Rate of transcription elongation and sequence-specific pausing by RNA polymerase I directly influence rRNA processing

Abstract

One of the first steps in ribosome biogenesis is transcription of the ribosomal DNA by RNA polymerase I (Pol I). Processing of the resultant rRNA begins cotranscriptionally, and perturbation of Pol I transcription elongation results in defective rRNA processing. Mechanistic insight regarding the link between transcription elongation and ribosome assembly is lacking because of limited in vivo methods to assay Pol I transcription. Here, we use native elongating transcript sequencing (NET-Seq) with a strain of Saccharomyces cerevisiae containing a point mutation in Pol I, rpa190-F1205H, which results in impaired rRNA processing and ribosome assembly. We previously demonstrated that this mutation caused a mild reduction in the transcription elongation rate of Pol I in vitro; however, transcription elongation by the mutant has not been characterized in vivo. Here, our findings demonstrate that the mutant Pol I has an increased pause propensity during processive transcription elongation both in vitro and in vivo. NET-Seq reveals that rpa190-F1205H Pol I displays alternative pause site preferences in vivo. Specifically, the mutant is sensitized to A/G residues in the RNA:DNA hybrid and at the last incorporated nucleotide position. Furthermore, both NET-Seq and EM analysis of Miller chromatin spreads reveal pileups of rpa190-F1205H Pol I throughout the ribosomal DNA, particularly at the 5′ end of the 35S gene. This combination of in vitro and in vivo analyses of a Pol I mutant provides novel insights into Pol I elongation properties and indicates how these properties are crucial for efficient cotranscriptional rRNA processing and ribosome assembly. One of the first steps in ribosome biogenesis is transcription of the ribosomal DNA by RNA polymerase I (Pol I). Processing of the resultant rRNA begins cotranscriptionally, and perturbation of Pol I transcription elongation results in defective rRNA processing. Mechanistic insight regarding the link between transcription elongation and ribosome assembly is lacking because of limited in vivo methods to assay Pol I transcription. Here, we use native elongating transcript sequencing (NET-Seq) with a strain of Saccharomyces cerevisiae containing a point mutation in Pol I, rpa190-F1205H, which results in impaired rRNA processing and ribosome assembly. We previously demonstrated that this mutation caused a mild reduction in the transcription elongation rate of Pol I in vitro; however, transcription elongation by the mutant has not been characterized in vivo. Here, our findings demonstrate that the mutant Pol I has an increased pause propensity during processive transcription elongation both in vitro and in vivo. NET-Seq reveals that rpa190-F1205H Pol I displays alternative pause site preferences in vivo. Specifically, the mutant is sensitized to A/G residues in the RNA:DNA hybrid and at the last incorporated nucleotide position. Furthermore, both NET-Seq and EM analysis of Miller chromatin spreads reveal pileups of rpa190-F1205H Pol I throughout the ribosomal DNA, particularly at the 5′ end of the 35S gene. This combination of in vitro and in vivo analyses of a Pol I mutant provides novel insights into Pol I elongation properties and indicates how these properties are crucial for efficient cotranscriptional rRNA processing and ribosome assembly. Ribosome biogenesis requires the intricate coordination of multiple biochemical processes. The first step is transcription by RNA polymerase I (Pol I), which synthesizes rRNA from a ribosomal DNA (rDNA) template. In Saccharomyces cerevisiae (yeast), Pol I transcribes a single rDNA gene, the 35S, which is organized in approximately 200 tandem repeats. Processing of the rRNA transcripts is complex and requires hundreds of transacting factors and RNAs, ultimately giving rise to the three largest mature rRNAs that serve as the backbone of the ribosome. The earliest studies on rRNA processing suggested that it could only occur post-transcriptionally, and this was supported by the detection of full-length rRNA products in the cell (1Udem S.A. Warner J.R. Ribosomal RNA synthesis in Saccharomyces cerevisiae.J. Mol. Biol. 1972; 65: 227-242Crossref PubMed Scopus (228) Google Scholar, 2Venema J. Tollervey D. Processing of pre-ribosomal RNA in Saccharomyces cerevisiae.Yeast. 1995; 11: 1629-1650Crossref PubMed Scopus (189) Google Scholar). However, over the past 2 decades, it was shown that rRNA processing begins cotranscriptionally. The first evidence of cotranscriptional rRNA processing was from Miller chromatin spreads, which allow for the visualization of engaged Pol I transcription elongation complexes with a DNA template in vivo. from that demonstrated that transcripts Pol I was with the rDNA Miller The of rRNA transcription in chromatin spreads are rRNA processing PubMed Scopus Google Scholar, ribosomal RNA is into the to from transcripts in Saccharomyces PubMed Scopus Google Scholar). Furthermore, it was that transcription elongation by Pol I is is a in rRNA processing and ribosome biogenesis elongation by RNA polymerase I is to efficient rRNA processing and ribosome PubMed Scopus Google Scholar). these findings demonstrate that cotranscriptional processing is for the efficient of the of this is not The by which transcription elongation the processing of RNA is not One that the rate of RNA synthesis the of RNA and the of cotranscriptional that as processing in RNA polymerase elongation PubMed Scopus Google Scholar, RNA during PubMed Scopus Google Scholar, in and and RNA polymerase elongation Biol. 11: PubMed Scopus Google Furthermore, a that are on Pol I transcription rate and rRNA Tollervey D. transcript a in RNA polymerase elongation PubMed Scopus Google Scholar). results demonstrated that Pol I rDNA at a with and that rRNA that the polymerase allow for which could transcription findings and of a during transcription and the of the elongation PubMed Scopus Google that polymerase the of RNA and In the of RNA has been shown to an on Pol transcription rate RNA during transcription on elongation rate and RNA PubMed Scopus Google that the of elongation RNA and RNA is the three at I and of studies the of polymerase elongation properties on RNA and this has been for Pol it was that the mutation of of the residues that in the largest of Pol I, impaired elongation rate in vitro and caused in rRNA processing and ribosome biogenesis elongation by RNA polymerase I is to efficient rRNA processing and ribosome PubMed Scopus Google Scholar). However, the of Pol I transcription elongation and how the of these properties to in rRNA and ribosome assembly is lacking in vivo. The cotranscriptional of rRNA as as the of Pol I this an to the of cotranscriptional on polymerase elongation properties Miller The of rRNA transcription in chromatin spreads are rRNA processing PubMed Scopus Google Scholar, ribosomal RNA is into the to from transcripts in Saccharomyces PubMed Scopus Google Scholar, Tollervey D. processing and occur PubMed Scopus Google and in Tollervey D. in ribosome PubMed Scopus Google the between Pol I elongation and cotranscriptional we characterized the of a Pol I mutant that a point mutation the The is a the site of of and is to transcription elongation rate and propensity in studies of the RNA polymerase elongation Biol. 65: PubMed Scopus Google Scholar, to transcription by RNA Biol. PubMed Scopus Google Scholar, of transcription PubMed Scopus Google We previously shown that this rpa190-F1205H, elongation rate by Pol I in vitro of site residues to transcription by RNA I and PubMed Scopus Google Scholar). Here, we the transcription elongation of the and mutant in vitro transcription with a of rDNA pause for the and by the in we native elongating transcript sequencing (NET-Seq) and EM of Miller chromatin methods reveal that this mutation to Pol I throughout the 35S gene, which could for the in rRNA processing and of ribosome these that increased by Pol I results in cotranscriptional processing because of in rRNA and ribosome assembly results the that transcription elongation is an of that not only the the of the RNA that is both the and rpa190-F1205H to our findings demonstrate that Pol I has a of transcription elongation properties that to the rDNA to efficient ribosome the of rDNA on Pol I we on a to point mutation in a in the rpa190-F1205H, and the of this as with in in vitro transcription We shown previously that the transcription elongation rate of rpa190-F1205H Pol I is of the rate of site residues to transcription by RNA I and PubMed Scopus Google however, this has not been in these we of the cerevisiae rDNA that of the Pol I in the and elongation by RNA polymerase I is to efficient rRNA processing and ribosome PubMed Scopus Google of the in both we a that not residues in the we transcription by with and in the of elongation complexes at the first to the transcription of site residues to transcription by RNA I and PubMed Scopus Google Scholar, analysis of transcription elongation by RNA polymerase I in Mol. 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In to our studies that was a reduction in the rate of transcription elongation by the mutant Pol I, we by the rpa190-F1205H Specifically, the of pause was by the mutant polymerase by and demonstrate that the mutation of the in Pol I by the on the rDNA to Pol I, in a The in vitro biochemical demonstrate that the rpa190-F1205H mutation not only Pol I transcription elongation rate of site residues to transcription by RNA I and PubMed Scopus Google polymerase on the native rDNA this is in we EM of Miller chromatin spreads and allow for the visualization of Pol I transcription on rDNA chromatin as as of the of Pol I throughout the rDNA at single nucleotide of Miller chromatin spreads reveal of rDNA transcription in vivo. We and previously this to a of of Pol I polymerase and transcript processing ribosomal RNA is into the to from transcripts in Saccharomyces PubMed Scopus Google Scholar, of site residues to transcription by RNA I and PubMed Scopus Google Scholar, The complex is for efficient transcription elongation by RNA polymerase PubMed Scopus Google Scholar, J. polymerase polymerase to the rRNA transcription Biol. 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We Miller chromatin spreads of rDNA from and rpa190-F1205H to the of the mutation on Pol I in vivo and The Pol I on the rDNA template from the rpa190-F1205H strain from on the 35S in the an increased pause propensity for Pol I in the mutant Miller spreads Pol I elongation complexes in the of the rDNA to the 5′ we that transcripts in the mutant as with particularly at the end of the rDNA template. 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