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Widespread Germline Genetic Heterogeneity of Human Ribosomal RNA Genes bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453267; this version posted July 25, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Widespread germline genetic heterogeneity of human ribosomal RNA genes Wenjun Fan1, Eetu Eklund1, Rachel M. Sherman2,#, Hester Liu1, Stephanie Pitts1, Brittany Ford3, Rajeshkumar NV1, Marikki Laiho1* 1 Department of Radiation Oncology and Molecular Radiation Sciences, and Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. 2 Department of Computer Science, Whiting School of Engineering, Johns Hopkins University, Baltimore, MD, USA. 3 Drug Research Program, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland. # Current affiliation: Illumina Inc. * Corresponding author: Marikki Laiho, Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21287 United States. Phone: +1-4105029748. Fax: +1-4105022821. Email: [email protected] Running title: Human rRNA gene heterogeneity Key words: rRNA, ribosome, rDNA array, 1000 Genomes Project Conflict of interest: M.L. holds patents on RNA polymerase I inhibitors, which are managed by the Johns Hopkins University. These are unrelated to the work described here. The other authors declare no competing interests. 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453267; this version posted July 25, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Abstract 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. We identified four rDNA copies on chromosome 21 (GRCh38) with 99% similarity to recently introduced reference sequence KY962518.1. Pairwise alignment of the rRNA coding sequences of these copies showed differences in sequence and length. We customized a GATK bioinformatics pipeline using the four rDNA loci, spanning a total 145 kb, for variant calling. We employed whole genome sequencing (WGS) data from the 1000 Genomes Project phase 3 and analyzed variants in 2,504 individuals from 26 populations. Using the pipeline, we identified a total of 3,790 variant positions. The variants positioned non-randomly on the rRNA gene. Invariant regions included the promoter, early 5’ ETS, 5.8S, ITS1 and certain regions of the 28S rRNA, and large areas of the intragenic spacer. 18S rRNA coding region had very few variants, while 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. These findings provide a genetic view for rRNA heterogeneity and raise the need to functional assess how the 28S rRNA variants affect ribosome functions. 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453267; this version posted July 25, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. INTRODUCTION Ribosomes translate mRNA templates to proteins. The human ribosome is a large protein-RNA complex that is composed of a large 60S subunit consisting of 28S, 5S and 5.8S ribosomal RNAs (rRNA) and 47 proteins and a small 40S subunit with 18S rRNA and 33 proteins (Anger et al. 2013; Khatter et al. 2015). The rRNAs are transcribed by RNA polymerases I (5.8S, 18S, 28S rRNAs) and III (5S rRNA). RNA polymerase I (Pol I) transcribes a long polycistronic 47S rRNA precursor that is processed and cleaved into the mature 5.8S, 18S and 28S rRNAs assisted by a multitude of rRNA biogenesis proteins and small nucleolar non-coding RNAs (Moss et al. 2007). The processing, folding and maturation requires also extensive posttranslational modification of the rRNAs (Sloan et al. 2017). Ribosomes are highly abundant in cells and their numbers reflect the translation rates of the host. However, this dependency may be nonlinear leading to defects and diseases states related to alterations in ribosomal protein stoichiometry or modifications of the ribosomal proteins and rRNAs (Emmott et al. 2019; Li and Wang 2020). The ensuing ribosome heterogeneity potentially leads to functional consequences such as abnormalities observed in ribosomopathies (Mills and Green 2017) . Functionally heterogeneous ribosomes are also referred to as specialized ribosomes. Specialized ribosomes translate subsets of mRNAs and are predicted to provide a dynamic response to stress or stimuli, or occur in a cell- or tissue-specific manner (Emmott et al. 2019). The human multicopy rRNA genes are organized in repeat arrays on five acrocentric chromosomes 13, 14, 15, 21 and 22 (Henderson et al. 1972). Copy numbers are estimated to vary between 100 – 600 per diploid genome, are unevenly distributed on the acrocentric chromosomes, and vary between individuals (McStay 2016; van Sluis et al. 2020). The rRNA gene loci contain not only the gene arrays, but are also highly repetitive due to presence of satellite repeats and other repetitive elements rendering them challenging to study and assess their genetic variability (McStay 2016). The Telomere-to-Telomere (T2T) Consortium very recently provided in-depth mapping of these previously unannotated gene arrays covering a total of 10 Mb rDNA sequence derived from a functionally haploid CHM13 hydatidiform mole cell line . This analysis, using a combination of PacBio HiFi, Oxford Nanopore and PCR-free sequencing and bioinformatic methods, indicated chromosome-dependent variation of the rDNA copies . 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453267; this version posted July 25, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. While substantial progress has been made in identifying the proximal and distal sequences of the arrays, consensus or reference sequences for the human rRNA gene have been lacking (McStay 2016). Only few reference sequences have existed until recently (U13369, AL353644.34) (Gonzalez and Sylvester 1995). The reference sequence U13369.1 was deposited in 1994 (Gonzalez and Sylvester 1995) and has been used in vast majority of studies since then. Yet, reports recognizing rRNA sequence variation have been published since 1980s (Gonzalez et al. 1985; Gonzalez et al. 1988; Worton et al. 1988; Kuo et al. 1996). Recent transformation-associated recombination (TAR) cloning and long-read coding efforts, using a single chromosome 21 mouse/human hybrid as source, enabled the accurate assembly of a new 45 kb rDNA sequence, identified numerous discrepancies to the earlier reference and substantially refined it, and introduced a reference sequence (KY962518.1) which was 1.8 kb longer than the previous (Kim et al. 2018). These sequencing efforts also identified single nucleotide variants (SNV) and insertion/deletions (INDEL) in the rRNA coding and non-coding intragenic spacer (IGS) sequences based on multiple sequenced clones of chromosome 21 (Kim et al. 2018). Several earlier studies have conducted variant calling approaches of the rDNA genes, but may be compromised by calling variants representing sequencing or alignment errors due to the use of the earlier reference U13369 (Babaian 2017; Xu et al. 2017; Parks et al. 2018). The identification of variants still requires refinement and their implications are yet to be determined. Here we identified three full and one partial rRNA gene copies on chromosome 21 (GRCh38) with 99% similarity to the most recent rDNA reference KY962518.1. We employed whole genome sequencing (WGS) data from the 1000 Genomes Project phase 3 including 2,504 individuals from 26 populations and performed variant calling against the identified rDNA sequences in GRCh38 to generate a comprehensive set of germline SNVs and INDEL variant positions (SNV/INDEL) of the rDNA arrays. We discovered a total of 3,790 variant positions on the rRNA genes. Moreover, the variants distinctly clustered on the rRNA gene coding and IGS regions, while regions for core ribosomal protein interactions and rRNA modification sites and large areas in the IGS were near invariant. A large number of variant positions, 380 SNVs and 90 INDELs, were detected on the mature 28S rRNA. The majority of the 28S rRNA variants located on the highly flexible human-expanded rRNA helical folds ES7L and ES27L. Collectively, our findings provide a genetic view for rRNA heterogeneity and suggest potential links between variants and ribosome functions. 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453267; this version posted July 25, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. RESULTS Genomic variation of rDNA copies in GRCh38 chromosome 21 To identify rDNA loci in GRCh38 human genome reference, we performed pairwise alignment of human rDNA reference sequence assembled by using TAR cloning and long-read sequencing (GenBank: KY962518.1) against current human reference genome GRCh38. We identified three full and one partial rDNA copies on chromosome 21 (GRCh38) with 99% similarity to the reference KY962518 (Figure 1A and Table S1).
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