Ribosomal RNA Biogenesis Research Articles

Dyskeratosis Congenita Caused by a 3′ Deletion: Germline and Somatic Mosaicism in a Female Carrier

AbstractX-linked dyskeratosis congenita (DC) is a bone marrow failure syndrome caused by mutations in the DKC1 gene located at Xq28. By 20 years of age, most affected boys develop bone marrow failure, whereas female carriers show a skewed pattern of X-chromosome inactivation. The gene product, dyskerin, is homologous to a yeast protein involved in ribosomal RNA biogenesis, providing a unique insight into a cause of aplastic anemia. Whereas most causative mutations are single amino acid substitutions, and nonsense or frameshift mutations have not been observed, we present here a case of DC caused by a 2-kb deletion that removes the last exon of the gene. Normal levels of mRNA are produced from the deleted gene, with the transcripts using a cryptic polyadenylation site in the antisense strand of the adjacent MPP1 gene, normally located 1 kb downstream of DKC1 in a tail to tail orientation. The predicted truncated protein lacks a lysine-rich peptide that is less conserved than the rest of the dyskerin molecule and is dispensable in yeast, supporting the contention that it may retain some activity and that null mutations at this locus may be lethal. The affected boy had an unaffected brother with the same haplotype around the DKC1 gene and a sister who was heterozygous for the deletion. We conclude therefore that the mother must be a germline mosaic with respect to this deletion. Investigation of her blood cells and other somatic tissues showed that a small proportion of these cells also carried the deletion, making her a somatic mosaic and indicating that the deletion took place early in development.

Development and application of an in vivo system to study yeast ribosomal RNA biogenesis and function

We have developed a system for mutational analysis of Saccharomyces cerevisiae ribosomal RNA in vivo in which yeast cells can be made completely dependent on mutant rRNA and ribosomes by a simple switch in carbon source. The system is based on a yeast strain defective in RNA polymerase I (Pol I) transcription [Nogi et al. (1991) Proc. Natl. Acad. Sci. USA 88, 3962-3966]. This normally inviable strain was rescued by integration of multiple copies of the complete 37S pre-rRNA operon under control of the inducible, Pol II-transcribed GAL7 promoter into the rDNA repeat on chromosome XII. The resulting YJV100 strain can only grow on medium containing galactose as the carbon source. A second, episomal vector was constructed in which the rDNA unit was placed under control of the constitutive PGK1 promoter. YJV100 cells transformed with this vector are now also able to grow on glucose-based medium making the cells completely dependent on plasmid-encoded rRNA. We show that the Pol II-transcribed pre-rRNA is processed and assembled similarly to authentic Pol I-synthesised pre-rRNA, making this 'in vivo Pol II system' suitable for the detailed analysis of rRNA mutations, even highly deleterious ones, affecting ribosome biogenesis or function. A clear demonstration of this is our finding that an insertion into variable region V8 in 17S rRNA, previously judged to be neutral with respect to processing of 17S rRNA, its assembly into 40S subunits and the polysomal distribution of these subunits [Musters et al. (1989), Mol. Cell. Biol. 9, 551-559], is in fact a lethal mutation.

Precursors of ribosomal RNA in the cellular slime mold Dictyostelium discoideum. Isolation and characterization.

The pathway of ribosomal RNA biogenesis in Dictyostelium discoideum has been defined through identification, isolation, and characterization of the rapidly labeled nuclear RNAs which are intermediates in the process. Comparison of the methylation patterns, base compositions, two-dimensional oligonucleotide maps, and hybridization properties of these intermediate RNAs with those of mature rRNAs has established clearly the precursor-product sequence relationships supporting the following scheme for rRNA production and processing: (formula: see text) The relationship of the 37 S RNA of Dictyostelium to primary rRNA transcripts of prokaryotes and other eukaryotes is discussed.

Ribosomal precursor particles in ascites tumor cell nucleoli

Ribonucleoprotein particles have been prepared from isolated nucleoli of ascites tumour cells. The release of nucleolar particles is obtained through the action of heparin. When analysed on sucrose gradients, most of the particles are assignable to the region of 80 S. When the same particles are processed in gradients containing EDTA, the maximum of absorbance at 260 nm is uniformly in the 55 S region. Detailed structural analysis of nucleolar particles was carried out under negative or positive staining in the electron microscope and compared with the finished cytoplasmic ribosomes. The 80 S nucleolar particle measures 230 × 180 Å and displays three striking features: 1. 1. They are often grouped by three or four particles linked by a fine strand of 10 Å. 2. 2. Isolated particles under positive staining are associated with a similar fine strand. 3. 3. Under negative contrast, they appear as tightly coiled irregular structures. On the other hand, the 55 S particle is a loose structure composed of subparticular elements: the average size of this particle is 200 × 170 Å. This 55 S particle is believed to be a dissociation of the 80 S by the action of EDTA. These result are discussed in view of the possible regulatory functions of the nucleolus in the biogenesis of ribosomal RNA and ribosomal peptides.