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Molecular cloning and sequence analysis of U3 snoRNA-associated mouse fibrillarin
S J Turley1, E M Tan, K M Pollard
1W.M. Keck Autoimmune Disease Center, Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037.
Abstract:
We have isolated and determined the sequence of a 1.1-kb cDNA from a murine WEHI-3 macrophage library which encodes the highly conserved, nucleolar protein, fibrillarin. The murine fibrillarin protein sequence displays 94.2% identity with human fibrillarin, 82.9% identity with amphibian fibrillarin and 74.0% identity with the yeast fibrillarin homolog, NOP1. Immunoprecipitation showed that anti-fibrillarin autoantibodies from human scleroderma sera and the monoclonal autoantibody 72B9 recognize the approx. 34-36 kDa in vitro transcribed and translated protein. Mouse fibrillarin contains a N-terminal glycine- and arginine-rich (GAR) domain which although conserved among the fibrillarins is not as strongly conserved as several regions in the carboxy tail of the protein. Specific amino acid residues in yeast NOP1 thought to be associated with the synthesis and maturation of ribosomes show strong conservation between the mouse, human, amphibian and yeast protein sequences.
Insights
Researchers identified the mouse fibrillarin protein, crucial for ribosome synthesis. This highly conserved nucleolar protein shares significant sequence identity with human, amphibian, and yeast homologs, aiding in understanding its function.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Fibrillarin is a highly conserved nucleolar protein essential for ribosome biogenesis.
- Understanding fibrillarin's structure and function across species can provide insights into cellular processes and disease mechanisms.
Purpose of the Study:
- To isolate and determine the sequence of murine fibrillarin.
- To analyze the sequence conservation of mouse fibrillarin with homologs in other species.
- To investigate the recognition of mouse fibrillarin by autoantibodies.
Main Methods:
- Isolation and sequencing of a 1.1-kb cDNA encoding murine fibrillarin from a WEHI-3 macrophage library.
- Sequence comparison of murine fibrillarin with human, amphibian, and yeast fibrillarin/NOP1.
- In vitro transcription and translation of mouse fibrillarin.
- Immunoprecipitation assays using human scleroderma sera and a monoclonal antibody (72B9).
Main Results:
- The murine fibrillarin cDNA sequence was determined.
- Murine fibrillarin exhibits high sequence identity (94.2%) with human fibrillarin, and significant identity with amphibian (82.9%) and yeast (74.0%) homologs.
- Autoantibodies from scleroderma patients and monoclonal antibody 72B9 recognized the translated murine fibrillarin protein (approx. 34-36 kDa).
- A conserved N-terminal glycine- and arginine-rich (GAR) domain and conserved regions in the carboxy tail were identified.
- Key residues in yeast NOP1 involved in ribosome synthesis are conserved in mouse fibrillarin.
Conclusions:
- The study successfully characterized murine fibrillarin, confirming its high conservation across species.
- The findings support fibrillarin's conserved role in ribosome biogenesis.
- The recognition by autoantibodies suggests potential relevance in autoimmune diseases like scleroderma.