N6-methyladenosine (m6A) is the most abundant epigenetic modification on eukaryotic mRNA and has important roles in gene expression. While m6A distribution and localization have been defined in zebrafish using antibody-based methods, no research has been published on the distribution of m6A during zebrafish embryogenesis with single-base precision. Here, we present the transcriptome-wide m6A landscape at single-nucleotide resolution during zebrafish early embryonic development using m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq). We identify over 36,000 high-confidence unique m6A sites that are primarily located in the DRACH consensus motif in long internal exons, 3’UTRs, and near stop codons. Our results show a > 4-fold decrease in m6A levels from 256-cell to 50% epiboly, consistent with the timing of the zebrafish maternal-to-zygotic transition. Analyses suggest that m6A-modified transcripts are predominantly associated with maternal classes at early developmental stages, with increased representation among zygotic-associated classes by later development. We highlight that differential methylation between developmental stages was observed in transcripts associated with mRNA metabolism, translational processing, and organ development.

Abstract Background The ribosomal DNA (rDNA) of the myxomycete Didymium iridis is located on a linear, multi-copy, non-Mendelian chromosome. Efforts to determine the complete sequence by short-read sequencing technologies have been prevented by the presence of highly repetitive regions. Here we use high coverage (~10,000 x) long-read Oxford Nanopore Technology to determine the rDNA chromosome sequence in haploid amoebae from telomere-to-telomere. Results The 20 kb rDNA chromosome, which is present at ~ 132 copies per haploid genome, is capped by regular TTAGGG telomeric repeats at both ends and carries an 11.3 kb pre-rRNA transcription unit coding for the small and large subunit rRNAs. The rRNA genes are further interrupted by autocatalytic group I introns, one of which encodes a homing endonuclease and two catalytic RNA domains with different functions in RNA processing. RNA mapping analyses from amoeba, microcyst, flagellate, and plasmodium stages, based on Illumina short-read sequencing, support the presence of a mature intron homing endonuclease mRNA both in haploid and diploid life stages in D. iridis. The non-transcribed sequence region upstream of the transcription unit contains several direct repeat arrays, including a highly complex upstream promoter region likely to be involved in pre-rRNA transcription regulation. Adjacent to the upstream telomere, a 4.2 kb palindromic region with potential for cruciform structure formation is found. Here, two putative replication origin candidates are located. Conclusions High coverage Oxford Nanopore Technology sequencing results in excellent resolution of complex sequence repeat feature in the D. iridis rDNA chromosome. The rRNA genes are interrupted by complex group I introns and RNA sequencing supports intron autocatalytic processing in haploid and diploid life stages. This study provides new insights into structural arrangements of nuclear rDNA in eukaryotic microorganisms.

N 6 -methyladenosine (m 6 A) is the most abundant epigenetic modification on eukaryotic mRNA and has important roles in gene expression. While m 6 A distribution and localization have been defined in zebrafish using antibody-based methods, no research has been published on the distribution of m 6 A during zebrafish embryogenesis with single-base precision. Here, we present the transcriptome-wide m 6 A landscape at single-nucleotide resolution during zebrafish early embryonic development using m 6 A-selective allyl chemical labeling and sequencing (m 6 A-SAC-seq). We identify over 36,000 high-confidence unique m 6 A sites that are primarily located in the DRACH consensus motif in long internal exons, 3’UTRs, and near stop codons. Our results show a >4-fold decrease in m 6 A levels from 256-cell to 50% epiboly, clearly linking this methylation to zebrafish maternal-to-zygotic transition. We highlight that differential methylation between developmental stages was observed in transcripts associated with mRNA metabolism, translational processing, and organ development.

N6-methyladenosine (m6A) is the most abundant epigenetic modification on eukaryotic mRNA and has important roles in gene expression. While m6A distribution and localization have been defined in zebrafish using antibody-based methods, no research has been published on the distribution of m6A during zebrafish embryogenesis with single-base precision. Here, we present the transcriptome-wide m6A landscape at single-nucleotide resolution during zebrafish early embryonic development using m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq). We identify over 36,000 high-confidence unique m6A sites that are primarily located in the DRACH consensus motif in long internal exons, 3’UTRs, and near stop codons. Our results show a >4-fold decrease in m6A levels from 256-cell to 50% epiboly, clearly linking this methylation to zebrafish maternal-to-zygotic transition. We highlight that differential methylation between developmental stages was observed in transcripts associated with mRNA metabolism, translational processing, and organ development. ### Competing Interest Statement The authors have declared no competing interest. The Research Council of Norway, https://ror.org/00epmv149, 275786

Marine threespine sticklebacks (Gasterosteus aculeatus) have colonized numerous freshwater lakes since the last ice age. The loss of body armor, such as reduced pelvic spines and lateral plate numbers, is a recurrent feature upon freshwater colonization and is attributed to parallel evolution. This study examines genome-wide genetic diversity and differentiation among sticklebacks from a marine site and two freshwater lakes of the same watercourse in subarctic Europe. The upper lake is unique in that it harbors a population of polymorphic sticklebacks, some with fully developed and others with reduced pelvic structures. Our results based on deep sequencing of pooled population samples showed common signatures of selection for freshwater sticklebacks in certain parts of the genome, such as the Eda containing region, but also evidence of differential selection, and the presence of large chromosomal inversions that seem to play an essential role in stickleback evolution. Pelvic reduction in sticklebacks has previously been linked to deletions in the enhancers of the pituitary homeobox transcription factor gene (Pitx1). While the genetics of Pitx1 seem unable to fully explain pelvic spine polymorphism in this population, we found differentiation between spined and spineless sticklebacks in several genomic regions, which harbor genes that might be involved in pelvic development. Most significantly, genetic differentiation between spined and spineless sticklebacks was noted in a region of chromosome 9 where the gene Hand2, previously implicated in limb development, is located. Our findings suggest that pelvic reduction in these sticklebacks involves multiple genetic factors, indicating parallel evolution through polygenic influences.

A subarctic Norwegian watercourse is known for its population of threespine sticklebacks (Gasterosteus aculeatus Linnaeus 1758) with unusual pelvic spine polymorphism; the upper lake contains a mixture of specimens that are normal-spined, asymmetric-spined, and spineless. In contrast, the downstream lakes and the nearby marine site contain only the normal spined morph. We investigated the maternal-line genetic structure in the watercourse using mitogenomics. A total of 242 sticklebacks representing two lakes and a marine site were assessed based on individual and pooled DNA sequencing. While two distinct mitogenome clades were detected in the upper lake (Lake Storvatnet), only one of these clades was present in the downstream lake. The marine site pooled DNA sample, however, contained several mitogenome haplotypes. We present mitogenome sequence features that include gene-specific single nucleotide polymorphisms, molecular phylogeny, and genetic differentiation assessments based on pairwise comparisons of pooled population samples from each site. The Lake Storvatnet mitogenomes belong to two distinct Euro-North American (ENA) clades; one of the ancestral lineages likely corresponds to the original maternal lineage in the watercourse, and the other was introduced more recently. We hypothesize that the second invader carried nuclear genomic features responsible for the observed present-day pelvic spine polymorphism in Lake Storvatnet.

PURPOSE:The purpose of this study was to compare the incidence of knee osteoarthritis (OA) between the anatomic single-bundle (SB) and anatomic double-bundle (DB) anterior cruciate ligament (ACL) reconstruction technique after 5-year follow-up (FU). Secondary objectives were to compare patient-reported outcome measures (PROMs), clinical examination, activity level, functional tests and graft failures between the two groups. METHODS:The study was a secondary analysis after 5-year FU of a randomized controlled trial (RCT) (Clinical Trials NCT01033188). One hundred and twenty patients between 18 and 40 years were randomized to either anatomic SB or anatomic DB reconstruction. The Kellgren-Lawrence (KL) classification grade ≥2 and the Osteoarthritis Research Society International (OARSI) atlas criteria score ≥2 were used for defining OA. Additionally, PROMs were obtained and clinical examinations of the knees were performed. Finally, the number of patients experiencing graft failure in each group was recorded. RESULTS:Radiographic imaging was performed in 39 patients in the SB group and in 37 patients in the DB group. Four patients (10%) in the SB group and two (5%) in the DB group developed osteoarthritis according to the KL classification (p = 0.28). Five (13%) in the SB group and three (8%) in the DB group developed osteoarthritis according to the OARSI atlas criteria (p = 0.59; difference 5.0% [95% confidence interval, CI: -0.10 to 0.20]). There were no significant differences in the PROMs, clinical examinations, activity levels, or functional tests when comparing the two groups. Of initially 62 SB patients, 14 (23%) experienced graft failure compared to 4 (7%) of the 58 DB patients (p = 0.015; difference 0.016 [95% CI: 0.03-0.29]). CONCLUSION:At 5-year FU, there were no significant differences in the incidence of OA, PROMS, or other clinical findings comparing the anatomic DB to anatomic SB ACL reconstructed patients. There were fewer graft failures among patients treated with anatomic DB ACL reconstruction. LEVEL OF EVIDENCE:Level II.

The subfamily Rasborinae is a species-rich group of freshwater fish related to zebrafish; however, its taxonomy remains unclear. We present the complete mitogenome and corresponding polyadenylated mitotranscriptome of Rasbora rasbora (Hamilton 1822) based on long-read and high-coverage Oxford Nanopore Technology sequencing. The mitogenome size, gene content, and gene organization correspond to the typical vertebrate composition, and the mitogenome generates 10 polyadenylated mRNAs. Two alternative polyadenylation sites of ND5 mRNA were detected, one with a 596 nt 3′untranslated region corresponding to the antisense ND6 gene. Polyadenylation also generates seven of the mRNA UAA stop codons. Complete mitogenome sequences, excluding the control region, were carefully aligned for RNA-coding and protein-coding features using 54 available species of the subfamily Rasborinae. The phylogenetic analyses based on maximum likelihood, Bayesian inference, and neighbor-joining tree building methods confirm the transfer of R. rasbora into the Sumatrana species group. The overall phylogeny of the subfamily Rasborinae supports with high confidence some previously observed changes within this subfamily, as well as contradicts some conclusions set by previous studies.

We sequenced and analyzed the complete mitogenome of a Norwegian isolate of the octocoral Alcyonium digitatum using the Ion Torrent sequencing technology. The 18,790 bp circular mitochondrial genome was found to harbor the same set of 17 genes, which encode 14 protein subunits, two structural ribosomal RNAs and one tRNA, as reported in other octocorals. In addition, we detected a new tRNAPro-like gene sequence nested within the MutS protein coding region. This putative tRNA gene feature appears to be conserved among the octocorals but has not been reported previously. The A. digitatum mitogenome was also shown to harbor an optional gene (ORFA) that encodes a putative protein of 191 amino acids with unknown function. A mitogenome-based phylogenetic analysis, presented as a maximum likelihood tree, showed that A. digitatum clustered with high statistical confidence with two other Alcyonium species endemic to the Mediterranean Sea and the Southeast Pacific Ocean.

RNAs originating from mitochondrial genomes are abundant in transcriptomic datasets produced by high-throughput sequencing technologies, primarily in short-read outputs. Specific features of mitochondrial small RNAs (mt-sRNAs), such as non-templated additions, presence of length variants, sequence variants, and other modifications, necessitate the need for the development of an appropriate tool for their effective identification and annotation. We have developed mtR_find, a tool to detect and annotate mitochondrial RNAs, including mt-sRNAs and mitochondria-derived long non-coding RNAs (mt-lncRNA). mtR_find uses a novel method to compute the count of RNA sequences from adapter-trimmed reads. When analyzing the published datasets with mtR_find, we identified mt-sRNAs significantly associated with the health conditions, such as hepatocellular carcinoma and obesity, and we discovered novel mt-sRNAs. Furthermore, we identified mt-lncRNAs in early development in mice. These examples show the immediate impact of miR_find in extracting a novel biological information from the existing sequencing datasets. For benchmarking, the tool has been tested on a simulated dataset and the results were concordant. For accurate annotation of mitochondria-derived RNA, particularly mt-sRNA, we developed an appropriate nomenclature. mtR_find encompasses the mt-ncRNA transcriptomes in unpreceded resolution and simplicity, allowing re-analysis of the existing transcriptomic databases and the use of mt-ncRNAs as diagnostic or prognostic markers in the field of medicine.

Loss of body armour, sometimes including a reduction in or loss of pelvic spines, is an adaptation observed in many isolated freshwater populations. Pelvic reduction in sticklebacks has previously been associated with recurrent, but variant, deletions within pelvic enhancer regions PelA and PelB, which regulate expression of the homeodomain transcription factor gene Pitx1. We investigated variation in nucleotide sequences of pelvic enhancers in sticklebacks collected from two small freshwater lakes in the same watercourse and a nearby marine site in subarctic Norway. Spineless, as well as asymmetrically spined and completely spined sticklebacks are present in the upper lake, while only specimens with complete spines are found at the other lake and the marine site. Observed variation at PelA between the three sites was mainly due to variable numbers of repeats at three fragile TG-repeat loci. The length of PelA, mainly at one of the TG-repeat loci, was consistently shorter among individuals in the upper lake compared with specimens from the two other sites. However, no obvious association was revealed between enhancer variants and pelvic status. No polymorphism was found at PelB. Thus, additional genetic factors and/or environmental cues need to be identified to fully explain the occurrence of pelvic reduction in sticklebacks in this lake.

Epithelial-mesenchymal transition (EMT) is a cellular plasticity program critical for embryonic development and tissue regeneration, and aberrant EMT is associated with disease including cancer. The high degree of plasticity in the mammary epithelium is reflected in extensive heterogeneity among breast cancers. Here, we have analyzed RNA-sequencing data from three different mammary epithelial cell line-derived EMT models and identified a robust mammary EMT gene expression signature that separates breast cancers into distinct subgroups. Most strikingly, the basal-like breast cancers form two subgroups displaying partial-EMT and post-EMT gene expression patterns. We present evidence that key EMT-associated transcription factors play distinct roles at different stages of EMT in mammary epithelial cells.

Background Mobile group I introns encode homing endonucleases that confer intron mobility initiated by a double-strand break in the intron-lacking allele at the site of insertion. Nuclear ribosomal DNA of some fungi and protists contain mobile group I introns harboring His-Cys homing endonuclease genes (HEGs). An intriguing question is how protein-coding genes embedded in nuclear ribosomal DNA become expressed. To address this gap of knowledge we analyzed nuclear L2066 group I introns from myxomycetes and ascomycetes. Results A total of 34 introns were investigated, including two identified mobile-type introns in myxomycetes with HEGs oriented in sense or antisense directions. Intriguingly, both HEGs are interrupted by spliceosomal introns. The intron in Didymium squamulosum , which harbors an antisense oriented HEG, was investigated in more detail. The group I intron RNA self-splices in vitro, thus generating ligated exons and full-length intron circles. The intron HEG is expressed in vivo in Didymium cells, which involves removal of a 47-nt spliceosomal intron (I-47) and 3′ polyadenylation of the mRNA. The D. squamulosum HEG (lacking the I-47 intron) was over-expressed in E. coli , and the corresponding protein was purified and shown to confer endonuclease activity. The homing endonuclease was shown to cleave an intron-lacking DNA and to produce a pentanucleotide 3′ overhang at the intron insertion site. Conclusions The L2066 family of nuclear group I introns all belong to the group IE subclass. The D. squamulosum L2066 intron contains major hallmarks of a true mobile group I intron by encoding a His-Cys homing endonuclease that generates a double-strand break at the DNA insertion site. We propose a potential model to explain how an antisense HEG becomes expressed from a nuclear ribosomal DNA locus.

Group I introns are mobile genetic elements encoding self-splicing ribozymes. Group I introns in nuclear genes are restricted to ribosomal DNA of eukaryotic microorganisms. For example, the myxomycetes, which represent a distinct protist phylum with a unique life strategy, are rich in nucleolar group I introns. We analyzed and compared 75 group I introns at position 516 in the small subunit ribosomal DNA from diverse and distantly related myxomycete taxa. A consensus secondary structure revealed a conserved group IC1 ribozyme core, but with a surprising RNA sequence complexity in the peripheral regions. Five S516 group I introns possess a twintron organization, where a His-Cys homing endonuclease gene insertion was interrupted by a small spliceosomal intron. Eleven S516 introns contained direct repeat arrays with varying lengths of the repeated motif, a varying copy number, and different structural organizations. Phylogenetic analyses of S516 introns and the corresponding host genes revealed a complex inheritance pattern, with both vertical and horizontal transfers. Finally, we reconstructed the evolutionary history of S516 nucleolar group I introns from insertion of mobile-type introns at unoccupied cognate sites, through homing endonuclease gene degradation and loss, and finally to the complete loss of introns. We conclude that myxomycete S516 introns represent a family of genetic elements with surprisingly dynamic structures despite a common function in RNA self-splicing.

BACKGROUND:Spliceosomal introns are parts of primary transcripts that are removed by RNA splicing. Although introns apparently do not contribute to the function of the mature transcript, in vertebrates they comprise the majority of the transcribed region increasing the metabolic cost of transcription. The persistence of long introns across evolutionary time suggests functional roles that can offset this metabolic cost. The teleosts comprise one of the largest vertebrate clades. They have unusually compact and variable genome sizes and provide a suitable system for analysing intron evolution.RESULTS:We have analysed intron lengths in 172 vertebrate genomes and show that teleost intron lengths are relatively short, highly variable and bimodally distributed. Introns that were long in teleosts were also found to be long in mammals and were more likely to be found in regulatory genes and to contain conserved sequences. Our results argue that intron length has decreased in parallel in a non-random manner throughout teleost evolution and represent a deviation from the ancestral state.CONCLUSION:Our observations indicate an accelerated rate of intron size evolution in the teleosts and that teleost introns can be divided into two classes by their length. Teleost intron sizes have evolved primarily as a side-effect of genome size evolution and small genomes are dominated by short introns (<256 base pairs). However, a non-random subset of introns has resisted this process across the teleosts and these are more likely have functional roles in all vertebrate clades.

Many severe inflammation conditions are complement-dependent with the complement component C5a-C5aR1 axis as an important driver. At the RNA level, the blood transcriptome undergoes programmed expression of coding and long non-coding RNAs to combat invading microorganisms. Understanding the expression of long non-coding RNAs containing Alu elements in inflammation is important for reconstructing cell fate trajectories leading to severe disease. We have assembled a pipeline for computation mining of new Alu-containing long non-coding RNAs by intersecting immune genes with known Alu coordinates in the human genome. By applying the pipeline to patient bulk RNA-seq data with sepsis, we found immune genes containing 48 Alu insertion as robust candidates for further study. Interestingly, 1 of the 48 candidates was located within the complement system receptor gene C5aR1 and holds promise as a target for RNA therapeutics.

A hallmark of sea anemone mitochondrial genomes (mitogenomes) is the presence of complex catalytic group I introns. Here, we report the complete mitogenome and corresponding transcriptome of the carpet sea anemone Stichodactyla haddoni (family Stichodactylidae). The mitogenome is vertebrate-like in size, organization, and gene content. Two mitochondrial genes encoding NADH dehydrogenase subunit 5 (ND5) and cytochrome c oxidase subunit I (COI) are interrupted with complex group I introns, and one of the introns (ND5-717) harbors two conventional mitochondrial genes (ND1 and ND3) within its sequence. All the mitochondrial genes, including the group I introns, are expressed at the RNA level. Nonconventional and optional mitochondrial genes are present in the mitogenome of S. haddoni. One of these gene codes for a COI-884 intron homing endonuclease and is organized in-frame with the upstream COI exon. The insertion-like orfA is expressed as RNA and translocated in the mitogenome as compared with other sea anemones. Phylogenetic analyses based on complete nucleotide and derived protein sequences indicate that S. haddoni is embedded within the family Actiniidae, a finding that challenges current taxonomy.

The lariat capping ribozyme (LCrz) constitutes an independent class of ribozymes. It is the defining feature of twin-ribozyme group I introns found in eukaryotic microorganisms. Its main function is to provide an intron-encoded homing endonuclease mRNA with a 5′-cap necessitated by the expression of the mRNA from within an intron in a ribosomal RNA gene. The cap is a tiny lariat in which the first and the third nucleotides are linked with a 2′, 5′ phosphodiester bond. A model for the origin of LCrz from group I intron splicing ribozymes by structural reorganization is well-supported. This shows the emergence of a branching reaction in a group I ribozyme scaffold and may be important in explaining the evolution of RNA splicing.

Nuclear group I introns are restricted to the ribosomal DNA locus where they interrupt genes for small subunit and large subunit ribosomal RNAs at conserved sites in some eukaryotic microorganisms. Here, the myxomycete protists are a frequent source of nuclear group I introns due to their unique life strategy and a billion years of separate evolution. The ribosomal DNA of the myxomycete Mucilago crustacea was investigated and found to contain seven group I introns, including a direct repeat-containing intron at insertion site S1389 in the small subunit ribosomal RNA gene. We collected, analyzed, and compared 72 S1389 group IC1 introns representing diverse myxomycete taxa. The consensus secondary structure revealed a conserved ribozyme core, but with surprising sequence variations in the guanosine binding site in segment P7. Some S1389 introns harbored large extension sequences in the peripheral region of segment P9 containing direct repeat arrays. These repeats contained up to 52 copies of a putative internal guide sequence motif. Other S1389 introns harbored homing endonuclease genes in segment P1 encoding His-Cys proteins. Homing endonuclease genes were further interrupted by small spliceosomal introns that have to be removed in order to generate the open reading frames. Phylogenetic analyses of S1389 intron and host gene indicated both vertical and horizontal intron transfer during evolution, and revealed sporadic appearances of direct repeats, homing endonuclease genes, and guanosine binding site variants among the myxomycete taxa.