Scientists from the Johns Hopkins Kimmel Cancer Center report that ribosomal RNA (rRNA) genes, which were thought to be similar among people, instead differed significantly based upon a person’s geographic ancestry. Particularly high variations were found on a segment called 28S rRNA, an essential component of the protein-translating ribosome.

The team, directed by Marikki Laiho, MD, PhD, director of molecular radiation sciences in the department of radiation oncology and molecular radiation sciences, then decided to focus on a basic biology concept they wanted to better understand. They had developed cancer drugs that target synthesis of ribosomal rRNAs, a unique process that drives cancer development. Without these, cancer cells cannot multiply. The researchers, who published their study (“Widespread genetic heterogeneity of human ribosomal RNA genes”) in RNA, wondered if the rRNA gene itself was altered in cancers, and how that could affect their targeting strategy. Despite the importance of this gene, there was no definitive reference sequence published to date.

“Polymorphism drives survival under stress and provides adaptability. Genetic polymorphism of ribosomal RNA (rRNA) genes derives from internal repeat variation of this multicopy gene, and from interindividual variation. A considerable amount of rRNA sequence heterogeneity has been proposed but has been challenging to estimate given the scarcity of accurate reference sequences,” write the investigators.

“We identified four rDNA copies on chromosome 21 (GRCh38) with 99% similarity to recently introduced reference sequence KY962518.1. We customized a GATK bioinformatics pipeline using the four rDNA loci, spanning a total 145 kb, for variant calling and employed high-coverage whole genome sequencing (WGS) data from the 1000 Genomes Project to analyze variants in 2,504 individuals from 26 populations. We identified a total of 3,791 variant positions.

“The variants positioned non-randomly on the rRNA gene. Invariant regions included the promoter, early 5’ ETS, most of 18S, 5.8S, ITS1, and large areas of the intragenic spacer. A total of 470 variant positions were observed on 28S rRNA. The majority of the 28S rRNA variants located on highly flexible human-expanded rRNA helical folds ES7L and ES27L, suggesting that these represent positions of diversity and are potentially under continuous evolution. Several variants were validated based on RNA-seq analyses.

Unexpected heterogeneity of ribosomal RNA genes in human populations revealed by genome studies suggests potential variation in protein translation by the ribosomes. [Wenjun Fan, PhD]
Population analyses showed remarkable ancestry-linked genetic variance, and presence of both high penetrance and frequent variants in the 5’ ETS, ITS2 and 28S regions segregating according to the continental populations. These findings provide a genetic view of rRNA gene array heterogeneity and raise the need to functional assess how the 28S rRNA variants affect ribosome functions.”

Team members set out to take a bioinformatics approach to rRNA gene sequences, using high-performance computers at the Maryland Advanced Research Computing Center, a joint venture managed by  Johns Hopkins University and the University of Maryland. To start charting cancer alterations, they had to understand whether variants existed in the human population. The rRNA gene sequence was considered “untouchable,” or so fundamental that it seemed unlikely to have any variations.

“However, when we started that analysis, we very quickly realized that the cancer genomes were highly aberrant,” Laiho says. “In order for us to understand whether that aberration is real, meaning that it changes in a particular cancer, we needed to better understand what a normal human gene looks like.”

Next, they used whole-genome sequencing data from the 1,000 Genomes Project  to analyze variants in 2,504 individuals from 26 populations. They identified 3,791 variant positions on the rRNA gene. This included 470 variant positions seen on 28S rRNA. Most of these variants were located on long protruding folds of the rRNA that vary among species. These represent positions of diversity, and are potentially under continuous evolution.

“The analysis results were beyond our imagination,” continues Laiho. “We saw perfect conservation of sequences across vast swaths of the gene, and then highly variable sites in the exact locations that we expected to be unaltered. This suggests that the way variant rRNAs are built into the ribosomes could lead to potential alterations in how the ribosomes function.”

Many of the variants observed were segregated by population. For example, some variants were far more frequent among African or Asian populations versus American or European populations, and vice versa. This raises the possibility that some of the variants are ancient, ancestry-dependent, yet have been retained in modern populations, according to Laiho.

“It’s too early to speculate what these variants mean, but what is fascinating is that they are conserved by population, and this means their retention is somehow important,” she explains, adding that the study findings suggest there is a need to functionally assess how the 28S rRNA variants affect ribosome functions, which could in turn help lead to more targeted therapies for cancer or other diseases.