Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press 1 Abundantly expressed class of non-coding RNAs conserved through 2 the multicellular evolution of dictyostelid social amoebae 3 Jonas Kjellin1, Lotta Avesson2,3, Johan Reimegård4, Zhen Liao1,5, Ludwig Eichinger6, Angelika Noegel6, 4 Gernot Glöckner6,†, Pauline Schaap7, and Fredrik Söderbom1 5 6 1Department of Cell and Molecular Biology, Uppsala University, Box 596 Uppsala, S-75124 Sweden, 7 2Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences, Box 8 590, S-75124 Uppsala, Sweden, 4Department of Cell and Molecular Biology, National Bioinformatics 9 Infrastructure Sweden, Science for Life Laboratory, Uppsala University, Box 596 Uppsala, S-75124 10 Sweden, 6Centre for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, 11 Cologne, Germany and 7College of Life Sciences, University of Dundee, Dundee DD1 5EH, United 12 Kingdom. 3Present address: Novo Nordisk Foundation Center for Protein Research, University of 13 Copenhagen, Blegdamsvej 3B, 2200 Copenhagen. 5Present address: Department of Plant Biology, Swedish 14 University of Agricultural Sciences, Box 7080 Uppsala, S-750 07 Sweden 15 16 † Deceased 17 18 Corresponding author. [email protected]. Phone: 46 184714901 19 20 Running title: Conserved ncRNAs in multicellular evolution 21 22 Keywords (3-10): social amoeba, Dictyostelium discoideum, non-coding RNA, evolution, Class I RNA, 23 dictyostelids, multicellularity, Dictyostelia Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press 24 Abstract 25 Aggregative multicellularity has evolved multiple times in diverse groups of eukaryotes, exemplified by 26 the well-studied development of dictyostelid social amoebae, e.g. Dictyostelium discoideum. However, it 27 is still poorly understood why multicellularity emerged in these amoebae while the great majority of 28 other members of Amoebozoa are unicellular. Previously a novel type of non-coding RNA, Class I RNAs, 29 was identified in D. discoideum and demonstrated to be important for normal multicellular 30 development. Here we investigated Class I RNA evolution and its connection to multicellular 31 development. We identified a large number of new Class I RNA genes by constructing a co-variance 32 model combined with a scoring system based on conserved up-stream sequences. Multiple genes were 33 predicted in representatives of each major group of Dictyostelia and expression analysis confirmed that 34 our search approach identifies expressed Class I RNA genes with high accuracy and sensitivity and that 35 the RNAs are developmentally regulated. Further studies showed that Class I RNAs are ubiquitous in 36 Dictyostelia and share highly conserved structure and sequence motifs. In addition, Class I RNA genes 37 appear to be unique to dictyostelid social amoebae since they could not be identified in outgroup 38 genomes, including their closest known relatives. Our results show that Class I RNA is an ancient class of 39 ncRNAs, likely to have been present in the last common ancestor of Dictyostelia dating back at least 600 40 million years. Based on previous functional analyses and the presented evolutionary investigation, we 41 hypothesize that Class I RNAs were involved in evolution of multicellularity in Dictyostelia. 42 43 Introduction 44 The role of RNA goes far beyond being an intermediate transmitter of information between DNA and 45 protein, in the form of messenger (m)RNAs. This has been appreciated for a long time for some non- 46 coding RNAs (ncRNAs), such as transfer (t)RNAs, ribosomal (r)RNAs, small nuclear (sn)RNAs, and small 47 nucleolar (sno)RNAs. Today, we know that ncRNAs are involved in regulating most cellular processes and 2 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press 48 the advent of high-throughput sequencing technologies have facilitated the identification of numerous 49 different classes of ncRNAs (Cech and Steitz 2014). These regulatory RNAs vary greatly in size from 21-24 50 nucleotides (nt), e.g. micro (mi)RNAs and small interfering (si)RNAs, to several thousands of nucleotides, 51 such as long non-coding (lnc)RNAs. Several classes of ncRNAs are ubiquitously present in all domains of 52 life while others are specific to certain evolutionary linages, contributing to their specific characteristics. 53 This can be exemplified by Metazoa, where an increase in number of ncRNAs, e.g. miRNAs, is associated 54 with increased organismal complexity and is believed to have been essential for the evolution of 55 metazoan multicellularity (Gaiti et al. 2017). 56 57 Multicellularity in plants and animals is achieved by clonal division and development originating from a 58 single cell. This is in contrast to aggregative multicellularity, were cells stream together to form 59 multicellular structures upon specific environmental changes. Aggregative multicellularity has evolved 60 independently multiple times and is found both among eukaryotes and prokaryotes (Kawabe et al. 2019; 61 Brown et al. 2009, 2012b; He et al. 2014; Tice et al. 2016; Lasek-Nesselquist and Katz 2001; Brown et al. 62 2011, 2012a). The complexity of the aggregative multicellular life stages varies for different organisms, 63 but they all share the transition from unicellularity to coordinated development upon environmental 64 stress, such as starvation, which eventually leads to formation of fruiting bodies containing cysts or 65 spores (Kawabe et al. 2019). Probably the most well-studied aggregative multicellularity is the 66 development of the social amoeba Dictyostelium discoideum belonging to the group Dictyostelia within 67 the supergroup Amoebozoa. Dictyostelia is a monophyletic group estimated to date back at least 600 68 million years (Heidel et al. 2011), which is similar to the age of Metazoa (dos Reis et al. 2015). 69 Dictyostelia is currently divided into four major groups (Group 1-4) where all members share the ability 70 to transition from uni- to multicellularity upon starvation (Schilde et al. 2019). However, the complexity 71 of the development and the morphology of the fruiting bodies varies between different dictyostelids, 3 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press 72 where the highest level of multicellular complexity is found among group 4 species, which includes D. 73 discoideum (Romeralo et al. 2013; Schilde et al. 2014). Recently a new taxonomy was proposed for many 74 dictyostelids (Sheikh et al. 2018). As this new taxonomy has not yet been fully adopted by the research 75 community, we choose to use the previous designations throughout this study (old and new names, 76 including NCBI accession numbers, are summarized in Supplemental Table S1). 77 78 Well-annotated genome sequences are available for representative species of all four major groups of 79 Dictyostelia (Heidel et al. 2011; Eichinger et al. 2005; Sucgang et al. 2011; Urushihara et al. 2015; 80 Glöckner et al. 2016) and multiple draft genome sequences are available for additional dictyostelids. 81 This has allowed for comparative genomics, which has provided information about protein coding genes 82 that are important for the diversification of Dictyostelia from other amoebozoans. Comparison between 83 genomes has also given insight into the genes required for the evolution of the distinct morphological 84 characteristics, which define each group (Heidel et al. 2011; Eichinger et al. 2005; Sucgang et al. 2011; 85 Glöckner et al. 2016; Hillmann et al. 2018). However, evolution of complex traits such as multicellularity 86 in Dictyostelia as well as other eukaryotic groups, cannot solely be explained by the appearance of novel 87 genes but also relies on an increased ability to regulate pre-existing genes and their products so that 88 they can function in novel genetic networks (Glöckner et al. 2016; Deline et al. 2018). This is also 89 supported by the major transcriptional reprogramming during multicellular development in D. 90 discoideum (Rosengarten et al. 2015). 91 92 D. discoideum harbors several classes of developmentally regulated ncRNAs with regulatory potential, 93 e.g. microRNAs (Avesson et al. 2012; Liao et al. 2018; Hinas et al. 2007; Meier et al. 2016), long non- 94 coding RNAs (Rosengarten et al. 2016) and long antisense RNAs (Rosengarten et al. 2016; Hildebrandt 95 and Nellen 1992). In addition, a large part of the ncRNA repertoire of D. discoideum is constituted by 4 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press 96 Class I RNAs, originally identified in full length cDNA libraries (Aspegren et al. 2004). So far, Class I RNAs 97 have only been experimentally validated in D. discoideum (Aspegren et al. 2004; Avesson et al. 2011), 98 but they have also been computationally predicted in Dictyostelium purpureum (Sucgang et al. 2011). 99 Both species belong to the same evolutionary group of Dictyostelia, i.e. group 4 (Schilde et al. 2019; 100 Sheikh et al. 2018). In D. discoideum, Class I RNAs are 42-65 nt long and are expressed at high levels 101 from a large number of genes. Members of Class I RNAs are characterized by a short stem structure, 102 connecting the 5’ and 3’ ends, and a conserved 11 nt sequence motif adjacent to the 5’part of the stem. 103 The remainder of the RNA is variable both in sequence and structure (Fig 1A). Class I RNAs mainly 104 localize to the cytoplasm (Aspegren et al. 2004) where one of the RNAs has been shown to associate 105 with four different proteins of which at least one, the RNA recognition motif (RRM) containing protein 106 CIBP (Class I Binding Protein), directly binds to the Class I RNA (Avesson et al. 2011). Furthermore, the 107 Class I RNAs appear to be involved in regulating multicellular development. This is based on the 108 observations that Class I RNAs are developmentally regulated and that cells where a single Class I RNA 109 gene has been knocked out show aberrant early development (Aspegren et al.