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A human protein required for the second step of pre-mRNA splicing is functionally related to a yeast splicing factor
1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
Abstract:
We have identified a human splicing factor required for the second step of pre-mRNA splicing. This new protein, hPrp18, is 30% identical to the yeast splicing factor Prp18. In HeLa cell extracts immunodepleted of hPrp18, the second step of pre-mRNA splicing is abolished. Splicing activity is restored by the addition of recombinant hPrp18, demonstrating that hPrp18 is required for the second step. The hPrp18 protein is bound tightly to the spliceosome only during the second step of splicing. hPrp18 is required for the splicing of several pre-mRNAs, making it the first general second-step splicing factor found in humans. Splicing activity can be restored to hPrp18-depleted HeLa cell extracts by yeast Prp18, showing that important functional regions of the proteins have been conserved. A 90-amino-acid region near the carboxyl terminus of hPrp18 is strongly homologous to yeast Prp18 and is also conserved in rice and nematodes. The homology identifies one region important for the function of both proteins and may define a new protein motif. In contrast to yeast Prp18, hPrp18 is not stably associated with any of the snRNPs. A 55-kD protein that cross-reacts with antibodies against hPrp18 is a constituent of the U4/U6 and U4/U6 x U5 snRNP particles.
Insights
Researchers identified hPrp18, a human splicing factor essential for the second step of pre-messenger RNA (pre-mRNA) splicing. This discovery reveals a conserved functional region important for splicing across species.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Pre-mRNA splicing is a fundamental process in gene expression.
- The second step of splicing is crucial for removing introns and joining exons.
- Specific protein factors are required to facilitate spliceosome activity.
Purpose of the Study:
- To identify and characterize human proteins involved in the second step of pre-mRNA splicing.
- To determine the role of a newly identified human splicing factor, hPrp18.
- To investigate the conservation of splicing factors between humans and other organisms.
Main Methods:
- Immunodepletion of hPrp18 from HeLa cell extracts.
- Restoration of splicing activity using recombinant hPrp18.
- Analysis of hPrp18 binding to the spliceosome.
- Cross-reactivity studies with antibodies against hPrp18.
Main Results:
- hPrp18 is essential for the second step of pre-mRNA splicing in human cells.
- hPrp18 binds to the spliceosome specifically during the second step.
- Yeast Prp18 can restore splicing activity to hPrp18-depleted extracts, indicating functional conservation.
- A conserved 90-amino-acid region suggests a critical functional domain.
Conclusions:
- hPrp18 is the first identified general second-step splicing factor in humans.
- Functional conservation of splicing mechanisms exists between humans and yeast.
- The conserved region in hPrp18 may represent a novel protein motif important for splicing.