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Correlation of mirtron and host gene expression. (A) We calculated the Pearson correlation coefficients of the accumulation of mouse mirtron-derived small RNAs and spliced RNA-seq reads directly flanking the mirtron across seven tissues. We also performed 100 control comparisons where the tissue origins were shuffled. The cumulative distribution function (CDF) of these correlations was plotted, and observed to be significantly positively correlated (by Mann-Whitney U-test). (B) The binned distribution of mirtron/mRNA Pearson correlation coefficients was plotted. This visualization emphasizes their positive correlation, but also highlights a subset of discordant loci. (C) Examples of correlated and discordant expression of mirtron-derived miRNAs and host mRNAs across tissues. We show host level gene expression as reads per kilobase of transcript per million mapped reads (RPKM) and the spliced exonic reads that directly cross the mirtronic locus as reads per million mapped reads (RPM). Mirtron-derived miRNAs are quantified as reads per million mapped miRNA reads (RPMM).
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Mirtrons are microRNA (miRNA) substrates that utilize the splicing machinery to bypass the necessity of Drosha cleavage for their biogenesis. Expanding our recent efforts for mammalian mirtron annotation, we use meta-analysis of aggregate datasets to identify ~500 novel mouse and human introns that confidently generate diced small RNA duplexes. The...
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... as our effort relied upon a large meta- analysis, a question arises how many loci are due to aggregation of a few reads in each of many libraries. To address this, we plotted the maximum number of reads mapped to mouse and human mirtrons across individual libraries (S3A and S3B Fig). These analyses showed a few dozen mirtrons fit the bill of having only single-digit reads in any particular dataset, even though all passed a 50 read minimum cutoff. ...
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... Analysis of mouse small RNAs shows similar enrichment of canonical miRNAs and mirtron-derived small RNAs in Ago1 and Ago2 complexes relative to control IgG complex. accumulate more broadly across many libraries (S3E and S3F Fig). The interpretation of this analysis may be biased by the overall lower accumulation of mirtrons relative to canonical miR- NAs. ...
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... the superior depth of the mouse data for spliced reads across mirtrons substantially improved the correlation of host gene-mirtron expression. As shown in the CDF plot (Fig 3A), there is a strong bias for well-correlated pairs (p<9.97E-12). ...
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... similar conclusion can be seen via the binned data plot ( Fig 3B). Interestingly, this visuali- zation makes apparent a subset of loci for which the accumulation of spliced flanking exon reads and mirtron-derived miRNAs are particularly uncorrelated ( Fig 3B). ...
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... similar conclusion can be seen via the binned data plot ( Fig 3B). Interestingly, this visuali- zation makes apparent a subset of loci for which the accumulation of spliced flanking exon reads and mirtron-derived miRNAs are particularly uncorrelated ( Fig 3B). We present some individual cases of highly correlated and uncorrelated host mRNA/mirtron mouse expression profiles in Fig ...
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... than bulk genes, but their averages are interme- diate to all classes of mirtrons. (F) Bar graphs that emphasize the individual properties of genes that host various classes of non-coding RNAs. It is evident that the all four classes of mirtrons have a broader distribution of intron numbers relative to other types of non-coding RNAs. (PDF) S7 Fig. 3' untemplated additions in mammalian canonical miRNA-3p and mirtron-3p spe- cies. Pie charts depicting the fractions of 3p-arm canonical miRNAs and mirtron-derived miR- NAs that match the genome directly, or following trimming of 3' untemplated nucleotides. For both 5'-tailed and conventional mirtrons, reads ending precisely at the AG ...
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... Representations of their length distribution and localization along the viral genome were both made in R using a custom Shiny application based on the ggplot2 (Wickham, 2009) and Bioconductor Gviz (Hahne and Ivanek, 2016) packages respectively. Furthermore, expressed human miRNAs (miRBase v22.1 (Kozomara and Griffiths-Jones, 2011), among which known mirtrons (Wen et al, 2015), were also identified and quantified in each library using BEDTools v2.30.0 (Quinlan and Hall, 2010) by comparing their genomic coordinates to those of the original aligned reads and by counting only reads showing at least 80% overlap with the miRNA sequence (command: bedtools intersect -a <sample>-hg38coreGencodeSINVGFP.bwtmap.bed -b hsamatmir.bed ...
In mammalian somatic cells, the relative contribution of RNAi and the type I interferon response during viral infection is unclear. The apparent inefficiency of antiviral RNAi might be due to self-limiting properties and mitigating co-factors of the key enzyme Dicer. In particular, the helicase domain of human Dicer appears to be an important restriction factor of its activity. Here, we study the involvement of several helicase-truncated mutants of human Dicer in the antiviral response. All deletion mutants display a PKR-dependent antiviral phenotype against certain viruses, and one of them, Dicer N1, acts in a completely RNAi-independent manner. Transcriptomic analyses show that many genes from the interferon and inflammatory response pathways are upregulated in Dicer N1 expressing cells. We show that some of these genes are controlled by NF-kB and that blocking this pathway abrogates the antiviral phenotype of Dicer N1. Our findings highlight the crosstalk between Dicer, PKR, and the NF-kB pathway, and suggest that human Dicer may have repurposed its helicase domain to prevent basal activation of antiviral and inflammatory pathways.
... Nucleic Acids Research , 2023 In some species, such as humans, tailed mirtrons outnumber conventional varieties however a role in gene expression has yet to be reported for any of these species ( 11 ) . Further, mirtrons as a class have several fold lower expressions relative to canonical miRNAs and are poorly conserved. ...
Thousands of atypical microRNAs (miRNAs) have been described in the genomes of animals; however, it is unclear if many of these non-canonical miRNAs can measurably influence phenotypes. Mirtrons are the largest class of non-canonical miRNAs that are produced from hairpins excised by splicing, which after debranching become substrates for Dicer and load into RISC. Most mirtrons require additional processing after splicing to remove ‘tail’ residues interposed between one of the host intron splice sites and base of the hairpin precursor structure. Despite most mirtrons requiring tail removal no function has been elucidated for a tailed species, indeed for all mirtrons identified function has only been assigned to a single species. Here we study miR-1017, a mirtron with a 3′ tail, which is well expressed and conserved in Drosophila species. We found that miR-1017 can extend lifespan when ectopically expressed in the neurons, which seems partly due to this miRNA targeting its host transcript, acetylcholine receptor Dα2. Unexpectedly we found that not only did miR-1017 function in trans but also in cis by affecting splicing of Dα2. This suggests a mechanism for mirtron evolution where initial roles of structural elements in splicing lead to secondary acquisition of trans-regulatory function.
... The dominant class of splicing-derived miRNAs in mammals comprises 5 0 -tailed mirtrons, followed by conventional mirtrons and then 3 0 -tailed mirtrons. 23 There are also rare ''dual-tailed mirtrons'' in which neither pre-miRNA hairpin terminus seems to be defined directly by splicing, but where one end resides too close to the splice site to represent a plausible Microprocessor substrate. 23,24 Beyond mirtrons, a wide variety of other non-canonical miRNA substrates bypass either Microprocessor or Dicer. ...
... 23 There are also rare ''dual-tailed mirtrons'' in which neither pre-miRNA hairpin terminus seems to be defined directly by splicing, but where one end resides too close to the splice site to represent a plausible Microprocessor substrate. 23,24 Beyond mirtrons, a wide variety of other non-canonical miRNA substrates bypass either Microprocessor or Dicer. 16 Curiously, very few non-canonical miRNA loci are conserved, with many alternative pathways supporting the biogenesis of just one or a couple of conserved miRNAs. ...
... These collectively total at least 500 in both the mouse and the human genomes, although <10 are conserved among mammals. 23 Thus, there are distinct evolutionary trajectories for different classes of miRNA substrates, even though their ultimate activities to program Ago complexes are seemingly identical. Accordingly, we speculated that there should be a biochemical basis to distinguish canonical pre-miRNAs from mirtron hairpins, leading to preferential suppression and evolutionary extinction of the latter. ...
Canonical microRNA (miRNA) hairpins are processed by the RNase III enzymes Drosha and Dicer into ∼22 nt RNAs loaded into an Argonaute (Ago) effector. In addition, splicing generates numerous intronic hairpins that bypass Drosha (mirtrons) to yield mature miRNAs. Here, we identify hundreds of previously unannotated, splicing-derived hairpins in intermediate-length (∼50-100 nt) but not small (20-30 nt) RNA data. Since we originally defined mirtrons from small RNA duplexes, we term this larger set as structured splicing-derived RNAs (ssdRNAs). These associate with Dicer and/or Ago complexes, but generally accumulate modestly and are poorly conserved. We propose they contaminate the canonical miRNA pathway, which consequently requires defense against the siege of splicing-derived substrates. Accordingly, ssdRNAs/mirtrons comprise dominant hairpin substrates for 3' tailing by multiple terminal nucleotidyltransferases, notably TUT4/7 and TENT2. Overall, the rampant proliferation of young mammalian mirtrons/ssdRNAs, coupled with an inhibitory molecular defense, comprises a Red Queen's race of intragenomic conflict.
... Сравнительное исследование репертуаров миртронов у человека и мыши [17] показало, что в этих геномах кодируются по меньшей мере 478 и 488 миртронов, соответственно, из них 13 общих. Из числа общих только 3 имеют консервативную последовательность, а 10 регулируют трансляцию одинаковых по функции генов у этих двух организмов. ...
... "Debranched" mirtrons are similar to canonical pre-miRNAs and are exported by Exportin-5 in the cytoplasm. Their further maturation is identical to the canonical manner [20][21][22]. ...
microRNAs (miRNAs) are small single strand non-coding RNAs and powerful gene expression regulators. They mainly bind to the 3′UTR sequence of targeted mRNA, leading to their degradation or translation inhibition. miR-140 gene encodes the pre-miR-140 that generates the two mature miRNAs miR-140-5p and miR-140-3p. miR-140-5p/-3p have been associated with the development and progression of cancers, but also non-neoplastic diseases. In aging-related diseases, miR-140-5p and miR-140-3p expressions are modulated. The seric levels of these two miRNAs are used as circulating biomarkers and may represent predictive tools. They are also considered key actors in the pathophysiology of aging-related diseases. miR-140-5p/-3p repress targets regulating cell proliferation, apoptosis, senescence, and inflammation. This work focuses on the roles of miR-140-3p and miR-140-5p in aging-related diseases, details their regulation (i.e., by long non-coding RNA), and reviews the molecular targets of theses miRNAs involved in aging pathophysiology.
... Treiber, N. Treiber, and Meister, 2018). This mechanism was proposed as ancient, that emerged before the miRNA processing machinery (Ruby, Jan, and Bartel, 2007) (see an extensive characterization of mammalian mirtrons in Wen et al. (2015)) In the same way, chimeric hairpins derived from other non-coding RNA (ncRNA) families or Pol III transcripts, as small nucleolar RNAs (snoRNAs) or transfer RNAs (tRNAs), that could be subsequently be processed by Dicer. In case of snoRNAs, they are processed into short-stable miRNA-like fragments, called small nucleolar RNA-derived RNAs (sdRNAs). ...
As described over 20 years ago with the discovery of RNA interference (RNAi), double-stranded RNAs occupied key roles in regulation and as defense-line in animal cells. This thesis focuses on metazoan microRNAs (miRNAs). These small non-coding RNAs are distinguished from their small-interfering RNA (siRNA) relatives by their tightly controlled, efficient and flexible biogenesis, together with a broader flexibility to target multiple mRNAs by a seed imperfect base-pairing. As potent regulators, miRNAs are involved in mRNA stability and post-transcriptional regulation tasks, being a conserved mechanism used repetitively by the evolution, not only in metazoans, but plants and unicellular organisms. Through a comprehensive revision of the current animal miRNA model, the canonical pathway dominates the extensive literature about miRNAs, and served as a scaffold to understand the scenes behind the regulatory landscape performed by the cell. The characterization of a diverse set of non-canonical pathways has expanded this view, suggesting a diverse, rich and flexible regulatory landscape to generate mature miRNAs. The production of miRNAs, derived from isolated or clustered transcripts, is an efficient and highly conserved mechanism traced back to animals with high fidelity at family level. In evolutionary terms, expansions of miRNA families have been associated with an increasing morphological and developmental complexity. In particular, the Chordata clade (the ancient cephalochordates, highly derived and secondary simplified tunicates, and the well-known vertebrates) represents an interesting scenario to study miRNA evolution. Despite clearly conserved miRNAs along these clades, tunicates display massive restructuring events, including emergence of highly derived miRNAs. As shown in this thesis, model organisms or vertebrate-specific bias exist in current animal miRNA annotations, misrepresenting more diverse groups, such as marine invertebrates. Current miRNA databases, such as miRBase and Rfam, classified miRNAs under different definitions and possessed annotations that are not simple to be linked. As an alternative, this thesis proposes a method to curate and merge those annotations, making use of miRBase precursor/mature annotations and genomes together with Rfam predicted sequences. This approach generated structural models for shared miRNA families, based on the alignment of their correct-positioned mature sequences as anchors. In this process, the developed structural curation steps flagged 33 miRNA families from the Rfam as questionable. Curated Rfam and miRBase anchored-structural alignments provided a rich resource for constructing predictive miRNA profiles, using correspondent hidden Markov (HMMs) and covariance models (CMs). As a direct application, the use of those models is time-consuming, and the user has to deal with multiple iterations to achieve a genome-wide non-overlapping annotation. To resolve this, the proposed miRNAture pipeline provides an automatic and flexible solution to annotate miRNAs. It combines multiple homology approaches to generate the best candidates validated at sequence and structural levels. This increases the achievable sensitivity to annotate canonical miRNAs, and the evaluation against human annotation shows that clear false positive calls are rare and additional counterparts lie in retained-introns, transcribed lncRNAs or repeat families. Further development of miRNAture suggests an inclusion of multiple rules to distinguish non-canonical miRNA families. This thesis describes multiple homology approaches to annotate the genomic information from a non-model chordate: the colonial tunicate Didemnum vexillum. Detected high levels of genetic variance and unexpected levels of DNA degradation were evidenced through a comprehensive analysis of genome-assembly methods and gene annotation. Despite those challenges, it was possible to find candidate homeobox and skeletogenesis- related genes. On its own, the ncRNA annotation included expected conserved families, and an extensive search of the Rhabdomyosarcoma 2-associated transcript (RMST) lncRNA family traced-back at the divergence of deuterostomes. In addition, a complete study of the annotation thresholds suggested variations to detect miRNAs, later implemented on the miRNAture tool. This chapter is a showcase of the usual workflow that should follow comprehensive sequencing, assembly and annotation project, in the light of the increasing research approaching DNA sequencing. In the last 10 years, the remarkable increment in tunicate sequencing projects boosted the access to an expanded miRNA annotation landscape. In this way, a comprehensive homology approach annotated the miRNA complement of 28 deuterostome genomes (including current 16 reported tunicates) using miRNAture. To get proper structural models as input, corrected miRBase structural alignments served as a scaffold for building correspondent CMs, based on a developed genetic algorithm. By this means, this automatic approach selected the set of sequences that composed the alignments, generating 2492 miRNA CMs. Despite the multiple sources and associated heterogeneity of the studied genomes, a clustering approach successfully gathered five groups of similar assemblies and highlighted low quality assemblies. The overall family and loci reduction on tunicates is notorious, showing on average 374 microRNA (miRNA) loci, in comparison to other clades: Cephalochordata (2119), Vertebrata (3638), Hemichordata (1092) and Echinodermata (2737). Detection of 533 miRNA families on the divergence of tunicates shows an expanded landscape regarding currently miRNA annotated families. Shared sets of ancestral, chordates, Olfactores, and specific clade-specific miRNAs were uncovered using a phyloge- netic conservation criteria. Compared to current annotations, the family repertories were expanded in all cases. Finally, relying on the adjacent elements from annotated miRNAs, this thesis proposes an additional syntenic support to cluster miRNA loci. In this way, the structural alignment of miR-1497, originally annotated in three model tunicates, was expanded with a clear syntenic support on tunicates.
... For example, with a 3´tailed mirtron, its 5´-end matches the 5´splice donor and its 3´-end is followed by and unstructured region. Such mirtron intermediates are subjected to tail resection by unclear mechanisms and thus converge with the canonical miRNA biogenesis (Wen et al., 2015). ...
Au cours des 15 dernières années, des centaines de microARN ont été identifiés et proposés comme impliqués dans le contrôle de nombreux processus biologiques, en condition saine ou dans des maladies. Nous avons étudié deux aspects de la biologie des microARN : le rôle biologique d'un microARN perçu comme suppresseur de tumeur, et le contrôle de la stabilité des microARN. Certains microARN ont été définis comme pro-oncogéniques ou suppresseurs de tumeur de par leur rôle dans le contrôle de voies cellulaires critiques dans l'établissement de cancer. Néanmoins, une définition consensuelle d'un microARN suppresseur de tumeur fait toujours défaut. Comme pour les gènes codants, nous proposons que les microARN suppresseurs de tumeur doivent démontrer des signes d'inactivation génétique ou épigénétique dans les cancers et présenter une activité anti-proliférative à des niveaux d'expression endogènes. Dans un premier projet, nous avons testé cette définition avec le microRNA miR-34a, qui a attiré beaucoup d'attention puisqu'il est directement régulé par le facteur de transcription suppresseur de tumeur p53 et est devenu le premier miARN testé en tant que médicament atteignant la phase 1 des essais cliniques en oncologie. Nous avons utilisé des données de séquençage de cancers pour évaluer le niveau d'expression et le statut génétique de miR-34a dans plusieurs types de cancer. Nous avons également réalisé une ablation génétique dans des lignées de cellules cancéreuses afin de mesurer la fonction endogène de ce microRNA sur la prolifération cellulaire et avons examiné en profondeur les études antérieures de surexpression montrant son effet anti-prolifératif pour expliquer les divergences avec nos résultats. En parcourant une grande diversité de types de cancer, il apparaît que miR-34a n'est pas inhibé dans les tumeurs primaires par rapport aux tissus normaux adjacents, et que le locus exprimant ce microARN ne présente pas d'accumulation de mutations dans les cancers. Notre travail montre également que l'activité anti-proliférative établie du miR-34a est basée sur des expériences de surexpression conduisant à des niveaux de microRNA irréalistement élevés. Nos données indiquent finalement que les niveaux endogènes de miR-34a n'ont pas un tel effet et plaident donc contre une fonction tumeur-suppressive pour miR-34a.Les microARN répriment les ARNm, mais réciproquement, les ARNm cibles peuvent également moduler la stabilité des microARN. Dans un second projet, nous avons examiné la régulation endogène des microARN par les ARNm via la dégradation des microARN dirigée par les ARN cibles ou ''target RNA-directed microRNA degradation'' (TDMD). Des cibles artificielles ainsi que des exemples in vivo (des transcrits viraux, l'ARNlnc libra chez le Poisson-zèbre, l'ARNlnc Cyrano chez la Souris) ont permis de démontrer qu'une complémentarité étendue entre un ARN cible et un microARN entraîne la protéolyse du complexe protéique associé au microARN ou ''microRNA-Induced Silencing Complex'' par la voie ubiquitine-protéasome, ce qui expose le microARN à la dégradation. Sur la base des données publiées sur les modèles d'ARN cibles conduisant au TDMD et à l'analyse de la conservation phylogénétique des génomes, nous avons développé un outil informatique permettant l'identification in silico des sites d'ARN qui induisent la dégradation des microARN par TDMD. En le complémentant avec des données de RNA-seq et de small-RNA-seq publiées, notre logiciel permet de faire ressortir les candidats inducteurs de TDMD spécifiques à un type cellulaire. Nos résultats ont permis d'identifier plusieurs candidats convaincants dans les neurones de souris et leur caractérisation moléculaire a été initiée.
... Some miRNAs, however, are generated without DROSHA action. They are encoded within so-called mirtrons, which arise from the spliced introns of the host gene mRNA [14]. In other words, such pre-miRNA is generated as a result of splicing. ...
miRNAs and lncRNAs do not encode proteins, but they play an important role in the regulation of gene expression. They differ in length, biogenesis, and mode of action. In this work, we focus on the selected miRNAs and lncRNAs involved in the regulation of myogenesis and muscle regeneration. We present selected miRNAs and lncRNAs that have been shown to control myogenic differentiation and show that manipulation of their levels could be used to improve myogenic differentiation of various types of stem and progenitor cells. Finally, we discuss how physical activity affects miRNA and lncRNA expression and how it affects muscle well-being.
... Сравнительное исследование репертуаров миртронов у человека и мыши [159] показало, что в этих геномах кодируются по меньшей мере 478 и 488 миртронов, соответственно, из них 13 общих. Из числа общих только 3 имеют консервативную последовательность, а 10 регулируют трансляцию одинаковых по функции генов у этих двух организмов. ...
... Рис. 4. Схема канонических и опосредованных сплайсингом путей биогенеза микроРНК. Заимствовано из [159]. ...
The genomes of large multicellular eukaryotes mainly consist of DNA that encodes not proteins, but RNAs. The unexpected discovery of approximately the same number of protein genes in Homo sapiens and Caenorhabditis elegans led to the understanding that it is not the number of proteins that determines the complexity of the development and functioning of an organism. The phenomenon of pervasive transcription of genomes is finding more and more confirmation. Data are emerging on new types of RNA that work in different cell compartments, are expressed at different stages of development, in different tissues and perform various functions. Their main purpose is fine regulation of the main cellular processes. The presence of a rich arsenal of regulators that can interact with each other and work on the principle of interchangeability determines the physiological complexity of the organism and its ability to adapt to changing environmental conditions. An overview of the currently known functional RNAs expressed in eukaryotic genomes is presented here. There is no doubt that in the near future, using high-tech transcriptome technologies, many new RNAs will be identified and characterized. But it is likely that many of the expressed transcripts do not have a function, but are an evolutionary reserve of organisms.
... Some differentially expressed regions are located inside gene promoters, which often also contain some small RNAs [43]. A significant proportion also comes from introns, which are also known to contain regulatory elements, as well miRNAs [44], and snoRNAs [45]. Fig 6 shows the size distribution of these regions. ...
Small RNAs (sRNAs) encompass a great variety of molecules of different kinds, such as microRNAs, small interfering RNAs, Piwi-associated RNA, among others. These sRNAs have a wide range of activities, which include gene regulation, protection against virus, transposable element silencing, and have been identified as a key actor in determining the development of the cell. Small RNA sequencing is thus routinely used to assess the expression of the diversity of sRNAs, usually in the context of differentially expression, where two conditions are compared. Tools that detect differentially expressed microRNAs are numerous, because microRNAs are well documented, and the associated genes are well defined. However, tools are lacking to detect other types of sRNAs, which are less studied, and whose precursor RNA is not well characterized. We present here a new method, called srnadiff, which finds all kinds of differentially expressed sRNAs. To the extent of our knowledge, srnadiff is the first tool that detects differentially expressed sRNAs without the use of external information, such as genomic annotation or additional sequences of sRNAs.
