Fibrillarin Is Essential for Early Development and Required for Accumulation of an Intron-Encoded Small Nucleolar RNA in the Mouse

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American Society for Microbiology

RESUMO

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 β-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.

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