RNA evolves by adding substructural parts to growing molecules. Molecular accretion history can be dissected with phylogenetic methods that exploit structural and functional evidence. Here, we explore the statistical behaviors of lengths of double-stranded and single-stranded segments of growing tRNA, 5S rRNA, RNase P RNA, and rRNA molecules. The reconstruction of character state changes along branches of phylogenetic trees of molecules and trees of substructures revealed strong pushes towards an economy of scale. In addition, statistically significant negative correlations and strong associations between the average lengths of helical double-stranded stems and their time of origin (age) were identified with the Pearson’s correlation and Spearman’s rho methods. The ages of substructures were derived directly from published rooted trees of substructures. A similar negative correlation was detected in unpaired segments of rRNA but not for the other molecules studied. These results suggest a principle of diminishing returns in RNA accretion history. We show this principle follows a tendency of substructural parts to decrease their size when molecular systems enlarge that follows the Menzerath–Altmann’s law of language in full generality and without interference from the details of molecular growth. At the e other” . Their oe other” ,12,13) we other” ,15,16,17e other” , RNase Pe other” , SINE RNe other” , and rRNe other” ,21. In oOperationally, geometrical or statistical features of substructures are coded into linearly ordered multistate characters, for example, with the help of a web server . GeometrThe validity of phylogenetic accretion models has been tested against algorithmic and theoretical models of rRNA and tRNA histories. A recent algorithm of rRNA growth assumes the universal ribosomal core evolved by gradual insertion of “branch” helices onto preexisting, coaxially-stacked, “trunk” helices, growing the rRNA molecules outwards (onion-like) from the peptidyl transferase center (PTC) and leaving behind “insertion fingerprint” constrictions in their junctions ,26. WhilIn proteins, the length of structural domains decreases with increasing numbers of domains in multidomain proteins . Here, whttp://trnadb.bioinf.uni-leipzig.de accessed on 26 May 2021), 5S rRNA Database (http://biobases.ibch.poznan.pl/5SData/ accessed on 26 May 2021), RNase P database (retired), and European rRNA database (http://bioinformatics.psb.ugent.be/webtools/rRNA/ accessed on 26 May 2021). Data matrices and rooted phylogenetic trees describing the evolution of tRNA, 5S rRNA, RNase P RNA, and rRNA were from published studies ,14,18,19CSRs were implemented using Mesquite ver. 3.2  and MacCy(x) decreases monotonically with the size of the RNA molecule x, measured by the number of substructures it contains,A and c representing fitting parameters. Data were fitted to a straight line by plotting ln y(x) = ln A − cx and fitting parameters with F statistics. We report dependencies that are most useful for biological interpretation.The time of origin (age) of RNA helical and unpaired segments were derived from published rooted trees of substructures of tRNA, 5S rRNA, RNase P RNA, and rRNA ,18,19,21The reconstruction of the history of character state changes along branches of optimal phylogenetic trees requires a phylogeny and an initial evolutionary hypothesis . This scn − 1 edge connections that are non-reticulated, where n represents the number of character states. These linear graphs with vertices of degree 2 define landscapes of character state transformation (‘transformation series’) that can be represented with alphanumeric strings and are therefore amenable to straightforward computation. Linearly connected characters of this type are known as ordered characters or Wagner characters. They are widely used to describe serial homologies and have more resolving power and are less prone to resolution artefacts than other type of widely used characters, such as unordered characters typical of sequence analyses [Phylogenetic characters that describe the length of helical and unpaired segments of RNA molecules are multistate characters. Their transformation is constrained by the simplest of minimally-connected character state graphs (CSGs) with analyses .We applied a generalized maximum parsimony framework to trace character change in published trees of molecules and produce step-matrices of transformation costs from the structure of tRNA, 5S rRNA, RNase P RNA, and rRNA . All of these RNA molecules have very ancient evolutionary origins. The relative frequencies of change derived from CSRs were plotted in bubble diagrams , which cx to character x + 1 or vice versa. Helical regions included all segments that form canonical base pairs  and non-canonical base pairs  in the secondary structure of RNAs. Unpaired regions included those in junctions, bulges, and loops of the molecules. Since character transformations are additive in ordered characters, character state reconstructions generated from individual structural features belonging to helical and unpaired segments produced bubble diagrams with contributing relative transformation costs. For example, the difference of changes in step-matrices of helical segments versus those that excluded non-canonical G:U wobble base pairs were accounted for by the step-matrix of changes in the wobble pairs (data not shown).The bubble diagrams of We also found a clear differential behavior of helical and unpaired regions. In helical regions, losses were consistently favored over gains for average lengths of paired segments of less than nine base pairs with a reverse trend for longer segments. We note, however, that the reverse trend was almost absent in the short tRNA and 5S rRNA molecules, with exceptions in 7-to-8 character state changes for tRNA and 9-to-10(A) to 12(C)-to-13(D) state changes for 5S rRNA. However, and despite consistent reductive tendencies of helical segments, changes favored retention of G:U wobble base pairs (data not shown). Conversely, the reverse trend was clear for the large RNase P RNA and rRNA molecules, with exceptions in 11(B)-to-10(A) state changes for RNase P RNA and 18(I)-to-19(J) state changes for rRNA molecules. The forward and reverse trends were also evident in the less frequent multistep transformations. For example, the 3-to-0, 2-to-0, and 1-to-0 state changes of tRNA, typically involving the loss of the variable arm of the molecule, are only counteracted by a less frequent 0-to-3 tendency of expansion. A more balanced multistep gain-and-loss interplay is evident in the larger RNase P RNA and rRNA molecules. This probably reflects a diversity of expansion segments in these larger molecules. Thus, small helical RNA segments tend to become smaller while large segments tend to become larger for all RNA molecules explored.In sharp contrast, gains were consistently favored over losses of average lengths of unpaired regions, with frequencies decreasing with nucleotide length. This behavior counters the stabilizing effect of helical regions of RNA. It depicts the ‘frustrated’ energetics of base pairing that drives the structural stability and folding of RNA . Again, changes occurred most frequently in single steps, though 2-step changes adding or eliminating 2–3 unpaired nucleotides were particularly significant for 5S rRNA and those adding 2–3 nucleotides for RNase P RNA. Single-step growth is typical of bulges and hairpin loops. Thus, helical and unpaired regions tend to become larger by expansion of unpaired segments in all RNA molecules explored.We note that a number of multi-step changes occurred at low frequency in the step-matrices, especially in the larger RNase P RNA and rRNA molecules. As expected, the incidence of these clouds diffusing from the double diagonal patterns of the bubble plots increased with average length of RNA. The number of array entries with significant frequencies of change was 20, 61, 97, and 316 for helical segments and 60, 75, 91, 370 for unpaired segments of tRNA, 5S rRNA, RNase P RNA, and rRNA, respectively. In all cases, entries represented about 30–50% of the array total. Larger molecules provided more opportunities for insertions–deletions causing changes in the length of paired and unpaired regions of the molecules. However, calculating expected cost-change graphs for multistate characters of these kinds is difficult. It depends on tree shape and character state frequency of leaves (taxa) as well as their number .To confirm patterns of change derived from trees of molecules, we traced character change in published trees of substructures describing the evolution of the small and large rRNA molecules . Patternit is made up of substructures with origins preceding ages ≤ it. Thus, the evolutionary heat maps constitute models of molecular evolution that portray the gradual addition of substructures to evolving molecules. We note that the time of origin of RNA segments is not a result of the model of character state change that is used to reconstruct phylogenetic trees, since changes can occur at equal frequencies in large or small substructures and in different parts of the trees. For example, tracing character state changes in the branches of trees of rRNA substructures dispelled the idea of longer stems being attracted to the base of the trees since changes in the length of stems were spread throughout branches of the trees , a distance in nodes from the hypothetical ancestor on a relative scale from 0 (origin of the RNA molecule) to 1 (the present molecule) . These ahe trees .p < 0.001) with strong association strengths (r ranging from −0.80 to 0.97). Note that Pearson’s correlations are independent of making assumptions of normality either in the marginal distributions or in the bivariate surface with reasonable sample sizes of n > 20. While n values were lower for tRNA and 5S rRNA, the null hypothesis that the data were normally distributed was not rejected for all RNA types  except for rRNA . Given that Pearson’s correlation is extremely sensitive to outliers, the nonparametric Spearman’s rho test confirmed significant association (p < 0.001) for helical segments of all RNA examined, including rRNA. We also detected statistically significant correlations between length and time for unpaired segments of 5S rRNA (p < 0.05), RNase P RNA (p < 0.001) and rRNA (p < 0.001), being negative in rRNA  and exhibited R coefficients (0.709–0.893) typical of strong associations  suggests an evolutionary principle of diminishing returns when RNA substructures accumulate in time as the molecules grow in evolution. Because extant RNA molecules do not appear in evolution fully formed, their structures grow in evolution by accumulating substructures. As they grow, they become bigger. The principle makes them accrete gradually smaller stems as they grow. Because the time of origin of substructures is correlated with stem accumulation, we can plot lengths of RNA segments in logarithmic scale against number of segments making a molecule at some point in time. The insert of ciations .“the greater the whole, the smaller its constituents” [Early in the 1900s, Paul Menzerath proposed a qualitative generality of language in which the duration of the articulation of sounds shortens in long syllables . The genituents” , was supituents” , and latituents”  and vocaituents” . More reituents” ,57,58 anituents” , showingWhen analyzing proteins, a large majority of molecules contain more than one structural domain . This alFirst, we previewed a diminishing returns principle in character state changes along the branches of phylogenetic trees of molecules and trees of substructures. We used CSR methodologies to build bubble charts describing the average frequency of changes between character states in helical and unpaired segments . We obsea principle of least effort or some not yet known principle of balance recompensating lengthening on one side with shortening on the other” [constituent’ parts, y(x), with increases of the size or length of ‘whole’ constructs x, according to Equation (2)A, b, and c representing fitting parameters. Note that the general formulation explains dependencies between the size-structure of parts and wholes of a system by adding the effect of system’s hierarchy typical of multilevel structure characteristic of language and biological organization. Two special cases of the general formulation are generally used to fit parameters [c = 0, the general law takes the most commonly used form because it follows a power-law that enables fitting parameters in log-log plots. This is the formulation used for example in the analysis of structural domains of proteins [b = 0, y(x) decreases monotonically with the system’s size measured by its length or number of parts, with parts and constructs being contiguous in the hierarchy of system’s organization. We use this formulation (see Equation (1)) to fit parameters of the law to RNA accretion history by simply defining x as the number of substructures making up molecules. Parameter fitting showed that the regression model was statistically significant and exhibited correlations typical of strong associations  was defined by two terms. The first term was a cost describing the energy–matter investment in the molecule (CP), which depends on x and the average length of substructures. The second term described the flexibility–robustness of the molecular system (FRP), which depends on A, x, and slope b. The derivative of P with respect to x, when set equal to zero, gives the power law form of the MA law, with intercept A, which can be considered the length of the first molecular construct and an upper bound for the MA law’s shortening principle. A is also a parameter that establishes a flexibility–robustness stratum. Mathematical elaboration also showed that the FRP/CP ratio is only controlled by exponent b, with steeper slopes implying increases in trade-offs benefitting flexibility–robustness over economy in a frustrated landscape of molecular persistence. While the proteome data used to fit the power law version of the MA law in [N, such as RNA length, decreases at a proportional rate such that it satisfies the ordinary first order differential equation dN/dt = −λN, with λ representing an exponential decay constant. Solving the equation results in Equation (3)N(t) is the quantity at time t, 0N = N(0) is the quantity at time 0, and λ > 0. This class of functions is useful because functions can be easily computed for sums and counts . The typical example is ‘nuclear decay’, the stochastic process by which an atomic nucleus that is unstable loses energy by radiation in the form of one or more subatomic particles or photons. The model has been applied to numerous problems in the natural, social, and computer sciences, including many in biology [x is approximately linearly proportional to t, with t measured as time of origin in a relative 0–1 nd scale  = y(x) is the length of substructures at time t, N0 = A is the length of the first molecular construct appearing in evolution at time 0 (the intercept), and λ is the constant that describes the rate of decay of molecular length (the slope in a loglinear plot), which is proportional to c. In nuclear decay, λ is a characteristic number for each nuclide, ranging from 0.69 × 10−24 yr−1 for the highly stable 28Te to 0.30 × 1023 sec−1 for the highly unstable 7H. It measures nuclide stability, an atomic population characteristic related to atomic persistence. Similarly, time decay of the length of RNA substructures translates into a range of c exponents, from 0.012 for rRNA to 0.460 for tRNA  or gyration (gR) radii of long RNA molecules measured with fluorescence correlation spectroscopy compared well with predictions from ensemble averaging methods that consider sequence-dependent molecular branching. Measurements revealed a general scaling law hR~gR~vN, with N representing the length of the RNA molecule in nucleotides and v a scaling exponent. While v for small compact molecules approaches 0.34, longer molecules such as those of viral RNA genomes that are under evolutionary pressure to fold into icosahedral viral capsid have scaling relationships with v ranging from 0.5 to 0.6 that deviate from simple monotonic behavior . These observations suggest junction-induced branching patterns  are strongly biased by the size, compact folding, and function of the molecules. In addition to these biophysical and evolutionary constraints, a number of stochastic processes involving biases in mutation, insertion, and deletion of RNA sequences will affect the size and function of RNA substructures. This putative decay force could also embody a molecular principle of diminishing returns. The c exponents of the MA law formulation reveal pushes towards economy as the sizes of RNA molecules increase and expand the flexibility–robustness stratum suggested by the length of the first ‘originating’ molecular construct (A). Conversely, smaller molecules could become independent of the economy push by exploring other mechanisms, including the use of modified bases, wobble base pairing, and pseudoknot lock-in configurations. The push towards increasing unpaired regions in all molecules except rRNA is also in line with a focus on flexibility/robustness. Such is the case of RNase P RNA. Recent analysis of atomic structure of the yeast RNase P complex [The decay of molecular length with evolutionary time likely involves biophysical and evolutionary culprits. Predicted and empirically measured sizes of long RNA molecules are vastly determined by branching patterns of their secondary structures . Hydrody complex  revealedWhat are the evolutionary agents of molecular change responsible for RNA accretion and the principle of diminishing returns we uncovered? We proposed a linkage theory  that useThe existence of MA law of syntax in RNA accretion history now provides an additional tool to reconcile phylogenetic, algorithmic, and theoretical models of molecular history. We have interpreted structural phylogenomic chronologies with global models of origin of proteins, cofactors, and protein biosynthesis , the gen


RNA polymerase (Pol) III transcribes small untranslated RNAs such as 5S ribosomal RNA, transfer RNAs, and U6 small nuclear RNA. Because of the functions of these RNAs, Pol III transcription is best known for its essential contribution to RNA maturation and translation. Surprisingly, it was discovered in the last decade that various inherited mutations in genes encoding nine distinct subunits of Pol III cause tissue-specific diseases rather than a general failure of all vital functions. Mutations in the POLR3A, POLR3C, POLR3E and POLR3F subunits are associated with susceptibility to varicella zoster virus-induced encephalitis and pneumonitis. In addition, an ever-increasing number of distinct mutations in the POLR3A, POLR3B, POLR1C and POLR3K subunits cause a spectrum of neurodegenerative diseases, which includes most notably hypomyelinating leukodystrophy. Furthermore, other rare diseases are also associated with mutations in genes encoding subunits of Pol III  and the BRF1 component of the TFIIIB transcription initiation factor. Although the causal relationship between these mutations and disease development is widely accepted, the exact molecular mechanisms underlying disease pathogenesis remain enigmatic. Here, we review the current knowledge on the functional impact of specific mutations, possible Pol III-related disease-causing mechanisms, and animal models that may help to better understand the links between Pol III mutations and disease. Transcription is essential to make genome-encoded information accessible, which is a basic condition for the creation of all life forms. It represents the first step in gene expression and is coordinated by regulatory mechanisms allowing cells to respond not only according to their own needs, but also, if necessary, to demands from neighboring cells or to differentiation programs.Nuclear RNA polymerases are responsible for the transcription of genomic DNA into RNA. In eukaryotes, up to five different nuclear DNA-dependent RNA polymerases (Pol I-V) have been described, each of which transcribes specific groups of genes. RNA polymerases I to III are expressed by all eukaryotes. RNA polymerase (Pol) I transcribes the large ribosomal gene, which is present in up to several hundred copies within eukaryotic genomes. The resulting ribosomal (r)RNAs represent the major constituents of ribosomes . Pol II Here, we will review recent discoveries connecting Pol III  transcription to diseases. We will focus on mutations in genes encoding subunits of the Pol III transcription system that have been associated with microbial infections or with neurodegenerative diseases, including Pol III-related hypomyelinating leukodystrophy (POLR3-HLD). These mutations are also referred to as pathogenic variants in medical genetics. First, we will give an overview of the regulation of Pol III expression. Subsequently, we will review mutations in genes encoding Pol III subunits that were shown to be altered in disease and discuss potential underlying pathophysiological mechanisms that may depend on altered expression of Pol III transcripts. Finally, we will discuss the role of Pol III in innate immunity and related diseases.Sec), the Epstein Barr virus EBER gene, the 7SL RNA gene, vault RNA genes  . The typNA genes  and the NA genes   gene are recognized by STAF/ZNF143 and OCT1 (DSE), as well as by SNAPc/PTF (PSE), which stimulate the recruitment of TFIIIB-α to the TSS, whereupon Pol III is recruited  and the transcription factor TFIIIA (type 1 promoter only) to recruit the transcription initiation factor TFIIIB-β . The regHuman genes transcribed by Pol III are composed of maximally ∼300–350 nucleotides, with the longest RNAs generated by transcription of SINEs as well as of 7SK and 7SL genes. Functional elements required for regulating gene expression rate are all found within less than 500 base pairs relative to the TSS. In contrast, enhancer elements are often distributed over tens or hundreds of kilobases in the case of Pol II genes  led to the suggestion that Pol III transcription fulfills primarily housekeeping functions in cells. These housekeeping functions supporting RNAs are thought to be essential for cell survival, but it was long assumed that their expression did not require any regulation since they are thought to be provided in excess compared to the needs of cells , caused by specific combinations of utations . These pPOLR3B mutation c.1568T>A , who may remain asymptomatic or paucisymptomatic until adulthood and even late adulthood  mutation had normal POLR3A protein levels  in genes encoding Pol III subunits suggests a hypomorphic disease mechanism, either through decreased protein abundance or because of abnormal interactions of the mutated subunit with other subunits, with DNA or with RNA . Given tutations , the majn levels . Second,his gene .POLR3A, POLR3B and POLR1C causing POLR3-HLD are located throughout the three genes without clear hotspots  or are located at the interface between subunits and have the potential to impair complex assembly (Type III), while a smaller number affect functional elements such as the bridge helix or the trigger loop (Type II). Li et al., mapped several mutations in Treacher Collins syndrome (TCS), WRS and POLR3-HLD. They suggest that these mutations may impair complex integrity or enzymatic activity.Missense mutations in hotspots  and affehotspots . Prior thotspots , the pottemplate . Similard POLR3B . Recentld POLR3B . First, in silico predictions, the effect of some mutations on Pol III complex assembly has been assessed experimentally , 7SK RNA and RNase MRP RNA  , 7SL RNAPOLR3K HLD-causing mutations  . AlthougPOLR3A mutations in oligodendrocytes. Nevertheless, these cells showed decreased expression of Myelin Basic Protein (MBP) mRNA upon differentiation into more mature oligodendroglial cells, indicating that the mild Pol III transcriptome alterations may be sufficient to alter oligodendrocyte differentiation and/or MBP expression  did not lead to any neurological or developmental abnormalities in homozygous animals (Polr3b c.308G>A (p.Arg103His) mutation, which has only been reported in compound heterozygosity with another missense mutation in humans, is embryonic lethal in mice  , while other RNAs, notably those encoding 7SL RNA and 5S rRNAs, were not affected. In vitro transcription experiments demonstrated a defect in both factor-independent and factor-dependent transcription for genes representative of the yeast Pol III transcriptome in this double mutant  . To achily672Glu . Double e mutant . Next, te mutant . Since he mutant . HomozygPOLR3H was recently found to cause primary ovarian failure  levels in animal models would also help understand the developmental and tissue-specific consequences of their depletion.A handful of other animal models with mutations in Pol III subunits have also been engineered. A mutation in  failure  and thismutation . In zebrmutation . This mupoptosis . Interessubunits . Both paAs described above, possible reasons for tissue-specific differences may reside in particular dependencies of individual cell types on Pol III transcription products. Below, we will focus on the major Pol III-transcribed RNAs that have shown altered expression in cells carrying mutations in genes associated with POLR3-related disorders. We will describe characteristics of tRNAs, 7SL RNA and BC200 RNA.tRNAs are short (76–90 nucleotides) non-coding RNAs that act as essential adapters during mRNA translation. Each tRNA is loaded at their 3’ end with the amino acid corresponding to its anticodon by cytoplasmic aminoacyl tRNA synthetases (aaRS1). tRNAs allow decoding of the genetic code by recognizing cognate codons in translating mRNA and providing the corresponding amino acid for addition to the nascent peptide  in the T stem loop in the C57BL/6J strain compared to other mouse strains. This results in accumulation of a precursor form of n-Tr20 and decreased levels of the mature form and leads to increased ribosome pausing on AGA codons. On its own, the n-Tr20 polymorphism was found to modulate seizure susceptibility and synaptic transmission  (n-Ti17) decreased total tRNA-Ile-UAU levels and similarly increased the ISR in mouse brains, indicating that this is not specific to n-Tr20 but rather a common response to deficient tRNA levels. Thus, specific tRNA isodecoders play essential roles in maintaining normal translation in mouse brains. Although isodecoders with CNS-specific expression have not yet been identified in humans, these results suggest that deficient expression of any single tRNA important for brain function could lead to translation deregulation. In the context of POLR3-HLD, reduced levels of specific isodecoder(s) important in certain spatio-temporal contexts could induce ribosome stalling at the corresponding codons and impair translation of proteins important for normal oligodendrocyte and/or neuronal function and underlie disease pathogenesis.Consistent with this hypothesis, the Ackerman group identified the first instance of a tissue-specific mammalian tRNA gene, ouse CNS . n-Tr20 smission . Togetheneration , suggestse (ISR) , an impose (ISR) . MoreovePOLR3A Met852Val mutation  the removal of the 5′ leader sequence by RNAse P , 2) procS. cerevisiae mutant strain carrying two POLR3-HLD mutations at the homologous positions in Rpc160 (yeast homolog of POLR3A)  modification efficiency  . Althoug POLR3A) , it indificiency . Global to occur , which m1G on tRNAs, also influences m6A deposition on mRNAs by interacting with FTO . In addition to 7SL RNA, the SRP is composed of the SRP9, 14, 54, 68 and 72 proteins. It is responsible for co-translational targeting of nascent secretory and transmembrane peptides to the endoplasmic reticulum (ER) through interaction with its SRP receptor . Pol IIIin vivo by TFIIIC, TFIIIB-β and Pol III  is a major myelin protein that is targeted to the ER and follows the secretory pathway to reach the site of myelination . ReducedIn neurons, protein trafficking through the ER is crucial for both dendritic and axonal function, including for synaptic plasticity and neurotransmitter trafficking  complex   . Reducedhy (SMA) , suggestr ataxia . GEMIN5 in vitro , it is pPOLR3K mutations  is a monomeric Alu RNA that is predominantly expressed in the brain of primates. It was discovered by northern blot analyses  employinon event , whereason event . As a coon event . Closer on event . In addion event . A recenon event .in vitro transcription of rodent BC1 gene  and in addition a TATA-like sequence upstream of the TSS (hybrid promoter). Sequences up to 100 nucleotides upstream of the TSS were suggested to be important for BC200 transcription efficiency in transient transfection experiments. Stepwise deletion of these sequences led to a gradual decline in transcription rate without changing the ability of Pol III to correctly recognize the TSS. In addition, mutation of gene internal A- or B-boxes abolished transcription of the BC200 gene . These rBC1 gene . In addiBC1 gene .in situ hybridization experiments did not detect BC200 RNA expression in adult brain white matter  , KO of Be matter , but thie matter , albeit in vitro, in tumor cell lines or by analogy with BC1 RNA. Nonetheless, the many identified interacting partners and potential functions for this non-coding RNA provide hypotheses as to how it may contribute to the pathogenesis of POLR3-related disorders.Alternatively or in addition, impaired expression of BC200 RNA may contribute to some of the neuronal phenotypes  observed in POLR3-related disorders. Functional studies on BC200 RNA have mostly been performed According to a structural model of BC200 RNA, the first 120 nucleotides at the 5′-end, together with nucleotides 175–200 of the C-terminal unique region, fold into an Alu-domain. The 5′ part of the Alu-domain and the unique C-rich domain at the 3′-end of BC200 RNA are separated by a loop-forming A-rich domain. The Alu-domain of BC200 is highly similar to that of 7SL RNA . Consequin vitro or only for BC1 RNA.These proteins were proposed to influence the stability and/or the export of BC200 from the nucleus to the cytoplasm (SRP9/14), mRNA transport in neuronal dendrites  and/or interfere with BC1/BC200 effects on translation. Whilst eIF4A and PABP are targets of translational inhibition by BC200/BC1 RNAs , hnRNP Ein vitro experiments or by analogy with BC1 RNA. Indeed, a recent report shows that depletion of BC200 in MCF-7 breast cancer cells resulted in the reduction of translation  translation during transport is mediated in part by hnRNP-E1 , which win vitro , providiThe data summarized here indicate that BC200 RNA plays important roles in post-transcriptional mRNA regulation, with most studies so far having focused on its function in postsynaptic translation. The Alu domain in BC200 RNA associated with SRP9/14 proteins resembles the translation arrest domain of the 7SL RNA-containing SRP, suggesting that it is likewise involved in translation inhibition. As a consequence, reduced BC200 RNA levels may lead to imbalanced postsynaptic translation. Although oligodendrocytes are the primary cell type affected in POLR3-HLD, a direct neuronal dysfunction is thought to be responsible for the neuronal loss observed in POLR3-HLD (e.g. cerebellum) and other POLR3-related disorders  . The rolShigella flexneri or Legionella pneumophilia), Gram-positive bacteria (Listeria monocytogenes) or from viral infection (herpes simplex virus 1), suggesting that both bacterial and viral sources can trigger the Pol III-dependent innate immune system AT-rich DNA in the cytoplasm and transcribes it into 5’ triphosphorylated RNA, which triggers the activation of retinoic acid-inducible gene I (RIG-I). RIG-I activation is dependent on the AT-content of the produced RNA since it can be abolished by insertion of GC sequences . Activate system . In addie system  or latene system .POLR3A, POLR3C) or of VZV encephalitis  , POLR3C (RPC3), POLR3F (RPC6) and POLR3E (RPC5) were shown to strongly impair immune response to varicella-zoster virus (VZV) infections in humans. This reduced immune response resulted in the development of VZV pneumonitis (mutations in  POLR3F) . Importa POLR3F) . TogethePOLR3A and POLR3C. These variants led to reduced interferon production in peripheral blood mononuclear cells (PBMCs) from patients, which could be rescued in vitro by introduction of wild type alleles of the mutated genes into these cells, suggesting defects in Pol III cytoplasmic function. Nuclear 5S rRNA gene transcription was not affected in PBMCs carrying POLR3A- and POLR3C mutations  was shown to be replaced by glutamine (Arg84Gln) in a patient who developed VZV-induced encephalitis . Interesin vitro . This fiPOLR3E (Asp40His) was recently identified in a child with recurrent and systemic viral infections and Langerhans cell histiocytosis, indicating a more severe immune deficiency than in individuals carrying heterozygous mutations conferring specific VZV susceptibility who are otherwise healthy. Induction of interferon expression was triggered by the CG-rich  and especially the Alu gene subgroup might also play a role in the development of POLR3-HLD, since their promoters and RNA products show sequence and structural similarities to 7SL and BC200 RNAs. However, to the best of our knowledge, their involvement in POLR3-related disorders has not yet been investigated.Surprisingly, and independently of POLR3-related neurological disorders, mutations in four Pol III subunits affecting innate immune defense have also been described. Apparently, the key factor in the development of these diseases is the activity of the Pol III enzyme in the cytoplasm rather than the transcription of specific Pol III target genes. It will also be of interest for this spectrum of diseases to determine the exact underlying mechanism in order to develop potential therapies to reduce the risk of life-threatening complications from viral infections.In summary, Pol III transcription has emerged as a key factor in the pathogenesis of several rare debilitating diseases. The establishment of molecular-pathological correlations will facilitate the development of rational therapies in the future.


Direct-sequencing technologies, such as Oxford Nanopore’s, are delivering long RNA reads with great efficacy and convenience. These technologies afford an ability to detect post-transcriptional modifications at a single-molecule resolution, promising new insights into the functional roles of RNA. However, realizing this potential requires new tools to analyze and explore this type of data.Here, we present Sequoia, a visual analytics tool that allows users to interactively explore nanopore sequences. Sequoia combines a Python-based backend with a multi-view visualization interface, enabling users to import raw nanopore sequencing data in a Fast5 format, cluster sequences based on electric-current similarities, and drill-down onto signals to identify properties of interest. We demonstrate the application of Sequoia by generating and analyzing ~ 500k reads from direct RNA sequencing data of human HeLa cell line. We focus on comparing signal features from m6A and m5C RNA modifications as the first step towards building automated classifiers. We show how, through iterative visual exploration and tuning of dimensionality reduction parameters, we can separate modified RNA sequences from their unmodified counterparts. We also document new, qualitative signal signatures that characterize these modifications from otherwise normal RNA bases, which we were able to discover from the visualization.https://github.com/dnonatar/Sequoia.Sequoia’s interactive features complement existing computational approaches in nanopore-based RNA workflows. The insights gleaned through visual analysis should help users in developing rationales, hypotheses, and insights into the dynamic nature of RNA. Sequoia is available at The online version contains supplementary material available at 10.1186/s12864-021-07791-z. In recent years, several studies have led to the discovery of dynamic chemical modifications of nucleotide RNA bases, which are changes that are increasingly seen as key switches in the metabolism of RNA –3. WhileSingle-molecule, long-read sequencing technologies from Oxford Nanopore Technologies (ONT), however, is enabling cost-effective RNA sequencing. The MinION from the ONT, for example, records changes in the current across the nanopore as an RNA molecule passes through a nanopore. The current disruption is sensitive to the characteristic of the moiety passing through the pore, making it possible to detect modifications at single-base level. However, developing tools that can automatically detect such modifications is a challenge, since it requires an understanding of signal features that discriminate modified from non-modified bases. Although such analyses can greatly benefit from visual inspection of signal features currently there are limited tools and resources which can facilitate the visual exploration of the signal space, impeding the development of computational models for single molecule mapping of modifications.Recently multiple packages and frameworks became available for analyzing and visualizing nanopore sequencing data  as a ‘signal instance’. The cumulative list of signals  is hereafter referred to as the ‘cumulative signals’ .Our use of DTW is motivated by the fact that it offers more tolerance in comparing signals, compared to a straight matching criterion. Additionally, DTW allows for measuring similarity between signals of different lengths, which is necessary in our case, given that 5-mers could have signals that vary in length, which is determined by the time needed for the molecule to pass through the nanopore. The constructed similarity matrix comprises the raw signal similarity between every pair of 5-mers found in the run. The matrix serves as an input to the clustering algorithm and visualization.The Sequoia interface consists of a multi-view visualization with several, interactive visual representations. The initial interface consists of a data selection textbox used to upload a preprocessed file and the 5-mer list displaying all 5 nucleotide sequences contained in the file. The charts include a t-SNE plot that visually displays the resemblance of the signal instances in selected 5-mers, a box or violin plot which depict the uniformity across the signal instance of a 5-mer, and a raw signal graph which superimposes signal instances from various 5-mers for comparison. Figure i)Data Selection Widget:The user initiates the visualization by providing the input directory containing the preprocessed Nanopore files, and then clicking the ‘choose’ button, as shown in Fig. ii)5-mer List:To illustrate the uniformity of signals within 5-mers, Sequoia plots distributions of time warping distances  in the form of box or violin plots, and ranks those plots in a scrollable list, as seen in Fig. iii)t-SNE plot:While the distribution plots in the 5-mer list serve as an overview, the t-SNE plot allows a user to study each 5-mer in more detail. The plot visualizes signal similarity on a 2D scatterplot, enabling the comparison of signals from one or more 5-mers. Once a 5-mer is selected from the list, points representing signals will appear in the t-SNE plot. The positioning of the points is such that similar signals appear relatively close to one another, as compared to signals that exhibit a different electric pattern, which are positioned further apart Signal plot:The signal plots Fig. K visualiUp to four 5-mers can be selected simultaneously from the 5-mer list to visualize associated data simultaneously. Once a 5-mer is selected by clicking, its set of signals are added to the t-SNE plot. The colors of points in the scatterplot are consistent with the selection highlight appearing in the box/violin plot. Brushing and linking are also enabled between the views; selecting items from one view will cause the other views to highlight as well. For example, hovering over 5-mers in the list will also cause their signals to be highlighted in the t-SNE and signal plots, thereby revealing the clustering of signals within 5-mer, as well as their general shape. Similarly, hovering over a point in the t-SNE plot will highlight its corresponding signal in the signal plot. Additionally, the t-SNE plot also supports multiple selections; the user can create a ‘brush’ by drawing a rectangular selection onto the plot, causing signals within the bounding box to be selected and highlighted.To demonstrate the application of Sequoia, we deployed it over two case studies. Each of the case studies illustrated the differential nature of the signal from ONT across m5C and m6A RNA modifications.More than 160 types of RNA modifications have been reported, with amalgamating evidence for their role in gene regulation, cell development, translation, metabolism and stress response , 15. HowWe used Sequoia to explore modified vs. unmodified samples in order to understand differential signals from ONT-based long-read sequencing. Corresponding visualizations were generated to establish the application, scalability and scope of the tool.Hela cell line was purchased from ATCC cell line collection and cultured in DMEM media supplemented with 10 % FBS and 0.5 % penicillin/streptomycin.Libraries were prepared following the Nanopore Direct RNA sequencing kit documented protocol (SQK-RNA002). Briefly, total RNA was isolated using Qiagen RNeasy Mini Kit , followed by PolyA enrichment using Thermo Fisher Dynabeads™ mRNA DIRECT™ Micro Purification Kit . 500 ng of poly (A) RNA was ligated to a poly (T) adaptor using T4 DNA ligase. Following adaptor ligation, the products were purified using Mag-Bind® TotalPure NGS Beads (M1378-00), following the NGS bead purification protocol. Sequencing adaptors preloaded with motor protein were then ligated onto the overhang of the previous adaptor using T4 DNA ligase followed by NGS bead purification protocol. The RNA library was eluted from the beads in 21 µl of elution buffer and quantified using a Qubit fluorometer using the manufacturer’s RNA assay. The final RNA libraries were loaded to FLO-MIN106 flow cells and run on MinION. Sequencing runs and base calling were performed using MinKNOW software (Oxford Nanopore Technologies Ltd.) The data output from MinKNOW for Hela cell line was 800k sequence reads using one flow cell. MinKnow generates data as pass and fail folders. FAST5 files from pass folder were again base called using Albacore 2.1.0 (Oxford Nanopore Technologies Ltd.) resulting in 500 K single-molecule reads for the Hela cell line which corresponded to full-length transcripts ranging from 50b to 8 kb. The direct RNA-sequencing data generated in this study are publicly available on SRA, under the project accession PRJNA604314.https://nanoporetech.com) to base call the Fast5 data generated and aligned the corresponding Fastq to the hg38 human genome using Graphmap (V 0.5.2) [To visually compare the ONT generated signal across modified and unmodified RNA bases, we extracted 8737 genomic locations for m5C and 84,149 m6A modifications in HeLa cells from previous RNA modification studies –26. AdhoV 0.5.2) . The resLong-read direct RNA sequencing promises a potential to answer fundamental questions around cellular components’ interaction and function from a transcriptomic and clinical point of view , 29. NanGiven the temporal nature of a signal instance, a tool like Sequoia, which can emphasize the underlying insights of a time series data is the need of the hour. Although there are tools that specialize in visualizing time series data, they are not necessarily suitable for nanopore sequencing data exploration. For instance, TimeSearcher is a visualization tool for exploring and forecasting time series data, but does not allow segregation of data into groups . SimilarTo demonstrate the extent of application and adaptability of Sequoia, we extracted Oxford Nanopore-based direct RNA-sequencing signal information for two RNA modifications, m6A and m5C to compare them with their respective unmodified nucleotide signals. Using these case studies, we also wanted to demonstrate and explore how various parameters dynamically interact and influence the signal visualization.To demonstrate the effect of one of Sequoia’s adjustable parameters called dynamic time warping penalty, which is the added distance when compression or expansion is applied to match a pair of signals, we compared the signals extracted from m5C and unmodified C in the HeLa cell line. The two datasets used in this study were from the m5C data consisting of 19,391 5-mer with modified ‘C’ in the middle and the unmodified data consisting of 537,697 5-mer with unmodified ‘C’ in the middle. In this study, we focused only on unique 5-mers that had at least 100 signals in the m5C dataset. The rest of the data was filtered out, with a final residue of 46 unique 5-mers. Further, for each of the 46 5-mers, we randomly sampled 100 signals from each dataset to be compared in the visualization.After the preprocessing, we fed the data into Sequoia’s backend and generated the dynamic visualizations shown in Fig. To explore the data further, we utilized the brushing tool for group selection, which is one of the functionalities provided by Sequoia. By brushing over a group of points on the t-SNE plot  and unmodified locations in HeLa cell line. After performing the sequencing, signal information for all m6A modified and unmodified locations were extracted (see methods 2.4.2). Since Nanopore generates signal corresponding to a 5-mer passing through the bioengineered pore, rather than single nucleotide, we extracted the signal in such a way that the modified or unmodified Adenosine falls in the middle of the 5-mer. A total of 41,867 5-mers with modified-A in the middle and 1,031,337 5-mers with unmodified-A were extracted. In this study, we focused only on unique 5-mers that had at least 100 signal instances in the m6A dataset. The rest of the data was filtered out, with a final set of 24 unique 5-mers. Furthermore, for each of the 24 5-mers, we randomly sampled 100 signals from modified-A and unmodified-A datasets, to visually compare the signal characteristics using Sequoia.To visualize the signal disparity across input datasets, Sequoia clusters the signals based on the similarity matrix see . The cumHere we demonstrate Sequoia’s ability to distinguish Nanopore sequencing based on signal differences across m6A and unmodified A, and provide the first reports of significant difference in the signal lengths of an m5C and unmodified C in human cell lines. Although applications we demonstrated in are specific to the RNA modifications investigated in this study, Sequoia is not limited to these modifications. Sequoia is developed in such a way that it can be employed to not only study the signal features of raw Nanopore sequencing datasets facilitating the understanding of various types of RNA modifications, such as the Adenosine-to-inosine (A-to-I), Pseudouridine, and 2’-O-methylated nucleotides (Nm), but also potentially to DNA sequencing data, and thereby DNA modifications as well. Sequoia can effectively be used to compare signals of any k-mers of interest across direct RNA, and DNA sequencing-based datasets. Using sequoia users can analyze publicly available Nanopore-based datasets on a large scale, enabling signal-based studies to understand RNA and DNA functional characteristics. Sequoia can also aid in the improvement of the current Nanopore Basecalling accuracy, by comparing various RNA and DNA modifications and discriminating signals to distinguish them from regular bases. Potentially, Sequoia can be developed into a more versatile platform by including a wider range of time-series features and integrating it with machine learning models that can automatically predict modification locations.Sequoia is a visual analytics platform that facilitates the exploration of nanopore sequencing datasets. The platform enables users to visualize and cluster signal instances and thereby aid in the identification of RNA modification patterns across datasets. Using Sequoia, users can input the raw Fast5 files of their interest and analyze k-mer specific signals, which can be further processed to generate visualizations that depict the characteristics of datasets as well as k-mers. Sequoia employs algorithms including dynamic time warping and t-SNE to highlight similarities and differences in the underlying nanopore current signal. This framework generates a set of intuitive visualizations, enabling users to pinpoint variations not only across cohorts and datasets but also individual k-mers themselves. We also demonstrate the usability, versatility, and empirical nature of Sequoia by deploying it on two case studies visualizing RNA modifications.Project name: Sequoia.https://github.com/dnonatar/Sequoia.Project home page: Operating system(s): Mac, Linux.Programming language: Python 3, JavaScript.Other requirements: web browsers.License: None.Any restrictions to use by non-academics: None.Additional file 1: Supplementary Figure 1. Dissection of data units used in the processing of the input FAST5 file. Separated sections showing composition of FAST5 files with Signal list and Events table. Using this Table, 1-mers across each read are annotated with pertaining signals: referred to as Signal instance. All signal instances of 5-mers across reads are condensed into cumulative signals.


Here, we report the genomic sequences of five severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains obtained from nasopharyngeal samples from five tested COVID-19-infected patients from the Lambayeque region in Peru during early April 2020. Here, we report the genomic sequences of five severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains obtained from nasopharyngeal samples from five tested coronavirus disease 2019 (COVID-19)-infected patients from the Lambayeque region in Peru during early April 2020. Coronaviridae; genus Betacoronavirus; subgenus Sarbecovirus) variants sequenced from five patients from whom samples were taken in different districts in the Lambayeque region of Peru , during early April 2020.Here, we present the genomes of five severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . This study has been registered in Proyectos de Investigación en Salud, del Instituto de Salud del Perú , with code 37EBF149-F123-447F-8970-680BF549DE1D.TC] values obtained by qPCR, <25) were sampled, and total RNA was isolated using the GenUP total RNA kit . rRNA depletion was carried out using the Ribozero rRNA removal kit (Illumina). The cDNA libraries were generated using the TruSeq stranded total RNA LT kit (Illumina), using a single index and a fragment size range of 388 to 523 bp. Sequencing for every sample was performed in an Illumina NextSeq 500 model (midoutput 300 cycles), generating paired-end reads (150 bases long). Illumina sequencing was performed at Genoma Mayor .Nasopharyngeal swabs from SARS-CoV-2-positive patients (cycle threshold [MN908947) as a reference. Read mapping was done using SAMtools version 1.10  , all fivMW185823, MW185824, MW185825, MW185826, and MW185827. Illumina raw reads for this sequencing project (PRJNA673055) can be accessed under accession numbers SRR12927887, SRR12927888, SRR12927889, SRR12927890, and SRR12927891.The sequences were deposited in GISAID with the codes EPI_ISL_593772, EPI_ISL_593773, EPI_ISL_593774, EPI_ISL_593775, and EPI_ISL_593776. Assembled genomic sequences were deposited in GenBank under accession numbers


Pocillopora verrucosa and 96 Acropora spp. fragments, divided into different groups . Eight fragment replicates from the same donor colony  were used in each group and monitored for one year. Our results show that fragment survival was high in both farming habitats (>90%), with P. verrucosa surviving significantly better in the lagoon and Acropora spp. surviving and growing significantly faster in the reef nursery. P. verrucosa growth rates were similar between reef and lagoon habitat. Different rearing depths in the reef nursery had no impact on the survival of P. verrucosa but coral growth decreased considerably with depth, reducing fragments’ ecological volume augmentation and growth rates by almost half from 5 to 15 m depth. Further, higher fish predation rates on fragments were recorded on the reef, which did not impact overall nursery performance. Mutualistic fauna, which correlated positively with fragment survival, was more frequently observed in the lagoon nursery. The occurrence of disease was noted in both habitats, even though implications for fragment health were more severe in the lagoon. Overall, our study demonstrates that lagoon and reef nurseries are suitable for rearing large numbers of coral fragments for transplantation. Nevertheless, we recommend considering the specific environmental conditions of the farming habitat, in particular water quality and year-round accessibility, in each case and to adjust the coral farming strategy accordingly. We hope that this novel research encourages the increased application of mid-water rope nurseries for ‘coral gardening’ to advance coral reef recovery and climate resilience in the Maldives.The need for comprehensive and effective coral restoration projects, as part of a broader conservation management strategy, is accelerating in the face of coral reef ecosystem decline. This study aims to expand the currently limited knowledge base for restoration techniques in the Maldives by testing the performance of mid-water rope nurseries in a lagoon and a reef habitat. We examined whether different coral farming habitats impacted fragment survival, health and growth of two coral genera and how the occurrence of mutualistic fauna, predation and disease influenced coral rearing success. Two nurseries were stocked with a total of 448  Coral reef restoration has become an increasingly applied tool and internationally adapted approach to counteract the worldwide degradation of coral reefs . While sAcropora species as well as an observed shift from mature populations towards small and medium sized colonies  was placed parallel to the island’s southern house reef, that exhibits a steep slope in this area. Here, the once abundant and diverse live coral cover has also been severely reduced to less than 5%, following the latest bleaching and an outbreak of the corallivorous seastar Acanthaster planci  in Alif Dhaal atoll, in the Republic of Maldives . Two midr planci , with sor planci .Pocillopora and Acropora.For the purpose of this study, the two nurseries were stocked with a total of 544 experimental fragments from two coral genera, namely Pocillopora verrucosa fragments derived from 14 donor colonies (12–18 cm diameter) that were previously reared in the two mid-water rope nurseries on Athuruga . These donors were originally collected in 2018 from two natal sites with similar conditions to Athuruga’s farming habitats. Pocillopora donors growing in the reef nursery originated from artificial substrate on Athuruga reef  and were reared between 7 and 18 m depth. Donors growing in the lagoon nursery at 5 m were originally collected from the shallow back reef of Thudufushi Island (3°47′05″N72°43′49″E), since Athuruga lagoon did not offer sufficient live corals for nursery stocking. All donor colonies were assumed to be of different genotype as they were initially collected as corals of opportunity spaced more than 10 m apart  according to nursery habitat and rearing depth. Each of the 14 donor colonies were fragmented in 32 similar sized fragments, ranging from 3–10 cm in diameter depending on the selected fragmentation size for each donor colony. Then, a subset of eight fragments was used for each study group, resulting in a total of 112 fragments per group with the same distribution of fragment genotypes and sizes. Fragmentation of P. verrucosa donor colonies from the RN and LN and restocking occurred on the nursery site and underwater using SCUBA equipment. To limit handling stress and damage, the stocked ropes were immediately reattached to the nurseries. Fragments that required translocation to a different rearing habitat were continuously submerged in separate containers and transported by divers the same day. Excess fragments were reared on separate ropes in the nurseries and excluded from the study.To compare coral farming performance between the lagoon and the reef habitat and for different depths in the RN, a total of 448 Acropora fragments were directly collected as corals of opportunity from a nearby reef (3°48′51″N72°45′10″E) from less than 5 m unshaded depth. Six suitable colonies were selected based on their fragmentable size (15–20 cm in diameter), similar arborescent branching morphology and distance between them (>30 m) to increase genetic diversity. As available Acropora spp. fragments possibly comprise more than one species, all comparisons are made at the genus level. The donor colonies were kept in shaded and spacious containers filled with fresh seawater and transported to Athuruga within one hour, followed by the same fragmentation and stocking procedure as for P. verrucosa. In the nurseries, Acropora spp. fragments represented two study groups of 48 fragments each, growing at 5 m depth in the LN and the RN . Again, subsets of 8 similar sized fragments (3–11 cm diameter) per donor colony were used, likely representing six different genotypes.A monthly monitoring and maintenance protocol was established for a one-year farming period. The protocol was interrupted due to Covid-19 from months three to eight, resulting in a total of seven surveys  and three growth measurements at stocking (T0), post interruption (T3) and after one year (T7) for all fragments. Water temperature was recorded at 5 m depth during each dive using a Suunto dive computer.The status of nursery-grown experimental fragments was analyzed applying the following parameters suggested by h’ representing the longest linear colony diameter of the three perpendicular measurements  by taking three measurements to the nearest mm using a Vernier caliper, where: P. verrucosa fragments was investigated using a Pearson correlation to obtain a better understanding of optimal stocking size for this species.In addition, the relationship between fragments’ initial size (EV at T0) and the subsequent growth rate for P. verrucosa fragments originating from ‘reef reared’ and ‘lagoon reared’ donors . Therefore, mean differences in growth rates between fragments originating from reef and lagoon farming habitats were compared within each study group using the Mann–Whitney test.The experimental design further allowed to test for any differences between All statistical analysis was performed using SPSS ver. 27  and all data is represented as arithmetic means ± standard error. Non-parametric test statistics were used when the normality assumption was violated.N = 544) was high (91%) after one year (T7) with differences between Pocillopora verrucosa  and Acropora spp.  fragment survival being marginally non-significant  = 3.59, p = 0.058). For P. verrucosa the survival rate was above 90% for all four groups with the highest survival recorded in the LN (99%), which was significantly different from the RN survival  = 6.95, p = 0.008). Here, the average survival rate for all depths was 92% and rearing depth had no significant effect on survival  = 1.334, p = 0.513; see Acropora spp. fragments, all growing at 5 m depth, was significantly higher in the RN (96%) than in the LN  = 5.031, p = 0.025).Overall, the survival of the experimental stock  were fully alive (H3) after one year (T7) with only a few partially alive corals (H2 and H1) found in each RN group  due to algae overgrowth. In the LN, the spread of ‘White Syndrome’ disease (see For ease see  had consease see  with no ease see .P. verrucosa fragments the average predation rate was significantly lower in the LN (37 ± 18%) than in the RN (47 ± 14%)  = 13.504, p < 0.001), where predation decreased significantly from 5 m  to 15 m depth  = 90.483, p < 0.001). In the RN, predation events were also more consistent throughout the study period (in 6 out of 7 surveys), while in the LN predation on fragments was only recorded in three surveys. Predation on Acropora spp. was only recorded once on two fragments in the RN. Corals only showed fish predation marks in both habitats, which never made up more than 5% of the fragment’s surface and visibly healed between surveys.On Trapezia were most frequently observed , while other small crabs, shrimps and fish made up the remaining 10%. Coral associated fauna was significantly higher in the LN  = 193.24, p < 0.001). Specifically, associated fauna was on average most frequently observed on P. verrucosa fragments in the LN (26 ± 7%), while only found in 7 ± 2% of RN fragments. Similarly, 23 ± 5% of Acropora spp. fragments in the LN were associated with fauna while in the RN it was only 4 ± 2%. A significant positive relationship between P. verrucosa survival and Trapezia crabs occurrence was found  = 9.674, p = 0.002).Of the fragment inhabiting fauna, guard crabs of the genus Sargrassum sp.) overgrowth was most noticeable on Acropora fragments in the RN, where 10 fragments had suffered partial tissue damage at T3 due to the interrupted maintenance schedule. In contrast, blue–green algae, identified in the field as mainly Schizothrix calcicola were prevalent on the LN structure, but did not overgrow living fragments.Temperature or stress induced bleaching was not an issue during the rearing period and water temperatures never exceeded 30 °C at 5 m depth in either habitat. Temporary bleaching of the upper fragment tissue was only observed in 3 fragments  during the study. Brown algae  = −9.15, p < 0.001; 3 to 780 ± 31 cm3; Z (N = 101) = −8.72, p < 0.001). The RN fragment size augmentation  = −8.77, p < 0.001) while at 15 m the EV increase was reduced to a 1127% increase  = −8.94, p < 0.001).Fragment size was calculated as ecological volume (EV), which increased significantly for all groups during the one-year survey period . The larentation  decreaseP. verrucosa varied significantly between fragment groups  = 36.284, p < 0.001). Post hoc testing revealed that there was no significant difference in daily growth rates between the lagoon (M = 0.08 ± .005) and the reef (M = 0.07 ± .004) at 5 m (p = 0.848). However, on the RN daily growth rates (see p ≤ 0.001).Therefore, daily growth rates for ates see  varied sAcropora spp. groups  = −5.37, p < 0.001) and in the RN by 1098%  = −5.91, p < 0.001). Size augmentation and daily growth rates (Z (N = 84) = 579, p = 0.008), with fragments growing much faster on the reef.EV also increased for both . groups  during tth rates  varied sP. verrucosa fragments was 5.22 ± 1.1 cm in diameter (h), ranged from 2.7 to 10.0 cm. A significant negative correlation between initial size EV and subsequent growth rate was found, with smaller fragments showing a faster growth rate (r(418) =  − 0.56; p < 0.001). This pattern was even more evident when analyzing treatment groups separately to account for the effect of depth (LN_5m: r(109) =  − 0.65; RN_5m: r(99) =  − 0.65; RN_10m: r(100) =  − 0.63; RN_15m: r(104) =  − 0.68; all p < 0.001; see Average initial size at stocking for all Z (N = 320) = 12419.5, p = 0.646).To investigate possible impacts of different donor farming habitats on fragments’ growth rates in the two nurseries, the observed effect of initial size had to be controlled for first. Therefore, fragments from two reef farmed donor colonies with the two smallest mean stocking sizes as well as fragments from two lagoon farmed donor colonies with the largest stocking means were removed from the analysis. The remaining 141 fragments from ‘reef farmed donors’ and 156 fragments from ‘lagoon farmed donors’ were non-significantly different in stocking size at T0 (P. verrucosa fragments that derived from reef donor colonies (MReef = 0.0597 ± .003) grew significantly faster than fragments from lagoon farmed donor colonies (MLag = 0.0478 ± .003) (Z (N = 297) = 8322, p < 0.001). This was also the case when comparing daily growth rates for each study group separately (Z (N = 79) = 577, p = 0.047).Growth rate comparison for these fragments at T7 revealed that parately . In all This study conducted a direct comparison and comprehensive assessment of mid-water rope nursery performance in a lagoon and a reef habitat in the Maldives for the first time. Our evaluations are based on fragment survival and growth as well as the occurrence of predation, disease and mutualistic fauna.Acropora cervicornis after 12 months in in-situ nurseries; Pocillopora damicornis after 10 months in a rope nursery; Pocillopora fragments’ survival was significantly higher in the LN, while Acropora fragments survived better in the RN. Closer inspection revealed that fragment survival and condition of both genera were affected differently by the spread of disease, which appeared to be coral genus and habitat specific, as only Pocillopora was affected on the reef while only Acropora was affected in the lagoon. For Acropora fragments, the negative effect of disease was also clearly noticeable when comparing growth, which was twice as fast in the disease-free RN stock. These findings highlight the need to investigate coral diseases in coral restoration further, in particular possible transmission routes, time and density dependences in nurseries and mitigation measures. In this context, water quality and human induced pollution, in particular when operating in a resort setting, also require further attention. Disease outbreaks can significantly impact coral farming success and there is an additional danger of introducing disease to transplantation sites  throughout the one-year study period. Similar survival rates have been reported, for example, from the Caribbean , when fragments had reached a suitable size and branch complexity to host guard crabs. The percentage of fauna hosting corals was significantly higher in the lagoon, for both Acropora and Pocillopora fragments. There could be two, not mutually exclusive explanations for this observation. First, Trapezia sp. predators such as small reef inhabiting wrasses were never encountered during the surveys in the lagoon, while they have been regularly observed on the RN during maintenance work, which could indicate a higher predator abundance on the reef. In fact, increased predation pressure has previously been linked to reduced abundance of mutualistic decapods in Pocillopora colonies  as coral weight increased over time, even if this was counteracted with additional buoyancy devices. Although rearing depth had no effect on survival, P. verrucosa growth rates decreased by 27% from 5 to 10 and another 21% from 10 to 15 m on the reef as light levels decrease. Light availability is an important environmental parameter determining coral growth and typically reflected in the abundance of fast-growing corals in shallow depths  at stocking increases growth rates for Pocillopora in both habitats, while increased rearing depth decreases fragment growth. We suggest that mutualistic fauna, here more abundant in the lagoon, could be increased by stocking fragments together with older colonies to facilitate transmission. Furthermore, apart from fish predation, our mid-water rope nurseries provided good protection from corallivory in the lagoon and reef habitat. How different farming habitats and rearing depths translate into outplanting success of coral gardening remains to be tested.This provides a good starting point for the application of the coral gardening approach, although increased species diversity should be included as a restoration goal. We also found some habitat and genus-specific differences, that are worth considering in future restoration projects. In direct comparison, the robust Finally, we consider reef mid-water rope nurseries a useful addition to the coral restoration tool kit in the Maldives, especially when lagoon farming habitats are not available, not easily accessible or conditions are unsuitable. Our streamlined rope nursery design withstood the high currents and fish abundance in the reef environment, while providing additional rearing space at depth. Therefore, we hope that this novel research provides some valuable insights for restoration practitioners and a step towards expanding restoration efforts in the Maldives.10.7717/peerj.12874/supp-1Figure S1Donor colonies were collected as fragments of opportunity in 2018 and reared in two different nursery habitats on Athuruga. Colonies were fragmented and reciprocally stocked in 2020 in the lagoon and reef nursery at different depths. After a one-year monitoring period differences in fragment parameters such as survival, condition, growth and interactions with mutualists or predators were analysed.Click here for additional data file.10.7717/peerj.12874/supp-2Supplemental Information 2Pocillopora and Acropora fragment health over the one-year study period in a lagoon and reef nursery. Furthermore 3 size measurements for each fragment are shown for three growth surveys.The data comprises detailed information on Click here for additional data file.