Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene.

Abstract

The transcriptional activity of spacer sequences flanking the rat 45S ribosomal DNA (rDNA) gene were studied. Nascent RNA labeled in in vitro nuclear run-on reactions hybridized with both 5' and 3' spacer regions. The highest level of hybridization was seen with an rDNA fragment containing tandem repeats of a 130-base-pair sequence upstream of the 45S rRNA initiation site. Synthesis of RNA transcripts homologous to this internally repetitious spacer region was insensitive to high levels of a-amanitin, suggesting that it is mediated by RNA polymerase I. Analysis of steady-state RNA showed that these transcripts were present at extremely low levels in vivo relative to precursor rRNA transcripts. In contrast, precursor and spacer run-on RNAs were synthesized at similar levels. This suggests that spacer transcripts are highly unstable in vivo; therefore, it may be the process of transcription rather than the presence of spacer transcripts that is functionally important. Transcription in this upstream rDNA region may be involved in regulation of 45S rRNA synthesis in rodents, as has been suggested previously for frog rRNA. In addition, the presence of transcriptional activity in other regions of the spacer suggests that some polymerase I molecules may transcribe through the spacer from one 45S gene to the next on rodent rDNA.Mammalian ribosomal DNA (rDNA) is organized in tandem repeats at a few chromosomal loci for a total of 100 to 250 copies per haploid genome (reviewed in reference 23). Each repeat consists of approximately 14 kilobases (kb) of DNA encoding the 45S precursor rRNA and 20 to 30 kb of spacer region termed nontranscribed spacer (NTS). Internally repetitious elements in the NTS flank both the 5' and 3' ends of the 45S gene in rodent rDNA (1,8,12,21). The 3' repeat pattern has been identified by the presence of multiple Sall restriction sites within 1 kb of the 28S rRNA coding region. It has recently been shown that rDNA transcription in mice terminates adjacent to this downstream repetitious region and that this region appears to contain information required for polymerase I (pol I) transcription termination (22). The 5' repeating element in both rats (12) and mice (28) is approximately 130 base pairs (bp) long and is bounded by variable-length tracts of T's. This repetitious region terminates approximately 210 bp upstream from the initiation site for 45S rRNA synthesis. Variation in the number of repeats in this region is responsible for major polymorphisms in both species (1)(2)(3)12). No function has yet been attributed to the 130-bp upstream elements, although it has been suggested that such repetitious regions may arise during recombination of tandemly repeated genes and, perhaps, facilitate this process (16; also see reference 48 for a discussion of NTS function).Repetitious regions flanking the initiation site for precursor rRNA synthesis have also been extensively described in nonmammals. In Xenopus spp., the repeated elements contain functional duplications of the RNA pol I promoter, as well a...

The promoter-proximal rDNA terminator augments initiation by preventing disruption of the stable transcription complex caused by polymerase read-in.

“…This is certainly its major effect in our assays, and it is also likely to be quite significant in vivo on the endogenous mammalian rRNA genes because polymerases clearly do approach the initiation region from upstream. These spacer polymerases include ones that initiated at promoters in the rDNA spacer (Cassidy et al 1987;Harrington and Chikaraishi 1987;Kuhn and Grummt 1987;Tower et al 1989) and possibly also ones that failed to terminate at the 3' end of the previous tandem rRNA gene. A terminator element could, however, have other positive effects on transcription as well.…”

An RNA polymerase I promoter located in the CHO and mouse ribosomal DNA spacers: functional analysis and factor and sequence requirements.

“…While mouse rDNA evidently does not contain spacer promoters within -1. 8 kilobase pairs (kbp) upstream from the gene promoter (46), nuclear run-on transcription data (15) in conjunction with in vitro runoff transcription data (13) suggest that an initiation site might exist within the adjoining several-hundred-basepair region. Furthermore, the identification of transcriptional terminators immediately upstream of mammalian gene promoters (13,16) has also indicated that transcripts read toward the gene promoter from the spacer.…”

Interaction between the yeast mitochondrial and nuclear genomes influences the abundance of novel transcripts derived from the spacer region of the nuclear ribosomal DNA repeat.

“…This result was surprising for at least two reasons. First, although it is now recognized that NTS sequences are transcribed in some organisms (9,23,25,35,36), those transcripts appear to be very unstable and have not been found to accumulate in cells to any appreciable extent. Second, the relationship between the relative abundance of NTS transcripts and the functional state of mitochondria or the mitochondrial genome was not immediately obvious.…”