Publications by authors named "Priscilla Braglia"

8 Publications

  • Page 1 of 1

Co-transcriptional RNA cleavage provides a failsafe termination mechanism for yeast RNA polymerase I.

Nucleic Acids Res 2011 Mar 23;39(4):1439-48. Epub 2010 Oct 23.

Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK.

Ribosomal RNA, transcribed by RNA polymerase (Pol) I, accounts for most cellular RNA. Since Pol I transcribes rDNA repeats with high processivity and polymerase density, transcription termination is a critical process. Early in vitro studies proposed polymerase pausing by Reb1 and transcript release at the T-rich element T1 determined transcription termination. However recent in vivo studies revealed a 'torpedo' mechanism for Pol I termination: co-transcriptional RNA cleavage by Rnt1 provides an entry site for the 5'-3' exonuclease Rat1 that degrades Pol I-associated transcripts destabilizing the transcription complex. Significantly Rnt1 inactivation in vivo reveals a second co-transcriptional RNA cleavage event at T1 which provides Pol I with an alternative termination pathway. An intact Reb1-binding site is also required for Rnt1-independent termination. Consequently our results reconcile the original Reb1-mediated termination pathway as part of a failsafe mechanism for this essential transcription process.
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http://dx.doi.org/10.1093/nar/gkq894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3045592PMC
March 2011

Role of the RNA/DNA kinase Grc3 in transcription termination by RNA polymerase I.

EMBO Rep 2010 Oct 3;11(10):758-64. Epub 2010 Sep 3.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

Transcription termination by RNA polymerase I in Saccharomyces cerevisiae is mediated by a 'torpedo' mechanism: co-transcriptional RNA cleavage by Rnt1 at the ribosomal DNA 3'-region generates a 5'-end that is recognized by the 5'-3' exonuclease Rat1; this degrades the downstream transcript and eventually causes termination. In this study, we identify Grc3 as a new factor involved in this process. We demonstrate that GRC3, an essential gene of previously unknown function, encodes a polynucleotide kinase that is required for efficient termination by RNA polymerase I. We propose that it controls the phosphorylation status of the downstream Rnt1 cleavage product and thereby regulates its accessibility to the torpedo Rat1.
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http://dx.doi.org/10.1038/embor.2010.130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2948184PMC
October 2010

Positive modulation of RNA polymerase III transcription by ribosomal proteins.

Biochem Biophys Res Commun 2009 Feb 29;379(2):489-93. Epub 2008 Dec 29.

Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, Viale G.P. Usberti 23/A, 43100 Parma, Italy.

A yeast nuclear fraction of unknown composition, named TFIIIE, was reported previously to enhance transcription of tRNA and 5S rRNA genes in vitro. We show that TFIIIE activity co-purifies with a specific subset of ribosomal proteins (RPs) which, as revealed by chromatin immunoprecipitation analysis, generally interact with tRNA and 5S rRNA genes, but not with a Pol II-specific promoter. Only Rpl6Ap and Rpl6Bp, among the tested RPs, were found associated to a TATA-containing tRNA(Ile)(TAT) gene. The RPL6A gene also emerged as a strong multicopy suppressor of a conditional mutation in the basal transcription factor TFIIIC, while RPL26A and RPL14A behaved as weak suppressors. The data delineate a novel extra-ribosomal role for one or a few RPs which, by influencing 5S rRNA and tRNA synthesis, could play a key role in the coordinate regulation of the different sub-pathways required for ribosome biogenesis and functionality.
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http://dx.doi.org/10.1016/j.bbrc.2008.12.097DOI Listing
February 2009

Budding yeast RNA polymerases I and II employ parallel mechanisms of transcriptional termination.

Genes Dev 2008 Apr;22(8):1082-92

Sir William Dunn School of Pathology, Oxford OX1 3RE, United Kingdom.

Both RNA polymerase I and II (Pol I and Pol II) in budding yeast employ a functionally homologous "torpedo-like" mechanism to promote transcriptional termination. For two well-defined Pol II-transcribed genes, CYC1 and PMA1, we demonstrate that both Rat1p exonuclease and Sen1p helicase are required for efficient termination by promoting degradation of the nascent transcript associated with Pol II, following mRNA 3' end processing. Similarly, Pol I termination relies on prior Rnt1p cleavage at the 3' end of the pre-rRNA 35S transcript. This is followed by the combined actions of Rat1p and Sen1p to degrade the Pol I-associated nascent transcript that consequently promote termination in the downstream rDNA spacer sequence. Our data suggest that the previously defined in vitro Pol I termination mechanism involving the action of the Reb1p DNA-binding factor to "road-block" Pol I transcription close to the termination region may have overlooked more complex in vivo molecular processes.
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http://dx.doi.org/10.1101/gad.463408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2335328PMC
April 2008

RNA polymerase I in yeast transcribes dynamic nucleosomal rDNA.

Nat Struct Mol Biol 2007 Feb 28;14(2):123-30. Epub 2007 Jan 28.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

RNA polymerase (Pol) I-transcribed ribosomal genes of budding yeast exist as a tandem array (about 150 repeats) with transcription units separated by spacer sequences. Half of these rDNAs are inactivated by repressive chromatin structure, whereas the rest exist in an open conformation transcribed by closely spaced Pol I elongation complexes. Whereas previous studies have suggested that active rDNA is devoid of nucleosomal structure, we demonstrate that active rDNA has nucleosomal structure, according to chromatin immunoprecipitation and biochemical fractionation. Using a yeast strain with reduced numbers of all actively transcribed rDNA repeats, we show that rDNA exists in a dynamic chromatin structure of unphased nucleosomes. Furthermore, it is associated with chromatin-remodeling enzymes Chd1p, Isw1p and Isw2p, whose inactivation causes defects in transcription termination. We suggest that Pol I transcription, like that of Pol II, may be modulated by specific chromatin structures.
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http://dx.doi.org/10.1038/nsmb1199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941936PMC
February 2007

Requirement of Nhp6 proteins for transcription of a subset of tRNA genes and heterochromatin barrier function in Saccharomyces cerevisiae.

Mol Cell Biol 2007 Mar 18;27(5):1545-57. Epub 2006 Dec 18.

Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, Viale G.P. Usberti 23A, 43100 Parma, Italy.

A key event in tRNA gene (tDNA) transcription by RNA polymerase (Pol) III is the TFIIIC-dependent assembly of TFIIIB upstream of the transcription start site. Different tDNA upstream sequences bind TFIIIB with different affinities, thereby modulating tDNA transcription. We found that in the absence of Nhp6 proteins, the influence of the 5'-flanking region on tRNA gene transcription is dramatically enhanced in Saccharomyces cerevisiae. Expression of a tDNA bearing a suboptimal TFIIIB binding site, but not of a tDNA preceded by a strong TFIIIB binding region, was strongly dependent on Nhp6 in vivo. Upstream sequence-dependent stimulation of tRNA gene transcription by Nhp6 could be reproduced in vitro, and Nhp6 proteins were found associated with tRNA genes in yeast cells. We also show that both transcription and silencing barrier activity of a tDNA(Thr) at the HMR locus are compromised in the absence of Nhp6. Our data suggest that Nhp6 proteins are important components of Pol III chromatin templates that contribute both to the robustness of tRNA gene expression and to positional effects of Pol III transcription complexes.
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http://dx.doi.org/10.1128/MCB.00773-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1820459PMC
March 2007

Sequence context effects on oligo(dT) termination signal recognition by Saccharomyces cerevisiae RNA polymerase III.

J Biol Chem 2005 May 22;280(20):19551-62. Epub 2005 Mar 22.

Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, Italy.

Eukaryotic RNA polymerase (Pol) III terminates transcription at short runs of T residues in the coding DNA strand. By genomic analysis, we found that T(5) and T(4) are the shortest Pol III termination signals in yeasts and mammals, respectively, and that, at variance with yeast, oligo(dT) terminators longer than T(5) are very rare in mammals. In Saccharomyces cerevisiae, the strength of T(5) as a terminator was found to be largely influenced by both the upstream and the downstream sequence context. In particular, the CT sequence, which is naturally present downstream of T(5) in the 3'-flank of some tDNAs, was found to act as a terminator-weakening element that facilitates translocation by reducing Pol III pausing at T(5). In contrast, tDNA transcription termination was highly efficient when T(5) was followed by an A or G residue. Surprisingly, however, when a termination-proficient T(5) signal was taken out from the tDNA context and placed downstream of a fragment of the SCR1 gene, its termination activity was compromised, both in vitro and in vivo. Even the T(6) sequence, acting as a strong terminator in tRNA gene contexts, was unexpectedly weak within the SNR52 transcription unit, where it naturally occurs. The observed sequence context effects reflect intrinsic recognition properties of Pol III, because they were still observed in a simplified in vitro transcription system only consisting of purified RNA polymerase and template DNA. Our findings strengthen the notion that termination signal recognition by Pol III is influenced in a complex way by the region surrounding the T cluster and suggest that read-through transcription beyond T clusters might play a significant role in the biogenesis of class III gene products.
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http://dx.doi.org/10.1074/jbc.M412238200DOI Listing
May 2005

A composite upstream sequence motif potentiates tRNA gene transcription in yeast.

J Mol Biol 2003 Oct;333(1):1-20

Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, Parco Area delle Scienze 23/A, 43100 Parma, Italy.

Transcription of eukaryotic tRNA genes relies on the TFIIIC-dependent recruitment of TFIIIB on a approximately 50 bp region upstream of the transcription start site (TSS). TFIIIC specifically interacts with highly conserved, intragenic promoter elements, while the contacts between TFIIIB and the upstream DNA have long been considered as largely non-specific. Through a computer search procedure designed to detect shared, yet degenerate sequence features, we have identified a conserved sequence pattern upstream of Saccharomyces cerevisiae tDNAs. This pattern consists of four regions in which particular sequences are over-represented. The most downstream of these regions surrounds the TSS, while the other three districts of sequence conservation (appearing as a centrally located TATA-like sequence flanked by T-rich elements on both sides) are located across the DNA region known to interact with TFIIIB. Upstream regions whose sequence conforms to this pattern were found to potentiate tRNA gene transcription, both in vitro and in vivo, by enhancing TFIIIB binding. A conserved pattern of DNA bendability was also revealed, with peaks of bending propensity centered on the TATA-like and the TSS regions. Sequence analysis of other eukaryotic genomes further revealed the widespread occurrence of conserved sequence patterns upstream of tDNAs, with striking lineage-specific differences in the number and sequence of conserved motifs. Our data strongly support the notion that tRNA gene transcription in eukaryotes is modulated by composite TFIIIB binding sites that may confer responsiveness to variation in TFIIIB activity and/or concentration.
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http://dx.doi.org/10.1016/j.jmb.2003.08.016DOI Listing
October 2003