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Multiple requirements for nematode spliced leader RNP function in trans-splicing
J A Denker1, P A Maroney, Y T Yu
1Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
RNA (New York, N.Y.)
|August 1, 1996
Summary
The SL RNA, crucial for nematode trans-splicing, requires specific protein interactions for function. Researchers identified four SL RNA-specific proteins that associate with core Sm proteins, impacting trans-splicing activity.
Area of Science:
- Molecular Biology
- RNA Biology
- Genetics
Background:
- The SL RNA is essential for nematode trans-splicing, a process similar to cis-splicing involving U snRNAs.
- Four key sequence elements in SL RNA are necessary for its function, but their roles and the protein composition of the functional SL RNP remain unclear.
Purpose of the Study:
- To elucidate the roles of essential sequence elements in SL RNA function.
- To determine the protein composition of the SL RNP and its role in trans-splicing.
Main Methods:
- Oligoribonucleotide affinity purification to isolate the SL RNP.
- In vitro assembly assays to study protein-RNA interactions.
- Mutational analysis and thiophosphate interference to investigate sequence requirements.
Main Results:
- The SL RNP contains core Sm proteins and four specific proteins (175, 40, 30, and 28 kDa).
- Association of 175- and 30-kDa SL-specific proteins correlates with trans-splicing function.
- Specific sequences within the Sm binding site and Stem-loop II are critical for SL RNA function.
- Activation of a cryptic 5' splice site suggests a role for U5 snRNA in splice site selection.
Conclusions:
- The identified SL-specific proteins are crucial for SL RNP function in trans-splicing.
- Both sequence and structural elements of SL RNA, along with specific protein binding, are vital for its activity.
- U5 snRNA may play a key role in specifying the 5' splice site during SL addition trans-splicing.