly with pre-mRNA
1Institute of Cell and Molecular Biology, University of Edinburgh, UK.
Abstract:
The PRP8 protein of Saccharomyces cerevisiae is required for nuclear pre-mRNA splicing. Previously, immunological procedures demonstrated that PRP8 is a protein component of the U5 small nuclear ribonucleoprotein particle (U5 snRNP), and that PRP8 protein maintains a stable association with the spliceosome during both step 1 and step 2 of the splicing reaction. We have combined immunological analysis with a UV-crosslinking assay to investigate interaction(s) of PRP8 protein with pre-mRNA. We show that PRP8 protein interacts directly with splicing substrate RNA during in vitro splicing reactions. This contact event is splicing-specific in that it is ATP-dependent, and does not occur with mutant RNAs that contain 5' splice site or branchpoint mutations. The use of truncated RNA substrates demonstrated that the assembly of PRP8 protein into splicing complexes is not, by itself, sufficient for the direct interaction with the RNA; PRP8 protein only becomes UV-crosslinked to RNA substrates capable of participating in step 1 of the splicing reaction. We propose that PRP8 protein may play an important structural and/or regulatory role in the spliceosome.
Insights
The PRP8 protein directly interacts with pre-mRNA during nuclear pre-mRNA splicing. This interaction is specific, ATP-dependent, and crucial for the first step of the splicing reaction.
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.