rRNA Minigene Research Articles

Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells

Most transcripts in growing cells are ribosomal RNA precursors (pre-rRNA). Here, we show that in mammals, aberrant pre-rRNA transcripts generated by RNA polymerase I (Pol I) are polyadenylated and accumulate markedly after treatment with low concentrations of actinomycin D (ActD), which blocks the synthesis of full-length rRNA. The poly(A) polymerase-associated domain-containing protein 5 is required for polyadenylation, whereas the exosome is partly responsible for the degradation of the short aberrant transcripts. Thus, polyadenylation functions in the quality control of Pol I transcription in metazoan cells. The impact of excessive aberrant RNAs on the degradation machinery is an unrecognized mechanism that might contribute to biological properties of ActD.

TAF12 Recruits Gadd45a and the Nucleotide Excision Repair Complex to the Promoter of rRNA Genes Leading to Active DNA Demethylation

Many studies have detailed the repressive effects of DNA methylation on gene expression. However, the mechanisms that promote active demethylation are just beginning to emerge. Here, we show that methylation of the rDNA promoter is a dynamic and reversible process. Demethylation of rDNA is initiated by recruitment of Gadd45a (growth arrest and DNA damage inducible protein 45 alpha) to the rDNA promoter by TAF12, a TBP-associated factor that is contained in Pol I- and Pol II-specific TBP-TAF complexes. Once targeted to rDNA, Gadd45a triggers demethylation of promoter-proximal DNA by recruiting the nucleotide excision repair (NER) machinery to remove methylated cytosines. Knockdown of Gadd45a, XPA, XPG, XPF, or TAF12 or treatment with drugs that inhibit NER causes hypermethylation of rDNA, establishes heterochromatic histone marks, and impairs transcription. The results reveal a mechanism that recruits the DNA repair machinery to the promoter of active genes, keeping them in a hypomethylated state.

Processing of Mammalian rRNA Precursors at the 3′ End of 18S rRNA

Molecular mechanisms involved in the nucleolytic cleavage at the 18S rRNA/internal transcribed spacer 1 (ITS 1) junction, a late step of small-subunit pre-rRNA processing in vertebrates, remain largely unknown, mostly due to the lack of faithful in vitro assays. To identify the minimal cis-acting signals required for this reaction, we studied the processing of truncated human rRNA gene transcripts transiently expressed upon transfection of rRNA minigenes into cultured mouse cells. We observed that processing at this site was faithfully reproduced with transcripts containing only 60 nucleotides of 18S rRNA and the adjacent 103 nucleotides of ITS 1, but was abolished or severely altered by further shortening of either sequence. Remarkably, this minimal transcript contains, within its 18S rRNA part, long sequences complementary to both U20 and U13 small nucleolar RNAs (snoRNAs). The cis-acting elements essential for the reaction were studied further by site-directed mutagenesis. The U20 snoRNA complementary region in 18S rRNA was not required for faithful processing at the 18S rRNA/ITS 1 junction. Also, processing at this site was not appreciably altered by random substitution of proximal ITS 1 sequences (including the 5' terminal nucleotide) or of the terminal nucleotide of mature 18S rRNA. Substitutions in the four-nucleotide loop of the 18S rRNA 3'-terminal stem-loop, including the two adenosine residues substrates of dimethylation, did not alter appreciably the formation of the 18S rRNA 3' end, showing that the (methyl)2A1850.(methyl)2A1851 doublet was not required for processing at this site. Two highly conserved 18S rRNA elements acted as major cis-acting signals for processing at the 3' end, the CAUU sequence immediately preceding the 3'-terminal nucleotide and the 3' strand of the 3'-terminal 18S rRNA helix, complementary to U13 snoRNA. Compensatory mutations, restoring the potential for helix formation, but not U13 snoRNA complementarity, did not restitute the cleavage at the 3' end of 18S rRNA. This suggests that U13 snoRNA may be a trans-acting factor in the nucleolytic cleavage at the 3' end of 18S rRNA.

Actinomycin D Stimulates the Transcription of rRNA Minigenes Transfected into Mouse Cells. Implications for the in vivo Hypersensitivity of rRNA Gene Transcription

The in vivo hypersensitivity of eukaryotic rRNA gene transcription to actinomycin D has long beenknown, but this effect could not be reproduced in model systems and its molecular mechanisms remain uncertain. We studied the action of actinomycin D using mouse rRNA minigenes (with RNA polymerase I promoter and terminator signals), carrying truncated mouse or human rDNA inserts, which are faithfully transcribed upon transient transfection into mouse cells. Low concentrations (0.01–0.08 μg/ml) of actinomycin D caused within 1–2 h a 2–7-fold stimulation of the transcription of rRNA minigenes which is inversely related to the size of the rDNA transcript. With transcripts longer than 3 kb the effect was reversed and at 4 kb a practically complete inhibition of the formation of full-length transcripts was observed, accompanied, however, by an enhanced accumulation of unfinished rDNA transcripts. The dependence of actinomycin D action on transcript length was also observed with lacZ gene segments of different size inserted into the mouse rRNA minigenes. The transcription initiation of endogenous rRNA genes was also stimulated by the low doses of actinomycin D as indicated by the enhanced synthesis of unfinished rDNA transcripts (spanning mainly the 5′ external transcribed spacer), whereas the synthesis of full-length transcripts was abolished. Removal of actinomycin D from the medium caused within 8–24 h a dramatic increase of the transcription from all rRNA minigenes tested. This stimulation was also inversely related to the size of the transcripts and varied from twofold to fivefold for the 3–4-kb transcripts to about 50–80-fold for the basic minigene transcript (395 nucleotides). The amount of endogenous aborted rDNA transcripts was also markedly increased, but the synthesis of full-length transcripts was not restored even 24 h after removal of the drug. The present results reproduce in a model cellular system the in vivo hypersensitivity of rRNA gene transcription to actinomycin D and reveal that the major factor involved is the size of the rRNA gene transcript. This effect requires only the basic rRNA gene promoter and terminator signals and does not depend on the G+C content of the RNA polymerase I transcripts. We suggest that at low concentrations, the intercalation of actinomycin D changes the conformation of DNA in the promoter region in a manner that stimulates the transcription of both endogenous and transfected rRNA genes. However, transcriptiondriven positive supercoiling of rDNA is enhanced by actinomycin D and above a critical transcript length it causes a blockage of transcription elongation and the accumulation of aborted rDNA transcripts.

Actinomycin D stimulates the transcription of rRNA minigenes transfected into mouse cells. Implications for the in vivo hypersensitivity of rRNA gene transcription.

The in vivo hypersensitivity of eukaryotic rRNA gene transcription to actinomycin D has long been known, but this effect could not be reproduced in model systems and its molecular mechanisms remain uncertain. We studied the action of actinomycin D using mouse rRNA minigenes (with RNA polymerase I promoter and terminator signals), carrying truncated mouse or human rDNA inserts, which are faithfully transcribed upon transient transfection into mouse cells. Low concentrations (0.01-0.08 micrograms/ml) of actinomycin D caused within 1-2 h a 2-7-fold stimulation of the transcription of rRNA minigenes which is inversely related to the size of the rDNA transcript. With transcripts longer than 3 kb the effect was reversed and at 4 kb a practically complete inhibition of the formation of full-length transcripts was observed, accompanied, however, by an enhanced accumulation of unfinished rDNA transcripts. The dependence of actinomycin D action on transcript length was also observed with lacZ gene segments of different size inserted into the mouse rRNA minigenes. The transcription initiation of endogenous rRNA genes was also stimulated by the low doses of actinomycin D as indicated by the enhanced synthesis of unfinished rDNA transcripts (spanning mainly the 5' external transcribed spacer), whereas the synthesis of full-length transcripts was abolished. Removal of actinomycin D from the medium caused within 8-24 h a dramatic increase of the transcription from all rRNA minigenes tested. This stimulation was also inversely related to the size of the transcripts and varied from twofold to fivefold for the 3-4-kb transcripts to about 50-80-fold for the basic minigene transcript (395 nucleotides). The amount of endogenous aborted rDNA transcripts was also markedly increased, but the synthesis of full-length transcripts was not restored even 24 h after removal of the drug. The present results reproduce in a model cellular system the in vivo hypersensitivity of rRNA gene transcription to actinomycin D and reveal that the major factor involved is the size of the rRNA gene transcript. This effect requires only the basic rRNA gene promoter and terminator signals and does not depend on the G + C content of the RNA polymerase I transcripts. We suggest that at low concentrations, the intercalation of actinomycin D changes the conformation of DNA in the promoter region in a manner that stimulates the transcription of both endogenous and transfected rRNA genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Analyses of the Extent of Immunological Relatedness between a Highly Purified Pea Chloroplast Functional RNA Polymerase and Escherichia coli RNA Polymerase

Summary The reported nucleotide sequence homology between the RNA polymerase (EC 2.7.7.6) coding (rpo) genes in Escherichia coli and their putative homologues in chloroplast DNA is not only limited to about 26-50 %, but also scattered. Utilising the homologous in vitro transcription system and immunological approach, we address as to which extent the chloroplast functional RNA polymerase and E. coli RNA polymerase are related. Highly purified RNA polymerase from pea chloroplasts has been demonstrated to transcribe both ribosomal and messenger RNA genes and the purified homologous antibodies inhibit the chloroplast gene promoter recognizing ability as well as in vitro transcriptional activity of the enzyme [Rajasekhar et al., 1991]. While recognizing all of the polypeptides of the chloroplast enzyme on Western blots, the chloroplast RNA polymerase antibodies did not cross-react with the sub-units of E. coli RNA polymerase or wheat germ RNA polymerase and vice versa. Antibodies against the 130 kDa,110 kDa, 75-95 kDa and 48 kDa polypeptides of the purified chloroplast RNA polymerase immobilized only homologous RNA polymerase on the affinity columns. The active enzyme eluted from the immunoaffinity columns showed sub-unit patterns similar to that of purified RNA polymerase. The specificity of these antibodies was confirmed with the lack of immobilization of E. coli RNA polymerase and vice versa with the E. coli RNA polymerase antibodies. The E. coli RNA polymerase also manifested transcription in vitro utilising the supercoiled 7A/30T recombinant containing the chloroplast 16S rRNA minigene as well as the supercoiled pCB 1-3 recombinant containing the upstream sequences of ribulose-1,5-bisphosphate carboxylase large sub-unit (rbc-L) gene. The transcriptional activities, however, were specifically affected only by homologous RNA polymerase antibodies, but not by heterologous RNA polymerase antibodies. While both the chloroplast and the E. coli RNA polymerases caused mobility shifts of the chloroplast 16S rRNA gene and rbc-L gene promoter fragments, only the homologous antibodies were effective in abolishing this response. The data obtained form the first functional evidence that the highly purified pea chloroplast RNA polymerase and the E. coli RNA polymerase are immunologically only distantly related.

The rRNA Enhancer Regulates rRNA Transcription in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the rRNA genes are organized as a tandem array of head-to-tail repeats. An enhancer of rRNA transcription is present just at the end of each transcription unit, 2 kb away from the next one. This enhancer is unusual for S. cerevisiae in that it acts both upstream and downstream of, and even across, genes. The role of the enhancer in the nutritional regulation of rRNA transcription was studied by introducing a centromere plasmid carrying two rRNA minigenes in tandem, flanking a single enhancer, into cells. Analysis of the transcripts from the two minigenes showed that the enhancer was absolutely required for the stimulation of transcription of rRNA that occurs when cells are shifted from a poor carbon source to a good carbon source. While full enhancer function is provided by a 45-bp region at the 3' end of the 190-bp enhancer, some activity was also conferred by other elements, including both a T-rich stretch and a region containing the binding sites for the proteins Reb1p and Abf1p. We conclude that the enhancer is composed of redundant elements and that it is a major element in the regulation of rRNA transcription.

The rRNA enhancer regulates rRNA transcription in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the rRNA genes are organized as a tandem array of head-to-tail repeats. An enhancer of rRNA transcription is present just at the end of each transcription unit, 2 kb away from the next one. This enhancer is unusual for S. cerevisiae in that it acts both upstream and downstream of, and even across, genes. The role of the enhancer in the nutritional regulation of rRNA transcription was studied by introducing a centromere plasmid carrying two rRNA minigenes in tandem, flanking a single enhancer, into cells. Analysis of the transcripts from the two minigenes showed that the enhancer was absolutely required for the stimulation of transcription of rRNA that occurs when cells are shifted from a poor carbon source to a good carbon source. While full enhancer function is provided by a 45-bp region at the 3' end of the 190-bp enhancer, some activity was also conferred by other elements, including both a T-rich stretch and a region containing the binding sites for the proteins Reb1p and Abf1p. We conclude that the enhancer is composed of redundant elements and that it is a major element in the regulation of rRNA transcription.

Activity of promoter mutants of the yeast ribosomal RNA gene with and without the enhancer

The promoter and enhancer of the rRNA gene of Saccharomyces cerevisiae have been studied using a nuclease S1 protection assay to detect transcripts of an rRNA minigene in transformed yeast. Analysis of 5' deletion mutants showed that DNA between -163 bp and -155 bp was important for promoter activity and that some DNA between -155 bp and -145 bp was essential. The importance of DNA far upstream from the initiation site was confirmed by showing that minigene expression was much reduced by linker scanner mutations clustered around -148 bp, -133 bp and -100 bp, and was abolished by mutations clustered around -118 bp. The enhancer for rRNA biosynthesis increased transcription from all of the five mutated promoters that were tested. The magnitude of the enhancer effects on weakly active promoters was two- to three-fold less than on the wild-type promoter. Expression of a minor transcript in a 5' deletion to -10 bp was substantially reduced by a mutation which altered two base pairs in the core sequence of the promoter-proximal REB1 binding site.

The yeast rRNA gene enhancer does not function by recycling RNA polymerase I and cannot act as a UAS

The mechanism of action of the yeast rRNA gene enhancer was investigated by measuring transcription of an rRNA minigene, cloned into a multicopy plasmid, in transformed yeast. Expression of the minigene was increased when the enhancer was cloned either upstream of or downstream from the minigene. When an enhancer was present both upstream and downstream of the minigene, the upstream element was functionally dominant. The upstream enhancer was active in this construct in the absence of detectable read-through by any RNA polymerase. In a construct containing tandem rRNA minigenes, an enhancer element located between the two promoters activated transcription from both independently. Therefore, the enhancer does not appear to activate transcription by recycling RNA polymerase I molecules to the promoter. The enhancer also failed to activate transcription from the intact promoter of the yeast CYC1 gene, and was unable to functionally substitute for the natural upstream activation sequences (UASs) of this gene. Therefore, the enhancer functions differently to UASs of RNA polymerase II genes, and is probably polymerase-specific.

Sequences within the spacer region of yeast rRNA cistrons that stimulate 35S rRNA synthesis in vivo mediate RNA polymerase I-dependent promoter and terminator activities.

Sequences within the spacer region of yeast rRNA cistrons stimulate synthesis of the major 35S rRNA precursor in vivo 10- to 30-fold (E. A. Elion and J. R. Warner, Cell 39:663-673, 1984). Spacer sequences that mediate this stimulatory activity are located approximately 2.2 kilobases upstream from sequences that encode the 5' terminus of the 35S rRNA precursor. By utilizing a centromere-containing plasmid carrying a 35S rRNA minigene, a 160-base-pair region of spacer rDNA was identified by deletion mapping that is required for efficient stimulation of 35S rRNA synthesis in vivo. A 22-base-pair sequence, previously shown to support RNA polymerase I-dependent selective initiation of transcription in vitro, was located 15 base pairs upstream from the 3' boundary of the stimulatory region. A 77-base pair region of spacer DNA that mediates transcriptional terminator activity in vivo was identified immediately downstream from the 5' boundary of the stimulatory region. Deletion mutations extending downstream from the 5' boundary of the 160-base-pair stimulatory region simultaneously interfere with terminator activity and stimulation of 35S rRNA synthesis from the minigene. The terminator region supported termination of transcripts initiated by RNA polymerase I in vivo. The organization of sequences that support terminator and promoter activities within the 160-base-pair stimulatory region is similar to the organization of rDNA gene promoters in higher organisms. Possible mechanisms for spacer-sequence-dependent stimulation of yeast 35S rRNA synthesis in vivo are discussed.

Sequences within the spacer region of yeast rRNA cistrons that stimulate 35S rRNA synthesis in vivo mediate RNA polymerase I-dependent promoter and terminator activities.

Sequences within the spacer region of yeast rRNA cistrons stimulate synthesis of the major 35S rRNA precursor in vivo 10- to 30-fold (E. A. Elion and J. R. Warner, Cell 39:663-673, 1984). Spacer sequences that mediate this stimulatory activity are located approximately 2.2 kilobases upstream from sequences that encode the 5' terminus of the 35S rRNA precursor. By utilizing a centromere-containing plasmid carrying a 35S rRNA minigene, a 160-base-pair region of spacer rDNA was identified by deletion mapping that is required for efficient stimulation of 35S rRNA synthesis in vivo. A 22-base-pair sequence, previously shown to support RNA polymerase I-dependent selective initiation of transcription in vitro, was located 15 base pairs upstream from the 3' boundary of the stimulatory region. A 77-base pair region of spacer DNA that mediates transcriptional terminator activity in vivo was identified immediately downstream from the 5' boundary of the stimulatory region. Deletion mutations extending downstream from the 5' boundary of the 160-base-pair stimulatory region simultaneously interfere with terminator activity and stimulation of 35S rRNA synthesis from the minigene. The terminator region supported termination of transcripts initiated by RNA polymerase I in vivo. The organization of sequences that support terminator and promoter activities within the 160-base-pair stimulatory region is similar to the organization of rDNA gene promoters in higher organisms. Possible mechanisms for spacer-sequence-dependent stimulation of yeast 35S rRNA synthesis in vivo are discussed.

Termination of transcription by yeast RNA polymerase I.

Analysis of the termination of transcription by yeast RNA polymerase I (Pol I) using in vitro run-on experiments in both isolated nuclei and permeabilized cells demonstrated that Pol I does not traverse the whole intergenic spacer separating consecutive 37S operons, but terminates transcription before reaching the 5S rRNA gene, that is within NTS 1. In order to discriminate between processing and termination at the 3'-end generating sites previously identified in vivo in NTS 1 (T1, T2 and T3), fragments containing these sites were inserted into the middle of the reporter DNA of an artificial rRNA minigene. RNA isolated from yeast cells transformed with these minigenes was analyzed for the presence of transcripts derived from sequences both up- and downstream of the insert by Northern blot hybridization, reverse transcription analysis and S1 nuclease mapping. In accordance with previously obtained results T1 (+15 to +50) was found to behave as a processing site. T2 (+210) however was concluded to be an efficient, genuine Pol I terminator. In addition to T2, two other terminators were identified in NTS 1: T3A (at +690) and T3B (at +950). Surprisingly, when the 3' terminal part of NTS 2 was tested for its capacity to generate 3'-ends, another terminator (Tp) was found to be present at a position 300 bp upstream of the transcription initiation site of the 37S-rRNA operon.

3'-End formation of transcripts from the yeast rRNA operon.

Deletion analysis of artificial rRNA minigenes transformed into Saccharomyces cerevisiae revealed that a 110 bp long fragment corresponding to positions -36 to +74 relative to the 3'-end of the 26S rRNA gene, is both necessary and sufficient for obtaining transcripts whose 3'-termini are identical to those of 26S and 37S (pre-)rRNA. These termini are produced via processing of longer transcripts because in an rna 82.1 mutant the majority of the minigene transcripts extend further downstream. Since the rna 82.1 mutation inactivates an endonuclease involved in the 3'-processing of 5S pre-rRNA it is concluded that the maturation of 37S- and that of 5S pre-rRNA requires a common factor. Comparison of the spacer sequences between Saccharomyces carlsbergensis, Saccharomyces rosei and Hansenula wingei revealed several conserved sequence blocks within the region between +10 and +55. These conserved sequence tracts, which are part of a longer region showing dyad symmetry, are supposed to be involved in the interaction with the processing component(s). Deletion of the sequences required for the formation of the 3'-ends of 26S rRNA and 37S pre-rRNA revealed a putative terminator for transcription by RNA polymerase I situated at position +210. This site maps within a DNA fragment that also contains the enhancing element for rDNA transcription by RNA polymerase I.

Upstream activation of ribosomal RNA biosynthesis in Saccharomyces cerevisiae.

Yeast was transformed with eight recombinants that contained an rRNA minigene and upstream elements of rDNA in different orientations in the multi-copy yeast-Escherichia coli shuttle vector, pJDB207. The effect of these elements of upstream rDNA on the initiation of transcription of the minigene at the site for rRNA biosynthesis was determined by using an S1 nuclease mapping procedure to measure the abundance of the minigene transcript in RNA from the yeast transformants. Transcription of the minigene was enhanced 3-fold by DNA within a 2.2 kb element more than 1.5 kb upstream from the initiation site. Inversion of the 2.2 kb element decreased expression of the minigene by 40%. This 2.2 kb element contained approx. 500 bp from the 25S rRNA coding region at the 3' end of the preceding rRNA gene and 1 kb of adjacent nontranscribed spacer rDNA. The enhancing activity was independent of interference from readthrough that might have contributed to the 7-fold decrease in minigene expression caused by removing all rDNA upstream from -209 bp.

Transcription of a yeast ribosomal RNA minigene in Saccharomyces cerevisiae.

A transcription system using intact yeast has been developed for investigating which sequences are implicated in the initiation of transcription of yeast rRNA genes. The system employs an rRNA minigene that consists of the initiation and termination sites for rRNA biosynthesis separated by approx. 700 base pairs of vector DNA in the Escherichia coli-yeast shuttle vector, pJDB207. Two recombinants containing this minigene were constructed; one retained all of the nontranscribed spacer DNA upstream from the initiation site, the other retained 208 base pairs of this DNA. Transcripts of this structurally unique minigene in RNA from yeast transformed with these recombinants were readily detected by nuclease S1 mapping. These transcripts were initiated at the site used by the host rRNA genes, were approx. 3-fold more abundant in the recombinant retaining all of the nontranscribed spacer and were less abundant when the yeast was not growing.