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The conserved pre-mRNA splicing factor U2AF from Drosophila: requirement for viability
R Kanaar1, S E Roche, E L Beall
1Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Science (New York, N.Y.)
|October 22, 1993
Summary
The Drosophila U2AF50 gene is essential for viability, as its protein product complements defective splicing in mammalian cells. This study enables in vivo analysis of conserved R/S domains in splicing factors.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The U2 small nuclear ribonucleoprotein auxiliary factor (U2AF) is crucial for spliceosome assembly in humans.
- The large subunit (hU2AF65) plays a vital role in this process.
- Conserved arginine- and serine-rich (R/S) domains are present in many splicing factors, but their function remains unclear.
Purpose of the Study:
- To isolate and characterize the Drosophila U2AF large subunit (dU2AF50).
- To investigate the essentiality of the dU2AF50 gene in vivo.
- To enable functional analysis of conserved R/S domains in splicing factors.
Main Methods:
- Isolation of a complementary DNA clone for dU2AF50.
- Expression and purification of recombinant dU2AF50 protein.
- Complementation assays with depleted mammalian splicing extracts.
- Germline transformation of Drosophila to rescue a lethal mutation.
Main Results:
- The dU2AF50 protein shares structural similarities with its mammalian counterpart, including RNA binding domains and an R/S domain.
- Recombinant dU2AF50 successfully restored splicing activity in mammalian extracts lacking U2AF.
- The dU2AF50 gene was confirmed as essential for Drosophila viability through genetic rescue.
Conclusions:
- The dU2AF50 gene is indispensable for life in Drosophila.
- The study provides a foundation for in vivo investigation of the function of R/S domains in splicing.
- This research highlights the conserved role of U2AF in mRNA splicing across species.