Research

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12 pages, 1652 KiB

Open AccessArticle

Defining the Influence of the A12.2 Subunit on Transcription Elongation and Termination by RNA Polymerase I In Vivo

by Andrew M. Clarke, Abigail K. Huffines, Yvonne J. K. Edwards, Chad M. Petit and David A. Schneider

Genes 2021, 12(12), 1939; https://0-doi-org.brum.beds.ac.uk/10.3390/genes12121939 - 30 Nov 2021

Cited by 5 | Viewed by 2102

Abstract

Saccharomyces cerevisiae has approximately 200 copies of the 35S rDNA gene, arranged tandemly on chromosome XII. This gene is transcribed by RNA polymerase I (Pol I) and the 35S rRNA transcript is processed to produce three of the four rRNAs required for ribosome [...] Read more.

Saccharomyces cerevisiae has approximately 200 copies of the 35S rDNA gene, arranged tandemly on chromosome XII. This gene is transcribed by RNA polymerase I (Pol I) and the 35S rRNA transcript is processed to produce three of the four rRNAs required for ribosome biogenesis. An intergenic spacer (IGS) separates each copy of the 35S gene and contains the 5S rDNA gene, the origin of DNA replication, and the promoter for the adjacent 35S gene. Pol I is a 14-subunit enzyme responsible for the majority of rRNA synthesis, thereby sustaining normal cellular function and growth. The A12.2 subunit of Pol I plays a crucial role in cleavage, termination, and nucleotide addition during transcription. Deletion of this subunit causes alteration of nucleotide addition kinetics and read-through of transcription termination sites. To interrogate both of these phenomena, we performed native elongating transcript sequencing (NET-seq) with an rpa12Δ strain of S. cerevisiae and evaluated the resultant change in Pol I occupancy across the 35S gene and the IGS. Compared to wild-type (WT), we observed template sequence-specific changes in Pol I occupancy throughout the 35S gene. We also observed rpa12Δ Pol I occupancy downstream of both termination sites and throughout most of the IGS, including the 5S gene. Relative occupancy of rpa12Δ Pol I increased upstream of the promoter-proximal Reb1 binding site and dropped significantly downstream, implicating this site as a third terminator for Pol I transcription. Collectively, these high-resolution results indicate that the A12.2 subunit of Pol I plays an important role in transcription elongation and termination. Full article

(This article belongs to the Special Issue Study of Regulatory Mechanisms Associated with Transcription of Ribosomal RNA)

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20 pages, 3940 KiB

Open AccessArticle

DNA Intercalators Inhibit Eukaryotic Ribosomal RNA Synthesis by Impairing the Initiation of Transcription

by William J. Andrews, Swagat Ray, Tatiana Panova, Christoph Engel and Konstantin I. Panov

Genes 2021, 12(9), 1412; https://0-doi-org.brum.beds.ac.uk/10.3390/genes12091412 - 14 Sep 2021

Cited by 8 | Viewed by 3555

Abstract

In eukaryotes, ribosome biogenesis is driven by the synthesis of the ribosomal RNA (rRNA) by RNA polymerase I (Pol-I) and is tightly linked to cell growth and proliferation. The 3D-structure of the rDNA promoter plays an important, yet not fully understood role in [...] Read more.

In eukaryotes, ribosome biogenesis is driven by the synthesis of the ribosomal RNA (rRNA) by RNA polymerase I (Pol-I) and is tightly linked to cell growth and proliferation. The 3D-structure of the rDNA promoter plays an important, yet not fully understood role in regulating rRNA synthesis. We hypothesized that DNA intercalators/groove binders could affect this structure and disrupt rRNA transcription. To test this hypothesis, we investigated the effect of a number of compounds on Pol-I transcription in vitro and in cells. We find that intercalators/groove binders are potent inhibitors of Pol-I specific transcription both in vitro and in cells, regardless of their specificity and the strength of its interaction with DNA. Importantly, the synthetic ability of Pol-I is unaffected, suggesting that these compounds are not targeting post-initiating events. Notably, the tested compounds have limited effect on transcription by Pol-II and III, demonstrating the hypersensitivity of Pol-I transcription. We propose that stability of pre-initiation complex and initiation are affected as result of altered 3D architecture of the rDNA promoter, which is well in line with the recently reported importance of biophysical rDNA promoter properties on initiation complex formation in the yeast system. Full article

(This article belongs to the Special Issue Study of Regulatory Mechanisms Associated with Transcription of Ribosomal RNA)

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12 pages, 1219 KiB

Open AccessArticle

Spt4 Promotes Pol I Processivity and Transcription Elongation

by Abigail K. Huffines, Yvonne J. K. Edwards and David A. Schneider

Genes 2021, 12(3), 413; https://0-doi-org.brum.beds.ac.uk/10.3390/genes12030413 - 12 Mar 2021

Cited by 6 | Viewed by 2007

Abstract

RNA polymerases (Pols) I, II, and III collectively synthesize most of the RNA in a eukaryotic cell. Transcription by Pols I, II, and III is regulated by hundreds of trans-acting factors. One such protein, Spt4, has been previously identified as a transcription factor [...] Read more.

RNA polymerases (Pols) I, II, and III collectively synthesize most of the RNA in a eukaryotic cell. Transcription by Pols I, II, and III is regulated by hundreds of trans-acting factors. One such protein, Spt4, has been previously identified as a transcription factor that influences both Pols I and II. Spt4 forms a complex with Spt5, described as the Spt4/5 complex (or DSIF in mammalian cells). This complex has been shown previously to directly interact with Pol I and potentially affect transcription elongation. The previous literature identified defects in transcription by Pol I when SPT4 was deleted, but the necessary tools to characterize the mechanism of this effect were not available at the time. Here, we use a technique called Native Elongating Transcript Sequencing (NET-seq) to probe for the global occupancy of Pol I in wild-type (WT) and spt4△ Saccharomyces cerevisiae (yeast) cells at single nucleotide resolution in vivo. Analysis of NET-seq data reveals that Spt4 promotes Pol I processivity and enhances transcription elongation through regions of the ribosomal DNA that are particularly G-rich. These data suggest that Spt4/5 may directly affect transcription elongation by Pol I in vivo. Full article

(This article belongs to the Special Issue Study of Regulatory Mechanisms Associated with Transcription of Ribosomal RNA)

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Review

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19 pages, 2718 KiB

Open AccessReview

Harnessing the Nucleolar DNA Damage Response in Cancer Therapy

by Jiachen Xuan, Kezia Gitareja, Natalie Brajanovski and Elaine Sanij

Genes 2021, 12(8), 1156; https://0-doi-org.brum.beds.ac.uk/10.3390/genes12081156 - 28 Jul 2021

Cited by 9 | Viewed by 4357

Abstract

The nucleoli are subdomains of the nucleus that form around actively transcribed ribosomal RNA (rRNA) genes. They serve as the site of rRNA synthesis and processing, and ribosome assembly. There are 400–600 copies of rRNA genes (rDNA) in human cells and their highly [...] Read more.

The nucleoli are subdomains of the nucleus that form around actively transcribed ribosomal RNA (rRNA) genes. They serve as the site of rRNA synthesis and processing, and ribosome assembly. There are 400–600 copies of rRNA genes (rDNA) in human cells and their highly repetitive and transcribed nature poses a challenge for DNA repair and replication machineries. It is only in the last 7 years that the DNA damage response and processes of DNA repair at the rDNA repeats have been recognized to be unique and distinct from the classic response to DNA damage in the nucleoplasm. In the last decade, the nucleolus has also emerged as a central hub for coordinating responses to stress via sequestering tumor suppressors, DNA repair and cell cycle factors until they are required for their functional role in the nucleoplasm. In this review, we focus on features of the rDNA repeats that make them highly vulnerable to DNA damage and the mechanisms by which rDNA damage is repaired. We highlight the molecular consequences of rDNA damage including activation of the nucleolar DNA damage response, which is emerging as a unique response that can be exploited in anti-cancer therapy. In this review, we focus on CX-5461, a novel inhibitor of Pol I transcription that induces the nucleolar DNA damage response and is showing increasing promise in clinical investigations. Full article

(This article belongs to the Special Issue Study of Regulatory Mechanisms Associated with Transcription of Ribosomal RNA)

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21 pages, 1323 KiB

Open AccessReview

Antagonising Chromatin Remodelling Activities in the Regulation of Mammalian Ribosomal Transcription

by Kanwal Tariq and Ann-Kristin Östlund Farrants

Genes 2021, 12(7), 961; https://0-doi-org.brum.beds.ac.uk/10.3390/genes12070961 - 24 Jun 2021

Cited by 2 | Viewed by 2721

Abstract

Ribosomal transcription constitutes the major energy consuming process in cells and is regulated in response to proliferation, differentiation and metabolic conditions by several signalling pathways. These act on the transcription machinery but also on chromatin factors and ncRNA. The many ribosomal gene repeats [...] Read more.

Ribosomal transcription constitutes the major energy consuming process in cells and is regulated in response to proliferation, differentiation and metabolic conditions by several signalling pathways. These act on the transcription machinery but also on chromatin factors and ncRNA. The many ribosomal gene repeats are organised in a number of different chromatin states; active, poised, pseudosilent and repressed gene repeats. Some of these chromatin states are unique to the 47rRNA gene repeat and do not occur at other locations in the genome, such as the active state organised with the HMG protein UBF whereas other chromatin state are nucleosomal, harbouring both active and inactive histone marks. The number of repeats in a certain state varies on developmental stage and cell type; embryonic cells have more rRNA gene repeats organised in an open chromatin state, which is replaced by heterochromatin during differentiation, establishing different states depending on cell type. The 47S rRNA gene transcription is regulated in different ways depending on stimulus and chromatin state of individual gene repeats. This review will discuss the present knowledge about factors involved, such as chromatin remodelling factors NuRD, NoRC, CSB, B-WICH, histone modifying enzymes and histone chaperones, in altering gene expression and switching chromatin states in proliferation, differentiation, metabolic changes and stress responses. Full article

(This article belongs to the Special Issue Study of Regulatory Mechanisms Associated with Transcription of Ribosomal RNA)

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17 pages, 724 KiB

Open AccessReview

The Ribosomal Gene Loci—The Power behind the Throne

by Konstantin I. Panov, Katherine Hannan, Ross D. Hannan and Nadine Hein

Genes 2021, 12(5), 763; https://0-doi-org.brum.beds.ac.uk/10.3390/genes12050763 - 18 May 2021

Cited by 11 | Viewed by 3626

Abstract

Nucleoli form around actively transcribed ribosomal RNA (rRNA) genes (rDNA), and the morphology and location of nucleolus-associated genomic domains (NADs) are linked to the RNA Polymerase I (Pol I) transcription status. The number of rDNA repeats (and the proportion of actively transcribed rRNA [...] Read more.

Nucleoli form around actively transcribed ribosomal RNA (rRNA) genes (rDNA), and the morphology and location of nucleolus-associated genomic domains (NADs) are linked to the RNA Polymerase I (Pol I) transcription status. The number of rDNA repeats (and the proportion of actively transcribed rRNA genes) is variable between cell types, individuals and disease state. Substantial changes in nucleolar morphology and size accompanied by concomitant changes in the Pol I transcription rate have long been documented during normal cell cycle progression, development and malignant transformation. This demonstrates how dynamic the nucleolar structure can be. Here, we will discuss how the structure of the rDNA loci, the nucleolus and the rate of Pol I transcription are important for dynamic regulation of global gene expression and genome stability, e.g., through the modulation of long-range genomic interactions with the suppressive NAD environment. These observations support an emerging paradigm whereby the rDNA repeats and the nucleolus play a key regulatory role in cellular homeostasis during normal development as well as disease, independent of their role in determining ribosome capacity and cellular growth rates. Full article

(This article belongs to the Special Issue Study of Regulatory Mechanisms Associated with Transcription of Ribosomal RNA)

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15 pages, 3194 KiB

Open AccessReview

The Mammalian and Yeast A49 and A34 Heterodimers: Homologous but Not the Same

by Rachel McNamar, Katrina Rothblum and Lawrence I. Rothblum

Genes 2021, 12(5), 620; https://0-doi-org.brum.beds.ac.uk/10.3390/genes12050620 - 22 Apr 2021

Cited by 3 | Viewed by 1639

Abstract

Ribosomal RNA synthesis is the rate-limiting step in ribosome biogenesis. In eukaryotes, RNA polymerase I (Pol I) is responsible for transcribing the ribosomal DNA genes that reside in the nucleolus. Aberrations in Pol I activity have been linked to the development of multiple [...] Read more.

Ribosomal RNA synthesis is the rate-limiting step in ribosome biogenesis. In eukaryotes, RNA polymerase I (Pol I) is responsible for transcribing the ribosomal DNA genes that reside in the nucleolus. Aberrations in Pol I activity have been linked to the development of multiple cancers and other genetic diseases. Therefore, it is key that we understand the mechanisms of Pol I transcription. Recent studies have demonstrated that there are many differences between Pol I transcription in yeast and mammals. Our goal is to highlight the similarities and differences between the polymerase-associated factors (PAFs) in yeast and mammalian cells. We focus on the PAF heterodimer A49/34 in yeast and PAF53/49 in mammals. Recent studies have demonstrated that while the structures between the yeast and mammalian orthologs are very similar, they may function differently during Pol I transcription, and their patterns of regulation are different. Full article

(This article belongs to the Special Issue Study of Regulatory Mechanisms Associated with Transcription of Ribosomal RNA)

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16 pages, 1829 KiB

Open AccessReview

Ribosomal RNA Transcription Regulation in Breast Cancer

by Cecelia M. Harold, Amber F. Buhagiar, Yan Cheng and Susan J. Baserga

Genes 2021, 12(4), 502; https://0-doi-org.brum.beds.ac.uk/10.3390/genes12040502 - 29 Mar 2021

Cited by 18 | Viewed by 4184

Abstract

Ribosome biogenesis is a complex process that is responsible for the formation of ribosomes and ultimately global protein synthesis. The first step in this process is the synthesis of the ribosomal RNA in the nucleolus, transcribed by RNA Polymerase I. Historically, abnormal nucleolar [...] Read more.

Ribosome biogenesis is a complex process that is responsible for the formation of ribosomes and ultimately global protein synthesis. The first step in this process is the synthesis of the ribosomal RNA in the nucleolus, transcribed by RNA Polymerase I. Historically, abnormal nucleolar structure is indicative of poor cancer prognoses. In recent years, it has been shown that ribosome biogenesis, and rDNA transcription in particular, is dysregulated in cancer cells. Coupled with advancements in screening technology that allowed for the discovery of novel drugs targeting RNA Polymerase I, this transcriptional machinery is an increasingly viable target for cancer therapies. In this review, we discuss ribosome biogenesis in breast cancer and the different cellular pathways involved. Moreover, we discuss current therapeutics that have been found to affect rDNA transcription and more novel drugs that target rDNA transcription machinery as a promising avenue for breast cancer treatment. Full article

(This article belongs to the Special Issue Study of Regulatory Mechanisms Associated with Transcription of Ribosomal RNA)

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