Fibrillarin is essential for early development and required for accumulation of an intron-encoded small nucleolar RNA in the mouse.
Fibrillarin, a protein component of C/D box small nucleolar ribonucleoproteins (snoRNPs), directs 2'-O-methylation of rRNA and is also involved in other aspects of rRNA processing. A gene trap screen in embryonic stem (ES) cells resulted in an insertion mutation in the fibrillarin gene. This insertion generated a fusion protein that contained the N-terminal 132 amino acids of fibrillarin fused to a beta-galactosidase-neomycin phosphotransferase reporter. As a result, the N-terminal GAR domain was present in the fusion protein but the methyltransferase-like domain was missing. The ES cell line with the targeted fibrillarin allele was transmitted through the mouse germ line, creating heterozygous animals. Western blot analyses showed a reduction in fibrillarin protein levels in the heterozygous knockout animals. Animals homozygous for the mutation were inviable, and massive apoptosis was observed in early Fibrillarin(-/-) embryos, showing that fibrillarin is essential for development. Fibrillarin(+/-) live-born mice displayed no obvious growth defect, but heterozygous intercrosses revealed a reduced ratio of +/- to +/+ mice, showing that some of the Fibrillarin heterozygous embryos die in utero. Analyses of tissue samples and cultured embryonic fibroblasts showed no discernible alteration in pre-rRNA processing or the level of the U3 snoRNA. However, the level of the intron-encoded box C/D snoRNA U76 was clearly reduced. This suggests a high requirement for snoRNA synthesis during an early stage in development.
Pubmed ID: 14612397 RIS Download
Amino Acid Sequence | Animals | Apoptosis | Base Sequence | Chromosomal Proteins, Non-Histone | Embryonic and Fetal Development | Female | Gene Expression Regulation, Developmental | Heterozygote | Introns | Male | Mice | Mice, Inbred C57BL | Mice, Knockout | Molecular Sequence Data | Mutagenesis, Insertional | Phenotype | Pregnancy | RNA Processing, Post-Transcriptional | RNA, Small Nucleolar | Recombinant Fusion Proteins