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Ancient Microrna Profiles of a 14,300-Year-Old Canid Samples Confirm

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Ancient Microrna Profiles of a 14,300-Year-Old Canid Samples Confirm Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Fromm et al 1 2 Title: Ancient microRNA profiles of a 14,300-year-old canid samples confirm 3 taxonomic origin and give glimpses into tissue-specific gene regulation from 4 the Pleistocene 5 6 Running title: Sequencing of ancient microRNAs. 7 8 Authors: Bastian Fromm1*, Marcel Tarbier1, Oliver Smith2,3, Emilio Mármol- 9 Sánchez1,4, Love Dalén4,5, M. Tom P. Gilbert2,6, Marc R. Friedländer1* 10 11 Affiliations: 12 1 Science for Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren 13 Institute, Stockholm University, Stockholm, Sweden 14 2 Section for Evolutionary Genomics, The Globe Institute, Faculty of Health and Medical 15 Sciences, University of Copenhagen, Copenhagen, Denmark 16 3 Micropathology Ltd, Venture Centre, University of Warwick Science Park, Coventry, UK 17 4 Centre for Palaeogenetics, Stockholm, Sweden 18 5 Department of Bioinformatics and Genetics, Swedish Museum of Natural History, 19 SE-10405 Stockholm, Sweden 20 6 Norwegian University of Science and Technology, University Museum, Trondheim, 21 Norway 22 23 24 * corresponding authors: [email protected] and [email protected] 25 26 Keywords: microRNAs, paleotranscriptomics, Pleistocene, canid, gene regulation Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Fromm et al. 27 Abstract 28 DNA sequencing is the current key technology for historic or ancient biological samples and has 29 led to many exciting discoveries in the field of paleogenomics. However, functional insights into 30 tissue identity, cellular composition or gene regulation cannot be gained from DNA. Recent 31 analyses have shown that, under favorable conditions, RNA can also be sequenced from ancient 32 samples, enabling studies at the transcriptomic and regulatory level. 33 Analyzing ancient RNA data from a Pleistocene canid, we find hundreds of intact microRNAs 34 that are taxonomically informative, show tissue-specificity and have functionally predictive 35 characteristics. With an extraordinary age of 14,300 years, these microRNA sequences are by far 36 the oldest ever reported. The authenticity of the sequences is further supported by a) the presence 37 of canid / Caniformia-specific sequences that never evolved outside of this clade, b) tissue- 38 specific expression patterns (cartilage, liver and muscle) that resemble those of modern dogs and 39 c) RNA damage patterns that are clearly distinct from those of fresh samples. 40 By performing computational microRNA-target enrichment analyses on the ancient sequences, 41 we predict microRNA functions consistent with their tissue pattern of expression. For instance, 42 we find a liver-specific microRNA that regulates carbohydrate metabolism and starvation 43 responses in canids. In summary, we show that straightforward paleotranscriptomic microRNA 44 analyses can give functional glimpses into tissue identity, cellular composition and gene 45 regulatory activity of ancient samples and biological processes that took place in the Pleistocene, 46 thus holding great promise for deeper insights into gene regulation in extinct animals based on 47 ancient RNA sequencing. 1 Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Fromm et al. 48 Introduction 49 Sequencing historic and ancient DNA from up to 780,000-year-old samples has become a 50 standard approach to infer genetic histories of extinct or extant species (Orlando et al. 2013). 51 While much has been learned from studying these genomes, it remains difficult to infer cellular 52 processes, such as in vivo genome function, from DNA directly. In contrast, the presence and 53 relative expression levels of RNA convey genetic information at the functional level, and can 54 thus give insights into the biological activity, the tissue identity, or even the cellular composition 55 of samples (Newman et al. 2015). The prospect of studying gene-expression, gene-regulation or 56 species-specific transcripts in extinct species would be a fantastic next step in the paleogenetics 57 field and, at least theoretically, could enable the discovery of extinct genes, typically overlooked 58 by aDNA-based approaches. However, given the putative relative lack of surviving material in 59 historic and ancient samples, due to the release of RNases during decomposition in most tissues 60 (Huynen et al. 2012), ‘ancient RNA’ (aRNA) has rarely been studied and is often disregarded as 61 a source for biological discoveries. On the other hand, preservative conditions for aDNA, such 62 as cold and drought, also inhibit RNAses, and many types of RNA molecules are present in 63 hundreds of thousands of copies per cell (see for microRNAs (Calabrese et al. 2007)). 64 65 With the availability of powerful and sensitive sequencing approaches, a small number of recent 66 studies has capitalized on early observations of very short RNA fragments surviving in some 67 archaeological plant and mummified materials (Rollo 1985; Venanzi and Rollo 1990), and have 68 recovered sequenceable amounts of ancient RNA (aRNA) from historic plant and feces samples. 69 In doing so, they have detected, for instance, viral RNA genomes (Smith et al. 2014; Ng et al. 70 2014), historical plant microRNAs (Smith et al. 2017) and fragments of plant protein-coding 2 Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Fromm et al. 71 transcripts (Fordyce et al. 2013). While initially only qPCR based methods were used on human 72 remains, such as the more than 5000 years old Tyrolean iceman (Keller et al. 2017), a recent 73 aRNA sequencing study detected disease-related microRNAs from about 1000 years old human 74 skeletons (Shaw et al. 2019). While these studies represent a proof of concept for aRNA 75 sequencing, or paleotranscriptomics, they are limited to relatively recent (~1000 years old) 76 samples and, except for one study, restricted to plant seed-material that conveys highly favorable 77 preservative conditions. 78 79 A recent sequencing study on three permafrost tissues samples from the ancient “Tumat-puppy” 80 (liver, cartilage and muscle) and two historical canid museum samples (95 and 151 years old 81 wolf-skins) showed that aRNA can survive for extended periods in mammalian samples (Smith 82 et al. 2019). With approximately 14,300 years of age for the Pleistocene permafrost samples, the 83 authors presented the oldest ever sequenced RNA to this date. In a paleotranscriptomic approach, 84 the presence of exon-exon junctions of protein-coding genes and high levels of ribosomal RNA 85 were detected, confirming the data’s authenticity. Using two custom bioinformatics methods 86 based on the quantitative comparison of detected short fragments of protein-coding genes 87 between ancient and recent tissue sample profiles, the aRNA profiles could resolve tissue identity 88 for two of the five samples (Smith et al. 2019). 89 90 MicroRNAs are ~22 nucleotide short RNA molecules that are key regulators of protein coding 91 genes with important functions in numerous biological processes including development and 92 disease (Bartel 2018). The so-called ‘mature microRNAs’ and their usually non-functional by- 93 products (‘star microRNAs’) derive from longer RNA hairpin structures (‘precursor 3 Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Fromm et al. 94 microRNAs’) by means of several well-studied processing steps that leave characteristic patterns 95 in sequencing data, which are useful for their annotation (Friedländer et al. 2012; Fromm 2016). 96 Many microRNAs also exhibit tissue-specific expression, making them excellent cell- and tissue 97 markers (McCall et al. 2017; de Rie et al. 2017; Christodoulou et al. 2010), also for difficult 98 samples (Xi et al. 2007; Liu et al. 2009; Courts and Madea 2010; Glynn 2019). Since it is well- 99 established that the number of times that a microRNA is detected by sequencing scales with its 100 molecular abundance in a sample - with certain caveats (Linsen et al. 2009) - it is possible to 101 apply sequencing to quantitatively measure microRNA compositions. Becasue microRNAs are 102 themselves molecules with regulatory functions, detecting them in the ancient Tumat puppy 103 tissues would give direct insights into gene regulation in the Pleistocene. Because the applied 104 RNA extraction method (adapted from Ambion miRvana kit) and library preparation strategy 105 (NEBNext Multiplex Small RNA Library Prep Set) clearly favors short RNAs (for details see 106 (Smith et al. 2019)), we were confident of detecting microRNAs among the sequenced aRNA 107 transcripts from the Tumat puppy (Smith et al. 2019), thus enabling us to expand previous 108 findings beyond protein-coding genes into the gene-regulatory level of ancient samples. 109 110 Furthermore, microRNAs are among the most conserved elements in metazoan genomes, giving 111 them great potential as phylogenetic (Sempere et al. 2006; Tarver et al. 2013; Kenny et al. 2015) 112 and taxonomic markers (Fromm et al. 2014). We have recently curated the microRNA 113 complements of 45 Metazoan organisms, including canid, in the curated microRNA gene 114 database MirGeneDB (Fromm et al. 2020) and shown that species-specific microRNAs can be 115 used to trace the organismic origin of samples using our user-friendly
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