A Sequence-Specific Interaction between the Saccharomyces cerevisiae rRNA Gene Repeats and a Locus Encoding an RNA Polymerase I Subunit Affects Ribosomal DNA Stability

Abstract

The spatial organization of eukaryotic genomes is linked to their functions. However, how individual features of the global spatial structure contribute to nuclear function remains largely unknown. We previously identified a high-frequency interchromosomal interaction within the Saccharomyces cerevisiae genome that occurs between the intergenic spacer of the ribosomal DNA (rDNA) repeats and the intergenic sequence between the locus encoding the second largest RNA polymerase I subunit and a lysine tRNA gene [i.e., RPA135-tK(CUU)P]. Here, we used quantitative chromosome conformation capture in combination with replacement mapping to identify a 75-bp sequence within the RPA135-tK(CUU)P intergenic region that is involved in the interaction. We demonstrate that the RPA135-IGS1 interaction is dependent on the rDNA copy number and the Msn2 protein. Surprisingly, we found that the interaction does not govern RPA135 transcription. Instead, replacement of a 605-bp region within the RPA135-tK(CUU)P intergenic region results in a reduction in the RPA135-IGS1 interaction level and fluctuations in rDNA copy number. We conclude that the chromosomal interaction that occurs between the RPA135-tK(CUU)P and rDNA IGS1 loci stabilizes rDNA repeat number and contributes to the maintenance of nucleolar stability. Our results provide evidence that the DNA loci involved in chromosomal interactions are composite elements, sections of which function in stabilizing the interaction or mediating a functional outcome. There are strong links between the spatial organization of a genome and its function (reviewed in references 1 to 3). This spatial organization is dynamic and specific for particular cell types and stages of the cell cycle (4-6). Genome spatial organization has been proposed to result from the physical properties of the chromosomes (e.g., volume and flexibility), the proteins (e.g., DNA binding and aggregation), and the nucleic acids (e.g., ability to form triplexes) that are present within the limited volume of the nucleus (7-10). While global genome structure contributes to cell identity (5) and differentiation and cycling (6), the contributions that individual contacts (i.e., interactions) between chromosomal loci make to genome function remain poorly understood. Here, we characterize a high-frequency interaction between the yeast rRNA genes (rDNA) and a locus encoding an RNA polymerase I subunit, RPA135 (Fig. 1A).In Saccharomyces cerevisiae, the rDNA is arranged as 150 to 200 tandem repeats on the long arm of chromosome XII (Chr XII) (11). Each of these tandemly arranged rDNA repeats consists of genes that encode the 35S and 5S rRNAs and two intergenic sequences, IGS1 and IGS2 (Fig. 1A). The rDNA is transcribed by two RNA polymerases: RNA polymerase I (RNAP I), which transcribes the 35S rRNA precursor, and RNAP III, which transcribes the 5S rRNA. Transcription of the rDNA by RNAP I is responsible for the majority of transcription in a cell (reviewed in reference 12).The high rate of transcription and the repetitive structure...

The long-range interaction map of ribosomal DNA arrays

“…This pattern of rDNA-gene contacts might partially explain the observation that genes whose expression was correlated with rDNA CN included several candidates encoding the protein components of the ribosome. Indeed, sequence specific inter-chromosomal interactions between the yeast rDNA array and an intergenic segment adjacent to the largest RNA pol I subunit has recently been demonstrated [ 30 ]. All in all, our study identified functionally coherent genes and GO categories that are depleted and enriched in direct rDNA contacts.…”

Multiplex Genome Engineering Methods for Yeast Cell Factory Development

“…Even though there are intergenic regions between the genes in rDNA cluster (see Figure 3A), IGS1 contains an origin of DNA replication (rARS) while IGS2 includes both the replication fork barrier (RFB) and a bidirectional RNA polymerase II-dependent promoter (Kobayashi and Sasaki, 2017). In addition, it has been revealed that IGS1 is also responsible for maintenance of nucleolar stability along with stabilization of rDNA repeat number (Cahyani et al, 2015). The disruption of even a few rDNA repeats among the hundreds of copies gained through evolutionary processes can therefore affect cell fitness and hinder strain development.…”

DNA replication stress restricts ribosomal DNA copy number

“…In yeast, only about half of the ~150 rDNA repeats are actively transcribed to meet the translational needs of the cell [ 7 ]. Although only about half of these repeats are actively transcribed, it is known that in budding yeast, reduced copy number makes cells more sensitive to DNA damage [ 3 ].The extra, untranscribed copies are thought to contribute to stability and integrity of the locus by acting as a ‘foothold’ for repair enzymes and for contacts with other regions of the genome [ 3 , 8 – 10 ]. Human population genome sequencing data analysis has revealed correlations between rDNA copy number and the expression of several genes encoding chromatin-modifying proteins [ 11 ], further supporting the idea that the effects of rDNA copy number go well beyond rRNA production for ribosome biogenesis.…”