Variant ribosomal RNA alleles are conserved and exhibit tissue-specific expression

Abstract

The ribosome, the integration point for protein synthesis in the cell, is conventionally considered a homogeneous molecular assembly that only passively contributes to gene expression. Yet, epigenetic features of the ribosomal DNA (rDNA) operon and changes in the ribosome's molecular composition have been associated with disease phenotypes, suggesting that the ribosome itself may possess inherent regulatory capacity. Analyzing whole-genome sequencing data from the 1000 Genomes Project and the Mouse Genomes Project, we find that rDNA copy number varies widely across individuals, and we identify pervasive intra- and interindividual nucleotide variation in the 5S, 5.8S, 18S, and 28S ribosomal RNA (rRNA) genes of both human and mouse. Conserved rRNA sequence heterogeneities map to functional centers of the assembled ribosome, variant rRNA alleles exhibit tissue-specific expression, and ribosomes bearing variant rRNA alleles are present in the actively translating ribosome pool. These findings provide a critical framework for exploring the possibility that the expression of genomically encoded variant rRNA alleles gives rise to physically and functionally heterogeneous ribosomes that contribute to mammalian physiology and human disease.

Fig. 1. rDNA operons in the human genome.

(A) Chromosomal locations of rDNA operons and organization of the rRNA genes within the 47S rDNA operon in the human genome. Together with the 5S rRNA, the 18S, 5.8S, and 28S rRNAs form the RNA core of the ribosome. tRNA, transfer RNA. (B) Per-individual rDNA copy number estimation in humans, grouped by population.

Fig. 2. High-frequency genomic rRNA variants detected in human.

Subunit interface views of the (A) small-subunit (40S) and (B) large-subunit (60S) tertiary structures of the human ribosome showing positions (red) with variant alleles exhibiting intraindividual AF > 20% in any individual analyzed. Structural features within the 40S and 60S subunits are indicated. PTC, peptidyl transferase center.

Fig. 3. rRNA variants with population-stratified intraindividual AF.

(A) Intraindividual AF of 28S rRNA sequence variant A2538G, by population (max V_st = 0.56). X axis has log₂ scaling. (B to D) Tertiary structures of the ribosome showing strongly population-stratified variants (V_st > 0.5) with high intraindividual AF (>20%) in any individual analyzed that are located (B) in expansion segments (ES6^S and ES3) in the surface near ribosomal protein eS6, (C) proximal to the binding site for ribosomal protein eS19, and (D) proximal to the binding site for ribosomal protein eL38. Ribosome tertiary structures [Protein Data Bank (PDB) ID 4V6X (67)] show rRNA (tan), ribosomal proteins (green), and key structural landmarks.

Fig. 4. Evolutionarily conserved rRNA variants in functionally important centers of the human ribosome.

(A) 18S C543U variant (red) of helix h16 contacts DHX29 (yellow), a component of translation initiation. (B) 18S G480A variant (red) of helix h5 occurs near contact points with residues 256 to 260 within domain 2 (yellow) of eEF1A (blue). (C) 28S G1764A variant (red) of helix H38 in the central protuberance. The structural models shown are based on EMD-3056-3058 (46) and PDB IDs 5LZS (87) and 4V6X (67), respectively, where eIF3 and DHX29 from EMD-3056-3058 were modeled onto PDB ID 5LVS.

Fig. 5. Tissue-specific expression of rRNA variants.

rRNA variant expression heat map and hierarchical clustering of the 26 variants detected to be differentially expressed among pairs of tissues. Each row represents a biological replicate. Rows are grouped by tissue source (three biological replicates, that is, rows, per tissue source). Each column represents an rRNA variant. Expression is normalized per rRNA variant (that is, by column) across all replicates and tissues (that is, 12 samples per each column). For example, the rRNA variant represented by the leftmost column has higher relative expression in brain, whereas the variant represented by the rightmost column has the lowest relative expression in liver.