Immobilization of proteins in the nucleolus by ribosomal intergenic spacer noncoding RNA

Cellular pathways are established and maintained by stochastic interactions of highly mobile molecules. The nucleolus plays a central role in the regulation of these molecular networks by capturing and immobilizing proteins. Here, we report a function for noncoding RNA (ncRNA) in the regulation of protein dynamics of key cellular factors, including VHL, Hsp70 and MDM2/PML. Stimuli-specific loci of the nucleolar intergenic spacer produce ncRNA capable of capturing and immobilizing proteins that encode a discrete peptidic code referred to as the nucleolar detention sequence (NoDS). Disruption of the NoDS/intergenic RNA interaction enables proteins to evade nucleolar sequestration and retain their dynamic profiles. Mislocalization of intergenic ncRNA triggers protein immobilization outside of the nucleolus, demonstrating that these ncRNA species can operate independently from the nucleolar architecture. We propose a model whereby protein immobilization by ncRNA is a posttranslational regulatory mechanism.     

The intergenic spacer region of the ribosomal RNA gene of Penicillium marneffei shows almost no DNA sequence diversity

The fungus Penicillium marneffei causes fatal systemic infections and is endemic in many parts of South-East Asia, especially Thailand. The intergenic spacer (IGS) region, the most variable region of rRNA genes, was found to be highly conserved among 58 P. marneffei strains. IGS analysis might not be suitable for molecular epidemiological analysis of P. marneffei infections.     

Human 28S ribosomal RNA sequence heterogeneity.

DNA sequencing of several cloned human 28S ribosomal RNA gene fragments has revealed sequence heterogeneity (1) but it was not clear whether these are inactive pseudogenes or are active genes that are transcribed and represented in ribosomes. S1 nuclease analysis allowed us to examine the population of ribosomal RNA molecules of a cell, and we found that 28S rRNA is a heterogeneous assortment of molecules in both mono- and polysomal preparations. Sequence variation, although largely concentrated in variable regions of the molecule, apparently also occurs in the conserved regions.     

Unmethylated regions in the intergenic spacer of maize and teosinte ribosomal RNA genes

The restriction endonucleases Hpa II and Msp I were used to examine cytosine methylation in the ribosomal RNA genes (rDNA) of inbred lines of maize and species of teosinte. In all of the rDNAs examined, Msp I (not sensitive to mCpG) digestion yielded a distribution of lower molecular weight fragments indicative of multiple recognition sites. The majority of the rDNA arrays in an individual were inaccessible to Hpa II (sensitive to mCpG) cleavage, but a significant fraction (10–25%) was cleaved at least once by Hpa II into repeat unit length fragments (9.1 kbp). In some maize inbred lines, one or two additional fragment populations (less than 9.1 kbp in length) were also produced by Hpa II digestion. All of the unmethylated Hpa II sites mapped to the intergenic spacer (IGS), and the major unmethylated site was located approximately 800 bp 5 to the start of the 18S RNA coding sequence. An Eco RI polymorphism, present in the 26S gene of certain inbred lines and hybrids, was utilized to investigate the organization of unmethylated repeat units in the rDNA array. In double digest experiments with Hpa II/Eco RI, the fragments from repeat units with two Eco RI sites were sensitive to Hpa II digestion, whereas, the fragments from repeat units with a single Eco RI site were almost completely resistant to Hpa II digestion. Similar digestion patterns were also observed in Eco RII (sensitive to mCNG)/Eco RI digests. These results suggest that unmethylated and Eco RI polymorphic sites occur in the same repeat units.     

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: intraspecific heterogeneity within the intergenic spacer region

The complete sequence of the nuclear ribosomal DNA gene complex of the phytopathogenic fungus Verticillium dahliae has been determined. The tandemly repeated unit was 7216 bp long and appears to be the shortest rDNA cluster described so far among filamentous fungi. Primer pairs were designed for amplification of the region spanning half of the 28S subunit, the intergenic spacer (IGS), and the 5' end of 18S subunit of a number of Verticillium strains, isolated from various hosts and geographic origins. Great heterogeneity was detected in the amplified products of the IGS region resulting in fragments varying from 1.6 to 2.0 kb. The majority of Verticillium isolates were classified into two groups with 1.6- and 1.7-kb amplified products, respectively. The former group included 31 V. dahliae, 7 V. longisporum, and 1 V. albo-atrum isolates, whereas the latter included 10 V. dahliae and 1 V. albo-atrum isolates. Sequence analysis of representative PCR products of the above groups identified a "hot-spot" region harboring most of larger insertions, whereas most of the small changes were due to transitions and transversions. One V. longisporum isolate with a 2.0-kb PCR product contained 13 perfectly conserved tandem repeats of 39 bp long. The presence of similar incomplete sequences in the corresponding regions of V. dahliae, V. longisporum, and V. albo-atrum isolates revealed a particular standard motif of insertions in the IGS region of the genus and is discussed.     

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: intraspecific heterogeneity within the intergenic spacer region

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: Intraspecific heterogeneity within the intergenic spacer region. Fungal Genetics and Biology 29, 19-27. The complete sequence of the nuclear ribosomal DNA gene complex of the phytopathogenic fungus Verticillium dahliae has been determined. The tandemly repeated unit was 7216 bp long and appears to be the shortest rDNA cluster described so far among filamentous fungi. Primer pairs were designed for amplification of the region spanning half of the 28S subunit, the intergenic spacer (IGS), and the 5' end of 18S subunit of a number of Verticillium strains, isolated from various hosts and geographic origins. Great heterogeneity was detected in the amplified products of the IGS region resulting in fragments varying from 1.6 to 2.0 kb. The majority of Verticillium isolates were classified into two groups with 1.6- and 1.7-kb amplified products, respectively. The former group included 31 V. dahliae, 7 V. longisporum, and 1 V. albo-atrum isolates, whereas the latter included 10 V. dahliae and 1 V. albo-atrum isolates. Sequence analysis of representative PCR products of the above groups identified a "hot-spot" region harboring most of larger insertions, whereas most of the small changes were due to transitions and transversions. One V. longisporum isolate with a 2.0-kb PCR product contained 13 perfectly conserved tandem repeats of 39 bp long. The presence of similar incomplete sequences in the corresponding regions of V. dahliae, V. longisporum, and V. albo-atrum isolates revealed a particular standard motif of insertions in the IGS region of the genus and is discussed.     

Human ribosomal RNA gene spacer sequences are found interspersed elsewhere in the genome

A cloned EcoRI fragment containing human 18 S rRNA gene sequences was used to screen a gene library to obtain a set of 8 overlapping cloned DNA segments extending into the non-transcribed spacer region of the human ribosomal RNA gene cluster. 19.4 kb of the approx. 43-kb rDNA repeat was obtained in cloned form and mapped with restriction endonucleases. None of the clones obtained extended into 28 S rRNA sequences. A 7-kb region of non-transcribed spacer DNA shared in common between five independent clones was subjected to comparative restriction digests. It was estimated that sequences among the five different spacer isolated varied by not more than 1.0%, if all the observed differences are assumed due to point mutation. HaeII-restriction fragments from within this same 7-kb region contain sequences carried not only within the tandem repeats of the gene cluster but interspersed elsewhere in the genome. Some of these sequences correspond to the Alu family of highly repeated interspersed sequences.     

Structure of the intergenic spacer region from the ribosomal RNA gene family of white spruce (Picea glauca)

Five genomic clones containing ribosomal DNA repeats from the gymnosperm white spruce (Picea glauca) have been isolated and characterized by restriction enzyme analysis. No nucleotide variation or length variation was detected within the region encoding the ribosomal RNAs. Four clones which contained the intergenic spacer (IGS) region from different rDNA repeats were further characterized to reveal the sub-repeat structure within the IGS. The sub-repeats were unusually long, ranging from 540 to 990 bp but in all other respects the structure of the IGS was very similar to the organization of the IGS from wheat, Drosophila and Xenopus.