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The Conservation Landscape of the Human Ribosomal RNA Gene Repeats

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The Conservation Landscape of the Human Ribosomal RNA Gene Repeats bioRxiv preprint doi: https://doi.org/10.1101/242602; this version posted April 28, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. 1 1 The conservation landscape of the human ribosomal RNA gene repeats 2 Saumya Agrawal1 and Austen R.D. Ganley1,2,* 3 1 Institute of Natural and Mathematical Sciences, Massey University, Private Bag 102-904, Auckland 4 0632, New Zealand 5 2 School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New 6 Zealand (current address) 7 * To whom correspondence should be addressed: Tel: +64- 9923 2906; Email: 8 [email protected] 9 Present address: Saumya Agrawal, Division of Genomic Technologies, RIKEN Center for Life 10 Science Technologies, Yokohama, Japan 11 12 ABSTRACT 13 Ribosomal RNA gene repeats (rDNA) encode ribosomal RNA, a major component of ribosomes. 14 Ribosome biogenesis is central to cellular metabolic regulation, and several diseases are associated 15 with rDNA dysfunction, notably cancer, However, its highly repetitive nature has severely limited 16 characterization of the elements responsible for rDNA function. Here we make use of phylogenetic 17 footprinting to provide a comprehensive list of novel, potentially functional elements in the human 18 rDNA. Complete rDNA sequences for six non-human primate species were constructed using de novo 19 whole genome assemblies. These new sequences were used to determine the conservation profile of 20 the human rDNA, revealing 49 conserved regions in the rDNA intergenic spacer (IGS). To provide 21 insights into the potential roles of these conserved regions, the conservation profile was integrated 22 with functional genomics datasets. We find two major zones that contain conserved elements 23 characterised by enrichment of transcription-associated chromatin factors, and transcription. 24 Conservation of some IGS transcripts in the apes underpins the potential functional significance of bioRxiv preprint doi: https://doi.org/10.1101/242602; this version posted April 28, 2018. 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. 2 25 these transcripts and the elements controlling their expression. Our results characterize the 26 conservation landscape of the human IGS, and suggest that noncoding transcription and chromatin 27 elements are conserved and important features of this unique genomic region. 28 29 Introduction 30 A characteristic feature of most eukaryote genomes is the presence of one or more tandem arrays of 31 gene repeats encoding ribosomal RNA (rRNA), a key building block of ribosomes. The major 32 eukaryotic rRNA gene repeat family is known as the ribosomal DNA (rDNA), with each repeat 33 encompassing a coding region encoding 18S, 5.8S and 28S rRNA, and an intergenic spacer (IGS) that 34 separates adjacent coding regions (Fig 1). In humans, each repeat unit is ~43 kb in length, with a ~13 35 kb rRNA coding region and a ~30 kb IGS [1], and there are approximately 200-600 rDNA copies 36 distributed amongst tandem arrays on the short arms of the five acrocentric chromosomes 37 (chromosomes 13, 14, 15, 21, and 22) [2-6]. The rDNA is transcribed by RNA Polymerase I (Pol-I) in 38 the nucleolus [7,8], and this primary role in ribosome biogenesis places the rDNA at the heart of 39 cellular metabolic homeostasis [9]. In addition, the rDNA has been found to mediate a number of 40 “extra-coding” functions, including roles in genome stability [10,11], cell cycle control [12-16], 41 protein sequestration [17], epigenetic silencing [18,19], and aging [20,21]. 42 43 Fig 1: Eukaryotic ribosomal DNA organization. A) Head-to-tail tandem arrangement of rDNA repeat 44 units. Typically there are more units in an array than depicted. B) Each rDNA unit has an rRNA coding 45 region (black) and an intergenic spacer (IGS; green). The coding region encodes the ~18S, 5.8S and 46 ~28S rRNAs (black boxes) separated by two internal transcribed spacers (ITS-1 and 2) and flanked by 47 two external transcribed spacers (5’- and 3’-ETS). 48 bioRxiv preprint doi: https://doi.org/10.1101/242602; this version posted April 28, 2018. 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. 3 49 A critical outcome of the central role the rDNA plays in the biology of the cell is an association with a 50 number of human diseases. The best characterized is the association between ribosome 51 biogenesis/rDNA and cancer, which dates back over 100 years and stems from observations of 52 nucleolar hypertrophy and upregulated rRNA expression in tumour cells [22-25]. rRNA dysfunction 53 is also associated with a group of genetic diseases that result from impaired ribosome biogenesis, 54 known as ribosomopathies [26,27]. In addition, there is growing evidence for the rDNA playing a role 55 in cellular differentiation [28-31]. Despite these strong connections to human pathology, the rDNA 56 remains poorly characterized. Although an rDNA unit sequence is available in Genbank (accession 57 U13369), it has not been placed in the human genome chromosomal assembly [32], and consequently 58 the rDNA is excluded from most genome-wide analyses. Moreover, the lack of tools to genetically 59 manipulate the highly repetitive rDNA in mammalian systems means that the human rDNA is not 60 well characterized at the molecular level. 61 Work in Saccharomyces cerevisiae has shown that many rRNA regulatory and rDNA extra-coding 62 functions are encoded in the IGS, where a number of functional elements have been characterized 63 [10,33-46]. In stark contrast, even though the human IGS is approximately ten times longer than 64 yeast, few functional elements have been defined to date. Those that have are restricted to the rRNA 65 promoter [47], 10 bp repeats (Sal boxes) some of which act as terminators of the primary rRNA 66 transcript [48], and two noncoding IGS transcripts that are associated with stress response [17]. Other 67 elements have been identified from their sequence composition, including several other repeat 68 elements [1], a cdc27 pseudogene [49], and putative c-Myc and p53 binding sites [50,51]. Pioneering 69 work characterizing the chromatin structure of the human IGS has provided evidence for regions with 70 distinct chromatin states, including states characteristic of transcriptional regulatory activity [52]. 71 Furthermore, there appears to be dynamic regulation of this rDNA chromatin structure [53,54]. 72 However, without further characterization, the functional significance of these human rDNA 73 chromatin elements is unclear. 74 Comparative genomics is a powerful method for the identification of functional elements that are 75 difficult to detect by traditional molecular approaches [55-58]. In particular, phylogenetic footprinting bioRxiv preprint doi: https://doi.org/10.1101/242602; this version posted April 28, 2018. 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. 4 76 is an effective way to identify potentially functional elements using orthologous sequence data alone. 77 The principle is that mutations in functional elements will be deleterious, therefore changes in the 78 sequences of functional elements are selected against and change at a slower rate than non-functional 79 elements over evolutionary time [59]. Thus, comparison of orthologous sequences from related 80 species results in the functional elements appearing as “phylogenetic footprints” - highly conserved 81 regions in a multiple sequence alignment against a background of non-functional, poorly conserved 82 sequences [59]. Application of this method to the rDNA of S. cerevisiae successfully identified both 83 known and novel functional elements in the IGS [10,44]. Given how little is known about functional 84 elements in the human IGS and the strong connections between rDNA biology and human pathology, 85 we decided to utilize phylogenetic footprinting to identify potential functional elements in the human 86 rDNA. 87 Here, we constructed complete rDNA sequences from six primate species for which these sequences 88 were previously unknown. Alignment of these sequences with a human rDNA sequence shows that 89 previously identified functional elements in the human IGS are evident as phylogenetic footprints, and 90 there are a number of other conserved regions not associated with any known functional element. 91 Building on the results characterizing the chromatin state of the human rDNA [52], we shed light on 92 the potential functions of these uncharacterized IGS conserved regions by overlaying publicly 93 available RNA-seq, CAGE, and ChIP-seq data onto the conservation profiles. These analyses suggest 94 that chromatin structure and the production/regulation of noncoding transcripts are major activities 95 associated with sequence conservation in the human IGS. This is reinforced by conservation of IGS 96 transcriptional activity in the apes, implying that these activities may be important for human rDNA 97 function. 98 99 Materials and Methods 100 Whole genome assemblies to obtain the primate rDNA sequences bioRxiv preprint doi: https://doi.org/10.1101/242602; this version posted April 28, 2018. 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.
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