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. 2017 May 3;18(1):75.
doi: 10.1186/s13059-017-1209-z.

Epistatic and Allelic Interactions Control Expression of Ribosomal RNA Gene Clusters in Arabidopsis Thaliana

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Free PMC article

Epistatic and Allelic Interactions Control Expression of Ribosomal RNA Gene Clusters in Arabidopsis Thaliana

Fernando A Rabanal et al. Genome Biol. .
Free PMC article

Abstract

Background: Ribosomal RNA (rRNA) accounts for the majority of the RNA in eukaryotic cells, and is encoded by hundreds to thousands of nearly identical gene copies, only a subset of which are active at any given time. In Arabidopsis thaliana, 45S rRNA genes are found in two large ribosomal DNA (rDNA) clusters and little is known about the contribution of each to the overall transcription pattern in the species.

Results: By taking advantage of genome sequencing data from the 1001 Genomes Consortium, we characterize rRNA gene sequence variation within and among accessions. Notably, variation is not restricted to the pre-rRNA sequences removed during processing, but it is also present within the highly conserved ribosomal subunits. Through linkage mapping we assign these variants to a particular rDNA cluster unambiguously and use them as reporters of rDNA cluster-specific expression. We demonstrate that rDNA cluster-usage varies greatly among accessions and that rDNA cluster-specific expression and silencing is controlled via genetic interactions between entire rDNA cluster haplotypes (alleles).

Conclusions: We show that rRNA gene cluster expression is controlled via complex epistatic and allelic interactions between rDNA haplotypes that apparently regulate the entire rRNA gene cluster. Furthermore, the sequence polymorphism we discovered implies that the pool of rRNA in a cell may be heterogeneous, which could have functional consequences.

Keywords: Dominance; Epistasis; Ribosomes; Transcription; rRNA genes.

Figures

Fig. 1
Fig. 1
Identification and annotation of polymorphisms along the 45S rRNA gene. a Schematic illustration of the positioning of the rDNA clusters (in black) at the distal end region of chromosomes 2 or 4 (in gray) in A. thaliana, the head-to-tail tandem arrangement of the 45S rRNA genes, and the structure of each ~10 kb long 45S rRNA gene. b Proportion of accessions in the population (1138 individuals; see “Methods”) carrying a variable site (present in > 5% of copies within an individual) along the 45S rRNA gene. Vertical lines represent SNPs or deletions in the minimal promoter (purple), 5’ETS (gray), 18S (red), ITSs (green), 5.8S (yellow), and 25S (blue) regions along the 45S rRNA gene. Black lines depict insertions. Inset shows the distribution of rRNA gene variants shared across accessions, where the number of accessions is displayed in log10 scale. c Example of linkage mapping of the abundance of an 18S variable site (position 2882, T to C) estimated by DNA-sequence coverage in 393 individuals of the MAGIC population (top). Estimated founder accession effect by multiple imputation using R/happy [47, 90] at the major quantitative trait locus from the top panel (bottom). d Similar to (c), but for a 25S variable site (position 6661, G to A)
Fig. 2
Fig. 2
rDNA cluster-specific expression in natural inbred lines. a The proportion of RNA-seq reads expressing a particular reporter variant (y-axis) against the proportion of DNA-seq reads accounting for the existence of the same variant (x-axis) for five natural inbred lines: Col-0 (6909), Sf-2 (7328), Bur-0 (7058), No-0 (7273), and Ct-1 (7067). Notice that no variants Bur-0 rDNA-2-specific passed the threshold (present in > 5% of copies within an individual) due to the small size of that rDNA cluster (Fig. 2b and [36]). Error bars represent standard deviations of three to seven biological replicates. The dashed line indicates the one-to-one ratio between DNA and RNA. b FISH results for the same accessions as in (a) showing that rDNA clusters carrying actively transcribed rRNA localize in proximity to the nucleolus, while silenced rDNAs are observed elsewhere in the nucleus. Images in black and white show DAPI-stained nuclei. Probes hybridizing the 45S rRNA gene cluster, chromosomes 2 and 4 are highlighted in yellow, red, and green, respectively. The nucleolus is marked by a dashed contour. Bar = 10 μm. For (a) and (b), purple and turquoise colored frames indicate accessions dominant for rDNA-2 and rDNA-4, respectively. c Relative frequency of nuclei with a particular rDNA configuration in relation to the nucleolus for the same parental accessions as in (a) and (b). The colored areas correspond to nuclei displaying exclusively two rDNA-2 (dark purple), one rDNA-2 (mid purple), two rDNA-2 per one rDNA-4 (light purple), two rDNA-4 (dark turquoise), one rDNA-4 (mid turquoise), two rDNA-4 per one rDNA-2 (light turquoise), one rDNA-2 per rDNA-4 (light gray), and two rDNA-2 per two rDNA-4 (light green) hybridization signals localized to the nucleolus. The number of nuclei (n) analyzed per accession is indicated at the top of each bar
Fig. 3
Fig. 3
The genotype at both rDNA clusters determines rRNA gene expression. a The proportion of RNA-seq reads expressing a particular reporter variant (y-axis) against the proportion of DNA-seq reads accounting for the existence of the same variant (x-axis) for MAGIC lines 261 and 485. b Similar to (a), but for MAGIC line 170. For both subfigures, error bars represent standard deviations of two biological replicates and the dashed line indicates the one-to-one ratio between DNA and RNA
Fig. 4
Fig. 4
“Every rDNA cluster for itself” in F1 crosses. a The mean proportion of RNA-seq reads expressing a particular reporter variant (y-axis) against the proportion of DNA-seq reads accounting for the existence of the same variant (x-axis) for F1: ♀ Sf-2 x ♂ Col-0. b Similar to (a), but for F1: ♀ Col-0 x ♂ Bur-0. c Similar to (a) and (b), but for F1: ♀ Bur-0 x ♂ Sf-2. The absence of reporter variants Bur-0 rDNA-2-specific is explained in the legend of Fig. 2b. For all subfigures, error bars represent standard deviations of four biological replicates. The dashed line indicates the one-to-one ratio between DNA and RNA
Fig. 5
Fig. 5
Genetic evidence of the interaction between rDNA clusters. a The proportion of RNA-seq reads expressing a particular reporter variant (y-axis) against the proportion of DNA-seq reads accounting for the existence of the same variant (x-axis) for parental accessions Algutsrum (8230) and TDr-9 (6195). Error bars represent standard deviations of two biological replicates. The dashed line represents the one-to-one ratio between DNA and RNA. b Linkage mapping of the expression of a TDr-9 rDNA-2-specific variant (position 1861 in the ETS, T to G) in 68 F2 individuals derived from the selfed F1 progeny of a cross between ♀ Algutsrum x ♂ TDr-9. c Similar to (b), but for a Algutsrum rDNA-4-specific variant (position 2445 in the 18S, T to C) in 183 F2 individuals. d Similar to (b) and (c), but for a Algutsrum rDNA-2-specific variant (position 3904 in the 18S, C to G) in 176 F2 individuals. e Similar to (bd), but for a TDr-9 rDNA-4-specific variant (position 4078 in the ITS, C to deletion) in 162 F2 individuals. For subfigures (be), black lines indicate Simple Interval Mapping (SIM), while orange and red lines indicate Multiple-QTL Mapping (MQM) additive and dominance models, respectively. The horizontal solid and dashed gray lines correspond to the permutation test at 10% and 5% significance levels, respectively. f Schematic representation of the rDNA cluster combinations inherited by F2 individuals (cross-matches between schemes outside the matrix), and the resulting mean expression pattern of their rRNA genes according to all available rDNA cluster-specific variants (squares inside the matrix). Error bars represent standard deviations across the number of F2 individuals (n, which varies between different reporter variants and is given as a range in the figures) in each rDNA cluster combination. Dashed lines indicate the one-to-one ratio between DNA and RNA

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