Molecular analyses of the functions of SL and U3 snRNPs.
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Animals↗
Biomedical subjects
B Sollner-Webb
Publications and source records attributed to B Sollner-Webb.
Half helical turn spacing changes convert a frog into a mouse rDNA promoter: a distant upstream domain determines the helix face of the initiation site.
Transcription of frog rDNA by mouse cell factors is the only documented exception to the observed species selectivity of rRNA gene expression. This heterologous transcription is authentic in that it uses the normal frog upstream and core promoter domains, as well as the normal mouse polymerase I transcription factors, but it initiates at residue -4. We now show that by introducing an insertion or deletion of approximately one-half helical turn anywhere within the 90-bp region between the upstream and core promoter domains, the initiation site moves to residue +1. Promoters bearing spacing changes of approximately one or two full helix turns do not initiate at residue +1, whereas a promoter with a one and one-half-turn helical turn spacing change again supports initiation at residue +1. Thus, the position of the upstream domain of the frog promoter shows a stereo-specific requirement relative to the core promoter domain and dictates the face of the DNA helix on which transcription initiates, 140 bp away. In contrast, relative to the core promoter domain, initiation can occur on either side of the DNA helix. Furthermore, the striking observation that several frog half helical turn spacing change mutants are stronger templates with the mouse factors than the homologous mouse rDNA suggests that the polymerase I transcriptional machinery of even distantly related species is far more similar than generally envisioned.
Animals↗
A rapidly rearranging retrotransposon within the miniexon gene locus of Crithidia fasciculata.
The tandemly arrayed miniexon genes of the trypanosomatid Crithidia fasciculata are interrupted at specific sites by multiple copies of an inserted element. The element, termed Crithidia retrotransposable element 1 (CRE1), is flanked by 29-base-pair target site duplications and contains a long 3'-terminal poly(dA) stretch. A single 1,140-codon reading frame is similar in sequence to the integrase and reverse transcriptase regions of retroviral pol polyproteins. Cloned lines derived from a stock of C. fasciculata have unique arrangements of CRE1s. In different cloned lines, CRE1s, in association with miniexon genes, are located on multiple chromosomes. By examining the arrangement of CRE1s in subclones, we estimate that the element rearranges at a rate of ca. 1% per generation. These results indicate that the C. fasciculata miniexon locus is the target for a novel retrotransposon.
Amino Acid Sequence↗
Enhancers for RNA polymerase I in mouse ribosomal DNA.
The intergenic spacer of the mouse ribosomal genes contains repetitive 140-base-pair (bp) elements which we show are enhancers for RNA polymerase I transcription analogous to the 60/81-bp repetitive enhancers (enhancers containing a 60-bp and an 81-bp element) previously characterized from Xenopus laevis. In rodent cell transfection assays, the 140-bp repeats stimulated an adjacent mouse polymerase I promoter when located in cis and competed with it when located in trans. Remarkably, in frog oocyte injection assays, the 140-bp repeats enhanced a frog ribosomal gene promoter as strongly as did the homologous 60/81-bp repeats. Mouse 140-bp repeats also competed against frog promoters in trans. The 140-bp repeats bound UBF, a DNA-binding protein we have purified from mouse extracts that is the mouse homolog of polymerase I transcription factors previously isolated from frogs and humans. The DNA-binding properties of UBF are conserved from the mouse to the frog. The same regulatory elements (terminators, gene and spacer promoters, and enhancers) have now been identified in both a mammalian and an amphibian spacer, and they are found in the same relative order. Therefore, this arrangement of elements probably is widespread in nature and has important functional consequences.
Animals↗
The first pre-rRNA-processing event occurs in a large complex: analysis by gel retardation, sedimentation, and UV cross-linking.
The first processing event that mouse pre-rRNA undergoes occurs within the external transcribed spacer and is efficiently reproduced in vitro. Analysis with nondenaturing polyacrylamide gels revealed the formation of heparin-resistant complexes of retarded electrophoretic mobility on the substrate rRNA. The specificity of these complexes was demonstrated by their elimination due to competition with processing-competent, but not with processing-incompetent, rRNAs. Furthermore, complex formation, like the processing cleavage, required only 28 nucleotides of rRNA sequence adjacent to the processing site but was stimulated by additional downstream conserved sequences. These processing complexes formed in a time-dependent manner, and once assembled, they were stable to challenge by competitor rRNA and remained on the processed rRNA. Their sedimentation coefficient was approximately 20S. UV cross-linking studies with 4-thiouridine-substituted rRNA have identified six polypeptides, 52 to 250 kilodaltons, that are specifically bound to the rRNA processing substrate.
Animals↗
The mouse ribosomal DNA promoter has more stringent requirements in vivo than in vitro.
Using mouse ribosomal DNA templates bearing polymerase I terminators to prevent transcriptional interference (S. L. Henderson, K. Ryan, and B. Sollner-Webb, Genes Dev. 3:212-223, 1989) and facilitate promoter analysis in intact cells, we demonstrate that a -140 promoter domain (as well as the core region) is essential for appreciable levels of initiation in vivo. This in vivo polymerase I promoter can also be detected in vitro but only under very stringent conditions.
Animals↗
The promoter-proximal rDNA terminator augments initiation by preventing disruption of the stable transcription complex caused by polymerase read-in.
We have examined the mechanism by which transcriptional initiation at the mouse rDNA promoter is augmented by the RNA polymerase I terminator element that resides just upstream of it. Using templates in which terminator elements are instead positioned at the opposite side of the plasmid rather than proximal to the promoter, or conditions where transcription is terminated elsewhere in the plasmid by UV-induced lesions, we show that the terminator's stimulatory effect is not position dependent. Mouse terminator elements therefore do not stimulate via the previously postulated 'read-through enhancement' model in which terminated polymerases are handed off to an adjacent promoter in a concerted reaction. The position independence and orientation dependence of the terminator also makes it unlikely that the terminator functions as a promoter element or as an enhancer. Instead, terminators serve to augment initiation by preventing polymerases from reading completely around the plasmid and through the promoter from upstream, an event which we show interferes with subsequent rounds of initiation. Notably, this transcriptional interference arises because polymerase passage across a promoter disrupts the otherwise stable transcription complex, specifically releasing the bound transcription factor D. These liberated D molecules can then bind to other templates and activate their expression. The rDNA transcriptional interference is not due to a steric impediment to the binding of new polymerase molecules, and it does not similarly liberate the initiation-competent polymerase (factor C). These studies have also convincingly demonstrated that multiple rounds of transcription are obtained from rDNA template molecules in vitro.
Animals↗
The Xenopus ribosomal DNA 60- and 81-base-pair repeats are position-dependent enhancers that function at the establishment of the preinitiation complex: analysis in vivo and in an enhancer-responsive in vitro system.
Although it is generally believed that the 60- and 81-base-pair (60/81-bp) repeats of the Xenopus laevis ribosomal DNA (rDNA) spacer are position-independent transcriptional enhancers, this has not been shown directly. We have now developed a critical assay which proves that the 60/81-bp repeats do, in fact, stimulate transcription from promoters in cis and that they function in both orientations and when up to 1 kilobase pair from the initiation site. However, contrary to the widely accepted view, these elements are found to be highly position dependent, for they have no net effect when downstream of the initiation site within the transcribed region and they behave as transcriptional silencers of promoters in cis when moved greater than 2 kilobase pairs upstream of the initiation site. The 60/81-bp elements therefore are position-dependent 5' enhancers. We also found that this rDNA enhancer was polymerase I specific and that it was composed of duplicated, individually functional elements. Finally, we report an in vitro system that reproduces both cis enhancement and trans competition by the 60/81-bp repeats. Sequential-addition studies in this system demonstrated that the rDNA enhancer functions in trans at or before establishment of the stable transcription complex, not subsequently at each round of transcription.
Animals↗
An RNA polymerase I promoter located in the CHO and mouse ribosomal DNA spacers: functional analysis and factor and sequence requirements.
We report results of experiments in which we demonstrated the existence of a polymerase I promoter within the ribosomal DNA spacer upstream from the rRNA initiation site in Chinese hamsters and mice. Transcription of the CHO spacer promoter was achieved by the same protein factors, C and D, that catalyzed transcription of the gene promoter, and these factors bound stably to the CHO spacer promoter in a preinitiation complex, just as they did to the gene promoter. In contrast to the CHO spacer promoter, which was transcribed in vitro nearly as efficiently as the gene promoter, the mouse spacer promoter was far less active; this low activity was attributable to the fact that the mouse spacer promoter bound factor D inefficiently. It is striking that the active CHO spacer promoter violated the otherwise universal rule that metazoan RNA polymerase I promoters all have a G residue at position -16. Sequence comparisons also revealed a great similarity between the CHO and mouse spacer promoter regions, yet there was much less similarity between the flanking sequences. There was also only limited homology between the spacer and gene promoter regions, but despite this the two kinds of initiation regions were organized similarly, both consisting of an essential core promoter domain and a stimulatory domain that extended upstream to approximately residue -135. Evolutionary considerations argue strongly that the presence of ribosomal DNA spacer promoters offers a significant selective advantage.
Animals↗
Sites of topoisomerase I action on X. laevis ribosomal chromatin: transcriptionally active rDNA has an approximately 200 bp repeating structure.
The sites at which topoisomerase I interacts with the transcriptionally active ribosomal chromatin of Xenopus oocytes were mapped by treating oocyte nuclei first with camptothecin to stabilize topoisomerase I-rDNA reaction intermediates, and then with SDS to resolve these adducts as protein-linked nicks in the DNA. Strikingly, the topoisomerase I sites are concentrated in the region encoding the 18S, 5.8S, and 28S rRNAs, where they are spaced with a periodicity of approximately 200 nucleotides. Plasmid rDNA that has assembled into a nucleoprotein structure in an oocyte nuclear extract yields this same restricted pattern of sites, while rDNA that is not in a chromatin structure exhibits a continuum of topoisomerase I cleavages. Thus, the approximately 200 bp spacing of sites of topoisomerase I interaction evidently reflects rDNA chromatin structure. These results indicate that the active rRNA genes are organized into nucleosome-like arrays, with topoisomerase I located in the linker regions.
Animals↗
Surprises in polymerase III transcription.
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Animals↗
Polymerase III transcription factor B activity is reduced in extracts of growth-restricted cells.
Extracts of cells that are down-regulated for transcription by RNA polymerase I and RNA polymerase III exhibit a reduced in vitro transcriptional capacity. We have recently demonstrated that the down-regulation of polymerase I transcription in extracts of cycloheximide-treated and stationary-phase cells results from a lack of an activated subform of RNA polymerase I which is essential for rDNA transcription. To examine whether polymerase III transcriptional down-regulation occurs by a similar mechanism, the polymerase III transcription factors were isolated and added singly and in pairs to control cell extracts and to extracts of cells that had reduced polymerase III transcriptional activity due to cycloheximide treatment or growth into stationary phase. These down-regulations result from a specific reduction in TFIIIB; TFIIIC and polymerase III activities remain relatively constant. Thus, although transcription by both polymerase III and polymerase I is substantially decreased in extracts of growth-arrested cells, this regulation is brought about by reduction of different kinds of activities: a component of the polymerase III stable transcription complex in the former case and the activated subform of RNA polymerase I in the latter.
Animals↗
Transcription of mouse rDNA is regulated by an activated subform of RNA polymerase I.
We have identified the species-nonspecific factor required for mouse rDNA transcription, factor C, as an activated subform of RNA polymerase I. C is an RNA polymerase I since it copurifies with bulk polymerase I activity on the three chromatographic columns used to achieve a virtually homogenous preparation of polymerase I, as well as on four additional matrices; it is quantitatively neutralized as well as immunoprecipitated by two different types of anti-polymerase I antibodies; and it has thermal lability identical to that of bulk polymerase I. However, C is clearly distinct from bulk polymerase I in its ability to participate in the stable rDNA transcription complex and to catalyze accurate initiation of rRNA synthesis. It also has a greater sedimentation coefficient than bulk polymerase I. Furthermore, this activated polymerase subform is specifically lacking in extracts of cells in which rDNA transcription was down-regulated because of cycloheximide treatment or attainment of stationary phase. These data suggest that regulation of rDNA transcription in vivo may involve modulation in availability of the activated polymerase I subform.
Animals↗
A combination of RNase H and S1 nuclease circumvents an artefact inherent to conventional S1 analysis of RNA splicing.
S1 nuclease mapping is commonly used to analyze transcription and processing of unlabelled RNAs. However, the S1 protocol that appears best suited to demonstrate splicing of a particular RNA (using an intronless probe that is 5' end-labelled in the downstream exon) is not diagnostic as expected. Rather, both intron-containing RNA and intronless RNA confer protection of probe across the splice juncture. To unambiguously demonstrate correctly spliced RNAs that begin at a specific initiation site, we present a procedure in which unspliced RNA molecules are first cleaved by RNase H following annealing to an intronic DNA fragment and the remaining RNA is then subjected to S1 analysis using an intronless probe present in vast excess. Only spliced, correctly initiated transcripts can protect the probe across the splice junction and up to residue +1. This RNase H/S1 method provides a broadly applicable technique with which to demonstrate splicing and initiation of a variety of transcripts, especially ones from transfected genes that can arise both from the normal and from activated cryptic initiation sites.
Endonucleases↗
Mouse and frog violate the paradigm of species-specific transcription of ribosomal RNA genes.
Transcription of ribosomal RNA genes by RNA polymerase I is generally accepted as being highly species specific, a conclusion based on numerous reports that rRNA genes of one species are not transcribed by factors of even closely related species. It thus was striking to find that cloned rDNA from the frog Xenopus laevis is specifically transcribed in extracts prepared from mouse cells. The data in this paper demonstrate that this heterologous transcription is due to a normal initiation process and not to a fortuitous event. Transcription of Xenopus rDNA in the mouse cell extract is directed by the same large promoter (residue-141 to +6) that is utilized to promote the synthesis of frog rRNA in homologous Xenopus systems. Moreover, the same factors of the mouse cell extract that transcribe the homologous mouse rDNA also catalyze transcription from the X. laevis rDNA promoter. We conclude that polymerase I transcriptional machinery does not evolve as rapidly as prior studies would suggest.
Animals↗
Efficient introduction of plasmid DNA into Trypanosoma brucei and transcription of a transfected chimeric gene.
Electroporation induces efficient transient transfection of Trypanosoma brucei, and the introduced DNA can be transcribed into RNA. When we delivered a high-voltage electric pulse to cells mixed with radiolabeled pBR322, approximately equal to 15% of the plasmid DNA was taken up by the parasites. When transfecting DNA contained a segment of T. brucei ribosomal DNA that included the 5' end of the rRNA gene, the introduced plasmid directed expression of RNA; this RNA expression was shown both by dot blots and by S1 nuclease protection assays carried out under conditions specific for probe hybridization to RNA. In the absence of the ribosomal region, analogous transcription did not occur. We optimized this trypanosomal expression system with regard to electric shock strength, concentration of input DNA, and incubation time after electric shock. This technique enabling specific trypanosome DNA expression in vivo should facilitate the molecular analysis of T. brucei gene expression.
Animals↗
Nucleotide sequence determining the first cleavage site in the processing of mouse precursor rRNA.
The first step in the processing of 47S precursor rRNA in mouse cells is reproduced in vitro in an S-100 transcription reaction and consists of an endonucleolytic cleavage at residue +650 of the primary transcript followed by rapid degradation of the fragment upstream from residue +650. An analogous processing occurs in human rRNA. The mouse and human rRNA sequences are approximately equal to 80% conserved for 200 nucleotides on the 3' side of these processing sites, suggesting that this conserved region may be important in specifying the processing. To test this hypothesis, we constructed a systematic series of deletion mutants approaching the mouse rDNA processing region from both the 5' and 3' directions and analyzed the processing of their transcripts in vitro. The 5' boundary of the region required for processing is quite sharp and corresponds to the rRNA cleavage site at the 5' end of the conserved sequence region. The 3' boundary is more complex: The 3' deletions extending to between 250 and 130 nucleotides beyond the processing site cause about a 50% decrease in the amount of the processed RNA. A 3' deletion that extends to 109 nucleotides beyond the processing site greatly reduces the processing efficiency. Deletions to or beyond 91 nucleotides on the 3' side of the processing site virtually eliminate processing. Under altered ionic conditions, transcripts of 3' deletions extending to only 41 nucleotides beyond the processing site can still direct a low level of accurate processing. These results demonstrate that the mouse/human conserved sequence just on the 3' side of the primary rRNA processing site consists of several domains that direct and/or augment both the initial endonucleolytic cleavage and the closely coupled selective degradation of the upstream fragment that together constitute the primary rRNA processing event.
Animals↗
Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation.
To analyze the specificity of RNA processing reactions, we constructed hybrid genes containing RNA polymerase III promoters fused to sequences that are normally transcribed by polymerase II and assessed their transcripts following transfection into human 293 cells. Transcripts derived from these chimeric constructs were analyzed by using a combined RNase H and S1 nuclease assay to test whether RNAs containing consensus 5' and 3' splicing signals could be efficiently spliced in intact cells, even though they were transcribed by RNA polymerase III. We found that polymerase III-derived RNAs are not substrates for splicing. Similarly, we were not able to detect poly(A)+ RNAs derived from genes that contained a polymerase III promoter linked to sequences that were necessary and sufficient to direct 3'-end cleavage and polyadenylation when transcribed by RNA polymerase II. Our findings are consistent with the view that in vivo splicing and polyadenylation pathways are obligatorily coupled to transcription by RNA polymerase II.
Animals↗