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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

Authors

  • Newton K
  • Petfalski E
  • Tollervey D
  • C├íceres JF

Journal

Molecular and cellular biology

Publication Data

December 12, 2003

Associated Grants

  • Agency: Medical Research Council, Id: MC_U127584479

Mesh Terms

  • 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