The ribosomal RNA of the trypanosomatid protozoan Crithidia fasciculata: physical characteristics and methylated sequences

Abstract

When extracted and analyzed under conditions which maintain noncovalently associated RNA-RNA complexes, the bulk cellular RNA of Crithidia fasciculata contains species of apparent molecular weights 1.3, 0.825, 0.08, 0.065, and 0.045 x 10(6) in addition to 5S rRNA and tRNA. Heat denaturation results in the disappearance of the 1.3 x 10(6) dalton RNA and the appearance of three new species having molecular weights of 0.67, 0.575, and 0.059 x 10(6). In addition, the apparent molecular weight of the 0.825 x 10(6) dalton component is reproducibly lowered to 0.81 x 10(6) after heat treatment. With the exception of tRNA, all of the RNA species are present in close to equimolar amounts in either undenatured or heat-denatured C. fasciculata bulk cellular RNA. On the basis of previous observations on the ribosomal RNA of the closely related organism, Crithidia oncopelti (Spencer, R. & Cross, G.A.M. (1976) J. Gen. Microbiol. 93, 82-88), the 1.3 and 0.825 x 10(6) dalton RNA's are considered to be components of the large and small subunits, respectively, of C. fasciculata ribosomes, but the subunit localization of the other RNA's described here has not yet been determined. O2'-Methylnucleosides account for about 1.4 mol% of the total nucleoside constituents of unfractionated C. fasciculata rRNA. Quantitative analysis suggests that the rRNA molecules in a C. fasciculata ribosome contain a total of 95-100 O2'-methyl groups, distributed in 80-85 Nm-Np sequences (including four 'hypermodified' Nm-Np, each containing a modification of a base or base-sugar linkage in addition to sugar methylation), six different Nm-Nm-Np sequences, and one Nm-Nm-Nm-Np sequence. While the specific pattern of O2'-methylation in the rRNA of C. fasciculata is distinct, both qualitatively and quantitatively, from the pattern observed in other organisms, Crithidia rRNA does contain certain 'universal' O2'-methylated sequences which appear to have been extensively conserved in evolution. The base-methylated nucleoside, N6,N6-dimethyladenosine (m26A), has been isolated from both C. fasciculata and wheat embryo rRNA in the form of the alkali-resistant dinucleotide, m26A-m26Ap. This dinucleotide and its enzymatic degradation products have been characterized by examination of their ultraviolet absorption spectra and electrophoretic and chromatographic properties.

A genome-wide analysis of C/D and H/ACA-like small nucleolar RNAs in Trypanosoma brucei reveals a trypanosome-specific pattern of rRNA modification

“…Only 84 Nms are predicted to exist on rRNA, based on this study, but early studies suggest the existence of as many as 100 Nms in Crithidia rRNA (Gray 1979). In addition, we have not yet identified any guide RNA that guides modification on trypanosome snRNAs such as scaRNA.…”

“…However, in yeast, there are only 55 such modifications. The estimated number of such modifications in trypanosomatids is ∼95-100 (Gray 1979) and resembles the number found in plants and vertebrates (Brown et al 2003a). Surprisingly, in Euglena, as in trypanosomes, the rRNA is extensively modified, and the estimated number of modifications is 150 Nms and 70 ⌿s (Russell et al 2004).…”

“…Interestingly, some modifications with no known guide RNAs (marked by dots) were also detected, suggesting that there are more modifications than we predicted. Indeed, earlier studies showed the existence of ∼100 Nms on Crithidia rRNA (Gray 1979).…”

“…Early studies suggest the existence of ∼100 2Ј-Omethylated nucleotides on the rRNA (Gray 1979). The first trypanosome C/D snoRNA (snoRNA-2) was identified in Leptomonas collosoma (Levitan et al 1998).…”

Expression Studies on Clustered Trypanosomatid Box C/D Small Nucleolar RNAs

“…This may be expected since ϳ100 sites of 2Ј-O-ribose methylation were mapped in Crithidia fasciculata, which is almost as numerous as in humans (48). The need to methylate so many sites and the relatively small genome size of the parasite may have forced many more snoRNAs to guide two sites.…”