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1
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Marturano G, Carli D, Cucini C, Carapelli A, Plazzi F, Frati F, Passamonti M, Nardi F. SmithHunter: a workflow for the identification of candidate smithRNAs and their targets. BMC Bioinformatics 2024; 25:286. [PMID: 39223476 PMCID: PMC11370224 DOI: 10.1186/s12859-024-05909-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2024] [Accepted: 08/21/2024] [Indexed: 09/04/2024] Open
Abstract
BACKGROUND SmithRNAs (Small MITochondrial Highly-transcribed RNAs) are a novel class of small RNA molecules that are encoded in the mitochondrial genome and regulate the expression of nuclear transcripts. Initial evidence for their existence came from the Manila clam Ruditapes philippinarum, where they have been described and whose activity has been biologically validated through RNA injection experiments. Current evidence on the existence of these RNAs in other species is based only on small RNA sequencing. As a preliminary step to characterize smithRNAs across different metazoan lineages, a dedicated, unified, analytical workflow is needed. RESULTS We propose a novel workflow specifically designed for smithRNAs. Sequence data (from small RNA sequencing) uniquely mapping to the mitochondrial genome are clustered into putative smithRNAs and prefiltered based on their abundance, presence in replicate libraries and 5' and 3' transcription boundary conservation. The surviving sequences are subsequently compared to the untranslated regions of nuclear transcripts based on seed pairing, overall match and thermodynamic stability to identify possible targets. Ample collateral information and graphics are produced to help characterize these molecules in the species of choice and guide the operator through the analysis. The workflow was tested on the original Manila clam data. Under basic settings, the results of the original study are largely replicated. The effect of additional parameter customization (clustering threshold, stringency, minimum number of replicates, seed matching) was further evaluated. CONCLUSIONS The study of smithRNAs is still in its infancy and no dedicated analytical workflow is currently available. At its core, the SmithHunter workflow builds over the bioinformatic procedure originally applied to identify candidate smithRNAs in the Manila clam. In fact, this is currently the only evidence for smithRNAs that has been biologically validated and, therefore, the elective starting point for characterizing smithRNAs in other species. The original analysis was readapted using current software implementations and some minor issues were solved. Moreover, the workflow was improved by allowing the customization of different analytical parameters, mostly focusing on stringency and the possibility of accounting for a minimal level of genetic differentiation among samples.
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Affiliation(s)
| | - Diego Carli
- Department of Biological, Geological and Environmental Sciences, University of Bologna, Via Selmi 3, 40126, Bologna, Italy
| | - Claudio Cucini
- Department of Life Sciences, University of Siena, 53100, Siena, Italy
| | - Antonio Carapelli
- Department of Life Sciences, University of Siena, 53100, Siena, Italy
- National Biodiversity Future Center (NBFC), 90133, Palermo, Italy
| | - Federico Plazzi
- Department of Biological, Geological and Environmental Sciences, University of Bologna, Via Selmi 3, 40126, Bologna, Italy
| | - Francesco Frati
- Department of Life Sciences, University of Siena, 53100, Siena, Italy
- National Biodiversity Future Center (NBFC), 90133, Palermo, Italy
| | - Marco Passamonti
- Department of Biological, Geological and Environmental Sciences, University of Bologna, Via Selmi 3, 40126, Bologna, Italy.
| | - Francesco Nardi
- Department of Life Sciences, University of Siena, 53100, Siena, Italy
- National Biodiversity Future Center (NBFC), 90133, Palermo, Italy
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2
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Muneretto G, Plazzi F, Passamonti M. Mitochondrion-to-nucleus communication mediated by RNA export: a survey of potential mechanisms and players across eukaryotes. Biol Lett 2024; 20:20240147. [PMID: 38982851 PMCID: PMC11283861 DOI: 10.1098/rsbl.2024.0147] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2024] [Revised: 05/16/2024] [Accepted: 06/06/2024] [Indexed: 07/11/2024] Open
Abstract
The nucleus interacts with the other organelles to perform essential functions of the eukaryotic cell. Mitochondria have their own genome and communicate back to the nucleus in what is known as mitochondrial retrograde response. Information is transferred to the nucleus in many ways, leading to wide-ranging changes in nuclear gene expression and culminating with changes in metabolic, regulatory or stress-related pathways. RNAs are emerging molecules involved in this signalling. RNAs encode precise information and are involved in highly target-specific signalling, through a wide range of processes known as RNA interference. RNA-mediated mitochondrial retrograde response requires these molecules to exit the mitochondrion, a process that is still mostly unknown. We suggest that the proteins/complexes translocases of the inner membrane, polynucleotide phosphorylase, mitochondrial permeability transition pore, and the subunits of oxidative phosphorylation complexes may be responsible for RNA export.
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Affiliation(s)
- Giorgio Muneretto
- Department of Biological, Geological and Environmental Sciences, University of Bologna, Bologna, Italy
| | - Federico Plazzi
- Department of Biological, Geological and Environmental Sciences, University of Bologna, Bologna, Italy
| | - Marco Passamonti
- Department of Biological, Geological and Environmental Sciences, University of Bologna, Bologna, Italy
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3
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Plazzi F, Le Cras Y, Formaggioni A, Passamonti M. Mitochondrially mediated RNA interference, a retrograde signaling system affecting nuclear gene expression. Heredity (Edinb) 2024; 132:156-161. [PMID: 37714959 PMCID: PMC10923801 DOI: 10.1038/s41437-023-00650-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2023] [Revised: 08/30/2023] [Accepted: 08/31/2023] [Indexed: 09/17/2023] Open
Abstract
Several functional classes of short noncoding RNAs are involved in manifold regulatory processes in eukaryotes, including, among the best characterized, miRNAs. One of the most intriguing regulatory networks in the eukaryotic cell is the mito-nuclear crosstalk: recently, miRNA-like elements of mitochondrial origin, called smithRNAs, were detected in a bivalve species, Ruditapes philippinarum. These RNA molecules originate in the organelle but were shown in vivo to regulate nuclear genes. Since miRNA genes evolve easily de novo with respect to protein-coding genes, in the present work we estimate the probability with which a newly arisen smithRNA finds a suitable target in the nuclear transcriptome. Simulations with transcriptomes of 12 bivalve species suggest that this probability is high and not species specific: one in a hundred million (1 × 10-8) if five mismatches between the smithRNA and the 3' mRNA are allowed, yet many more are allowed in animals. We propose that novel smithRNAs may easily evolve as exaptation of the pre-existing mitochondrial RNAs. In turn, the ability of evolving novel smithRNAs may have played a pivotal role in mito-nuclear interactions during animal evolution, including the intriguing possibility of acting as speciation trigger.
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Affiliation(s)
- Federico Plazzi
- Department of Biological, Geological and Environmental Sciences, University of Bologna, via Selmi, 3 - 40126, Bologna, BO, Italy.
| | - Youn Le Cras
- Department of Biological, Geological and Environmental Sciences, University of Bologna, via Selmi, 3 - 40126, Bologna, BO, Italy
- Magistère Européen de Génétique, Université Paris Cité, 85 Boulevard Saint Germain, 75006, Paris, Italy
| | - Alessandro Formaggioni
- Department of Biological, Geological and Environmental Sciences, University of Bologna, via Selmi, 3 - 40126, Bologna, BO, Italy
| | - Marco Passamonti
- Department of Biological, Geological and Environmental Sciences, University of Bologna, via Selmi, 3 - 40126, Bologna, BO, Italy
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4
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Lubośny M, Śmietanka B, Lasota R, Burzyński A. Confirmation of the first intronic sequence in the bivalvian mitochondrial genome of Macoma balthica (Linnaeus, 1758). Biol Lett 2022; 18:20220275. [PMID: 36196553 PMCID: PMC9532982 DOI: 10.1098/rsbl.2022.0275] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2022] [Accepted: 09/12/2022] [Indexed: 11/12/2022] Open
Abstract
In 2020, the first male-type mitochondrial genome from the clam Macoma balthica was published. Apart from the unusual doubly uniparental inheritance of mtDNA, scientists observed a unique (over 4k bp long) extension in the middle of the cox2 gene. We have attempted to replicate these data by NGS DNA sequencing and explore further the expression of the long cox2 gene. In our study, we report an even longer cox2 gene (over 5.5 kbp) with no stop codon separating conserved cox2 domains, as well as, based on the rtPCR, a lower relative gene expression pattern of the middle part of the gene (5' = 1; mid = 0.46; 3' = 0.89). Lastly, we sequenced the cox2 gene transcript proving the excision of the intronic sequence.
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Affiliation(s)
- Marek Lubośny
- Department of Genetics and Marine Biotechnology, Institute of Oceanology Polish Academy of Sciences, Sopot 81-712, Poland
| | - Beata Śmietanka
- Department of Genetics and Marine Biotechnology, Institute of Oceanology Polish Academy of Sciences, Sopot 81-712, Poland
| | - Rafał Lasota
- Faculty of Oceanography and Geography, Division of Marine Ecosystems Functioning, University of Gdańsk, Gdynia 81-378, Poland
| | - Artur Burzyński
- Department of Genetics and Marine Biotechnology, Institute of Oceanology Polish Academy of Sciences, Sopot 81-712, Poland
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5
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Mito-nuclear coevolution and phylogenetic artifacts: the case of bivalve mollusks. Sci Rep 2022; 12:11040. [PMID: 35773462 PMCID: PMC9247169 DOI: 10.1038/s41598-022-15076-y] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2022] [Accepted: 06/17/2022] [Indexed: 11/08/2022] Open
Abstract
Mito-nuclear phylogenetic discordance in Bivalvia is well known. In particular, the monophyly of Amarsipobranchia (Heterodonta + Pteriomorphia), retrieved from mitochondrial markers, contrasts with the monophyly of Heteroconchia (Heterodonta + Palaeoheterodonta), retrieved from nuclear markers. However, since oxidative phosphorylation nuclear markers support the Amarsipobranchia hypothesis instead of the Heteroconchia one, interacting subunits of the mitochondrial complexes ought to share the same phylogenetic signal notwithstanding the genomic source, which is different from the signal obtained from other nuclear markers. This may be a clue of coevolution between nuclear and mitochondrial genes. In this work we inferred the phylogenetic signal from mitochondrial and nuclear oxidative phosphorylation markers exploiting different phylogenetic approaches and added two more datasets for comparison: genes of the glycolytic pathway and genes related to the biogenesis of regulative small noncoding RNAs. All trees inferred from mitochondrial and nuclear subunits of the mitochondrial complexes support the monophyly of Amarsipobranchia, regardless of the phylogenetic pipeline. However, not every single marker agrees with this topology: this is clearly visible in nuclear subunits that do not directly interact with the mitochondrial counterparts. Overall, our data support the hypothesis of a coevolution between nuclear and mitochondrial genes for the oxidative phosphorylation. Moreover, we suggest a relationship between mitochondrial topology and different nucleotide composition between clades, which could be associated to the highly variable gene arrangement in Bivalvia.
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6
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Lubośny M, Śmietanka B, Arculeo M, Burzyński A. No evidence of DUI in the Mediterranean alien species Brachidontes pharaonis (P. Fisher, 1870) despite mitochondrial heteroplasmy. Sci Rep 2022; 12:8569. [PMID: 35595866 PMCID: PMC9122905 DOI: 10.1038/s41598-022-12606-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2021] [Accepted: 05/13/2022] [Indexed: 01/05/2023] Open
Abstract
Two genetically different mitochondrial haplogroups of Brachidontes pharaonis (p-distance 6.8%) have been identified in the Mediterranean Sea. This hinted at a possible presence of doubly uniparental inheritance in this species. To ascertain this possibility, we sequenced two complete mitogenomes of Brachidontes pharaonis mussels and performed a qPCR analysis to measure the relative mitogenome copy numbers of both mtDNAs. Despite the presence of two very similar regions composed entirely of repetitive sequences in the two haplogroups, no recombination between mitogenomes was detected. In heteroplasmic individuals, both mitogenomes were present in the generative tissues of both sexes, which argues against the presence of doubly uniparental inheritance in this species.
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Affiliation(s)
- Marek Lubośny
- Department of Genetics and Marine Biotechnology, Institute of Oceanology Polish Academy of Sciences, Sopot, Poland.
| | - Beata Śmietanka
- Department of Genetics and Marine Biotechnology, Institute of Oceanology Polish Academy of Sciences, Sopot, Poland
| | - Marco Arculeo
- Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Palermo, Italy
| | - Artur Burzyński
- Department of Genetics and Marine Biotechnology, Institute of Oceanology Polish Academy of Sciences, Sopot, Poland
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7
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OUP accepted manuscript. Zool J Linn Soc 2022. [DOI: 10.1093/zoolinnean/zlac047] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022]
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8
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Maeda GP, Iannello M, McConie HJ, Ghiselli F, Havird JC. Relaxed selection on male mitochondrial genes in DUI bivalves eases the need for mitonuclear coevolution. J Evol Biol 2021; 34:1722-1736. [PMID: 34533872 DOI: 10.1111/jeb.13931] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2021] [Revised: 08/21/2021] [Accepted: 09/05/2021] [Indexed: 12/31/2022]
Abstract
Mitonuclear coevolution is an important prerequisite for efficient energy production in eukaryotes. However, many bivalve taxa experience doubly uniparental inheritance (DUI) and have sex-specific mitochondrial (mt) genomes, providing a challenge for mitonuclear coevolution. We examined possible mechanisms to reconcile mitonuclear coevolution with DUI. No nuclear-encoded, sex-specific OXPHOS paralogs were found in the DUI clam Ruditapes philippinarum, refuting OXPHOS paralogy as a solution in this species. It is also unlikely that mt changes causing disruption of nuclear interactions are strongly selected against because sex-specific mt-residues or those under positive selection in M mt genes were not depleted for contacting nuclear-encoded residues. However, M genomes showed consistently higher dN /dS ratios compared to putatively ancestral F genomes in all mt OXPHOS genes and across all DUI species. Further analyses indicated that this was consistently due to relaxed, not positive selection on M vs. F mt OXPHOS genes. Similarly, selection was relaxed on the F genome of DUI species compared to species with strict maternal inheritance. Coupled with recent physiological and molecular evolution studies, we suggest that relaxed selection on M mt function limits the need to maintain mitonuclear interactions in M genomes compared to F genomes. We discuss our findings with regard to OXPHOS function and the origin of DUI.
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Affiliation(s)
- Gerald P Maeda
- Department of Integrative Biology, The University of Texas at Austin, Austin, Texas, USA
| | - Mariangela Iannello
- Department of Biological, Geological, and Environmental Sciences, University of Bologna, Bologna, Italy
| | - Hunter J McConie
- Department of Integrative Biology, The University of Texas at Austin, Austin, Texas, USA
| | - Fabrizio Ghiselli
- Department of Biological, Geological, and Environmental Sciences, University of Bologna, Bologna, Italy
| | - Justin C Havird
- Department of Integrative Biology, The University of Texas at Austin, Austin, Texas, USA
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9
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Lubośny M, Przyłucka A, Śmietanka B, Burzyński A. Semimytilus algosus: first known hermaphroditic mussel with doubly uniparental inheritance of mitochondrial DNA. Sci Rep 2020; 10:11256. [PMID: 32647112 PMCID: PMC7347871 DOI: 10.1038/s41598-020-67976-6] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2020] [Accepted: 06/11/2020] [Indexed: 11/08/2022] Open
Abstract
Doubly uniparental inheritance (DUI) of mitochondrial DNA is a rare phenomenon occurring in some freshwater and marine bivalves and is usually characterized by the mitochondrial heteroplasmy of male individuals. Previous research on freshwater Unionida mussels showed that hermaphroditic species do not have DUI even if their closest gonochoristic counterparts do. No records showing DUI in a hermaphrodite have ever been reported. Here we show for the first time that the hermaphroditic mussel Semimytilus algosus (Mytilida), very likely has DUI, based on the complete sequences of both mitochondrial DNAs and the distribution of mtDNA types between male and female gonads. The two mitogenomes show considerable divergence (34.7%). The presumably paternal M type mitogenome dominated the male gonads of most studied mussels, while remaining at very low or undetectable levels in the female gonads of the same individuals. If indeed DUI can function in the context of simultaneous hermaphroditism, a change of paradigm regarding its involvement in sex determination is needed. It is apparently associated with gonadal differentiation rather than with sex determination in bivalves.
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Affiliation(s)
- Marek Lubośny
- Department of Genetics and Marine Biotechnology, Institute of Oceanology Polish Academy of Sciences, Sopot, Poland.
| | - Aleksandra Przyłucka
- Department of Genetics and Marine Biotechnology, Institute of Oceanology Polish Academy of Sciences, Sopot, Poland
| | - Beata Śmietanka
- Department of Genetics and Marine Biotechnology, Institute of Oceanology Polish Academy of Sciences, Sopot, Poland
| | - Artur Burzyński
- Department of Genetics and Marine Biotechnology, Institute of Oceanology Polish Academy of Sciences, Sopot, Poland
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10
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Clues of in vivo nuclear gene regulation by mitochondrial short non-coding RNAs. Sci Rep 2020; 10:8219. [PMID: 32427953 PMCID: PMC7237437 DOI: 10.1038/s41598-020-65084-z] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2019] [Accepted: 04/22/2020] [Indexed: 12/15/2022] Open
Abstract
Gene expression involves multiple processes, from transcription to translation to the mature, functional peptide, and it is regulated at multiple levels. Small RNA molecules are known to bind RNA messengers affecting their fate in the cytoplasm (a process generically termed ‘RNA interference’). Such small regulatory RNAs are well-known to be originated from the nuclear genome, while the role of mitochondrial genome in RNA interference was largely overlooked. However, evidence is growing that mitochondrial DNA does provide the cell a source of interfering RNAs. Small mitochondrial highly transcribed RNAs (smithRNAs) have been proposed to be transcribed from the mitochondrion and predicted to regulate nuclear genes. Here, for the first time, we show in vivo clues of the activity of two smithRNAs in the Manila clam, Ruditapes philippinarum. Moreover, we show that smithRNAs are present and can be annotated in representatives of the three main bilaterian lineages; in some cases, they were already described and assigned to a small RNA category (e.g., piRNAs) given their biogenesis, while in other cases their biogenesis remains unclear. If mitochondria may affect nuclear gene expression through RNA interference, this opens a plethora of new possibilities for them to interact with the nucleus and makes metazoan mitochondrial DNA a much more complex genome than previously thought.
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11
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Lucentini L, Plazzi F, Sfriso AA, Pizzirani C, Sfriso A, Chiesa S. Additional taxonomic coverage of the doubly uniparental inheritance in bivalves: Evidence of sex‐linked heteroplasmy in the razor clam
Solen marginatus
Pulteney, 1799, but not in the lagoon cockle
Cerastoderma glaucum
(Bruguière, 1789). J ZOOL SYST EVOL RES 2020. [DOI: 10.1111/jzs.12386] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Affiliation(s)
- Livia Lucentini
- Department of Chemistry, Biology and Biotechnologies University of Perugia Perugia Italy
| | - Federico Plazzi
- Department of Biological, Geological and Environmental Sciences University of Bologna Bologna Italy
| | - Andrea Augusto Sfriso
- Department of Chemical and Pharmaceuticals Sciences University of Ferrara Ferrara Italy
| | - Claudia Pizzirani
- Department of Chemistry, Biology and Biotechnologies University of Perugia Perugia Italy
| | - Adriano Sfriso
- Department of Environmental Sciences, Informatics and Statistics Ca' Foscari University of Venice Venice Italy
| | - Stefania Chiesa
- Department of Molecular Sciences and Nanosystems Ca' Foscari University of Venice Venice Italy
- ISPRA Institute for Environmental Protection and Research Rome Italy
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