A novel promoter in the mouse rDNA spacer is active in vivo and in vitro.

Kuhn, Anne; Grummt, Ingrid

doi:10.1002/j.1460-2075.1987.tb02673.x

Cited by 78 publications

(46 citation statements)

References 38 publications

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“…6, lane 5). Mixing the two rDNA templates pMrSP and pMrWT resulted in equal transcription of both DNAs (lane 6), which was further reduced by the presence of pMrE13 in trans (lane 7). However, when the enhancercontaining fragment was linked to the promoter (pMrE-WT), transcription of the reference plasmid pMrSP was almost completely suppressed whereas the other template was pref- (8,9,15,18,19).…”

Section: Methodsmentioning

confidence: 99%

A 140-base-pair repetitive sequence element in the mouse rRNA gene spacer enhances transcription by RNA polymerase I in a cell-free system.

Kuhn¹,

Deppert²,

Grummt³

1990

Proc. Natl. Acad. Sci. U.S.A.

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We show that the repetitive 140-base-pair (bp) elements present in the spacer of mouse rRNA genes function as enhancers for RNA polymerase I. Attachment of these elements to the rDNA promoter stimulates rRNA synthesis both in vivo and in vitro. The cis-activating effect of the spacer repeats is orientation-independent and increases with increasing numbers of the 140-bp elements. Competition experiments demonstrate that the spacer repeats bind one or more of the transcription factors interacting with the rDNA promoter. Both the 140-bp elements and the core promoter act cooperatively and thus are functionally linked. The 60/81-bp enhancer repeats from Xenopus laevis rDNA compete for a murine transcription factor(s) and stimulate transcription when fused to the mouse rDNA promoter. The results indicate that despite the marked species specificity of rDNA transcription initiation, common factors may interact with both the rDNA promoter and the enhancer.The intergenic spacers between individual rRNA transcription units have rapidly diverged between different species, varying in length from 2 kilobases (kb) in yeast to >30 kb in mammals. In spite of this marked sequence divergence, several regulatory elements that affect the readout of rRNA genes have been maintained. Functionally important sequence elements within the spacer include promoter and terminator elements, spacer promoters, promoter-proximal terminators, and origins of replication. Furthermore, repetitive blocks of interspersed 60-or 81-base-pair (bp) elements that behave in some respect like enhancer elements have been identified in the intergenic spacer of Xenopus laevis (1, 2). They confer a competitive transcriptional advantage on promoters located in cis but compete against promoters located in trans (3,4). This cis stimulation of transcription is orientation-and position-independent and, therefore, these elements behave like transcriptional enhancers. To date, RNA polymerase I enhancers in rDNA spacers have been rigorously demonstrated only in yeast and in X. laevis and its relative X. borealis. In the mouse spacer there is a cluster of repetitive 140-bp sequence elements (5, 6) that lies between the spacer promoter (7) and the upstream terminator, To (8,9). Both their repetitive nature and their localization between the spacer promoter and the upstream terminator suggest that these elements may be functionally analogous to the frog 60/81-bp element. The results presented here demonstrate that this assumption is correct. We show that the 140-bp repeats stimulate in cis transcription from the rDNA promoter both in a cell-free system and after transfection into mouse cells. This activation appears to be mediated by transcription factor(s) that interact with both the promoter and the enhancer elements. METHODSPlasmid Constructions. See Fig. 1. Plasmid pMrWT contains a 324-bp mouse rDNA promoter fragment (positions -169 to + 155; ref. 10). pMrSP is identical to pMrWT except that the coding region extends to a Pvu II site at +292. The 5'-deletion pl...

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Section: Methodsmentioning

confidence: 99%

A 140-base-pair repetitive sequence element in the mouse rRNA gene spacer enhances transcription by RNA polymerase I in a cell-free system.

Kuhn¹,

Deppert²,

Grummt³

1990

Proc. Natl. Acad. Sci. U.S.A.

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“…In Drosophila, 5.8S and 28S rRNAs have additional cleavage sites and produce two pieces of rRNAs from each (5.8S/2S and 28S-1/-2 RNAs) (Tautz et al 1988). The DNA sequence encoding 45S pre-rRNA is flanked by intergenic spacers (IGS) (Morgan et al 1983;Kuhn and Grummt 1987;Paalman et al 1995). The rDNA is present as tandem direct repeats that are organized in one or several clusters in the genome (Long and Dawid 1980).…”

Section: Introductionmentioning

confidence: 99%

A deeply conserved, noncanonical miRNA hosted by ribosomal DNA

Chak

Mohammed

Lai

et al. 2015

RNA

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Advances in small RNA sequencing technologies and comparative genomics have fueled comprehensive microRNA (miRNA) gene annotations in humans and model organisms. Although new miRNAs continue to be discovered in recent years, these have universally been lowly expressed, recently evolved, and of debatable endogenous activity, leading to the general assumption that virtually all biologically important miRNAs have been identified. Here, we analyzed small RNAs that emanate from the highly repetitive rDNA arrays of Drosophila. In addition to endo-siRNAs derived from sense and antisense strands of the prerRNA sequence, we unexpectedly identified a novel, deeply conserved, noncanonical miRNA. Although this miRNA is widely expressed, this miRNA was not identified by previous studies due to bioinformatics filters removing such repetitive sequences. Deep-sequencing data provide clear evidence for specific processing with precisely defined 5 ′ and 3 ′ ends. Furthermore, we demonstrate that the mature miRNA species is incorporated in the effector complexes and has detectable trans regulatory activity. Processing of this miRNA requires Dicer-1, whereas the Drosha-Pasha complex is dispensable. The miRNA hairpin sequence is located in the internal transcribed spacer 1 region of rDNA and is highly conserved among Dipteran species that were separated from their common ancestor ∼100 million years ago. Our results suggest that biologically active miRNA genes may remain unidentified even in well-studied organisms.

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“…Finally, upstream of the frog, mouse, Chinese hamster, and rat enhancer repeats is a so-called spacer promoter, which has been the only other known RNA polymerase I promoter besides that for the pre-rRNA (21,34,52). (A very recent report suggests that an RNA polymerase I promoter also resides within an intron of the human ribosomal protein S14 gene [50].)…”

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confidence: 99%

Stimulation of the Mouse rRNA Gene Promoter by a Distal Spacer Promoter

Paalman

Henderson

Sollner‐Webb

1995

Molecular and Cellular Biology

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We show that the mouse ribosomal DNA (rDNA) spacer promoter acts in vivo to stimulate transcription from a downstream rRNA gene promoter. This augmentation of mammalian RNA polymerase I transcription is observed in transient-transfection experiments with three different rodent cell lines, under noncompetitive as well as competitive transcription conditions, over a wide range of template concentrations, whether or not the enhancer repeats alone stimulate or repress expression from the downstream gene promoter. Stimulation of gene promoter transcription by the spacer promoter requires the rDNA enhancer sequences to be present between the spacer promoter and gene promoter and to be oriented as in native rDNA. Stimulation also requires that the spacer promoter be oriented toward the enhancer and gene promoter. However, stimulation does not correlate with transcription from the spacer promoter because the level of stimulation is not altered by either insertion of a functional mouse RNA polymerase I transcriptional terminator between the spacer promoter and enhancer or replacement with a much more active heterologous polymerase I promoter. Further analysis with a series of mutated spacer promoters indicates that the stimulatory activity does not reside in the major promoter domains but requires the central region of the promoter that has been correlated with enhancer responsiveness in vivo.Of the three classes of eukaryotic RNA polymerase studied to date, only RNA polymerase I is committed to transcribing a single type of DNA: the ribosomal DNA (rDNA) that ultimately gives rise to the mature 18S, 5.8S, and 28S rRNAs of the ribosome. Study of the organization of the tandem rRNA genes and of the RNA polymerase I transcription machinery has led to the identification of cis-acting elements and transacting factors involved in the regulated expression of these genes (40,44,46). The best-studied rDNA elements are those of Xenopus laevis, but the rDNA of other metazoans, including mice, rats, Chinese hamsters, and Drosophila melanogaster, appears quite conserved in organization, although greatly diverged in sequence. Fig. 1A illustrates the similarity in the organizations of the X. laevis and mouse rDNA repeats.The conserved organization of rDNA begins with the promoter, the most important regions of which are an ϳ40-bp core element extending upstream from the initiation site and an upstream element from approximately residue Ϫ140 to approximately residue Ϫ120. Sequences upstream of the core element can be made dispensable in vitro but are critical in vivo. Just upstream of this complete gene promoter is a promoter-proximal transcriptional terminator element that is required for efficient initiation at the gene promoter in vivo, evidently by preventing polymerase read-in (2, 18) but possibly also by providing a position-dependent stimulation (28, 29). Immediately upstream of the promoter and terminator region in most animal and plant species examined are multiple copies of a repetitive sequence. In frogs and mice (Fig. 1A), t...

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A novel promoter in the mouse rDNA spacer is active in vivo and in vitro.

Cited by 78 publications

References 38 publications

A 140-base-pair repetitive sequence element in the mouse rRNA gene spacer enhances transcription by RNA polymerase I in a cell-free system.

A 140-base-pair repetitive sequence element in the mouse rRNA gene spacer enhances transcription by RNA polymerase I in a cell-free system.

A deeply conserved, noncanonical miRNA hosted by ribosomal DNA

Stimulation of the Mouse rRNA Gene Promoter by a Distal Spacer Promoter

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