| 1. |
- Aslanzadeh, Morteza, et al.
(författare)
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Malat1 affects transcription and splicing through distinct pathways in mouse embryonic stem cells
- 2024
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Ingår i: NAR Genomics and Bioinformatics. - 2631-9268. ; 6:2
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Tidskriftsartikel (refereegranskat)abstract
- Malat1 is a long-noncoding RNA with critical roles in gene regulation and cancer metastasis, however its functional role in stem cells is largely unexplored. We here perform a nuclear knockdown of Malat1 in mouse embryonic stem cells, causing the de-regulation of 320 genes and aberrant splicing of 90 transcripts, some of which potentially affecting the translated protein sequence. We find evidence that Malat1 directly interacts with gene bodies and aberrantly spliced transcripts, and that it locates upstream of down-regulated genes at their putative enhancer regions, in agreement with functional genomics data. Consistent with this, we find these genes affected at both exon and intron levels, suggesting that they are transcriptionally regulated by Malat1. Besides, the down-regulated genes are regulated by specific transcription factors and bear both activating and repressive chromatin marks, suggesting that some of them might be regulated by bivalent promoters. We propose a model in which Malat1 facilitates the transcription of genes involved in chromatid dynamics and mitosis in one pathway, and affects the splicing of transcripts that are themselves involved in RNA processing in a distinct pathway. Lastly, we compare our findings with Malat1 perturbation studies performed in other cell systems and in vivo.
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| 2. |
- Behm, Mikaela, 1986-, et al.
(författare)
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Synaptic expression and regulation of miRNA editing in the brain
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- In the brain, sophisticated networks of RNA regulatory events tightly control gene expression in order to achieve proper brain function. We and others have previously shown that several miRNAs, encoded within the miR-379-410 cluster, are subjected to A-to-I RNA editing. In the present study we conclude these edited miRNAs to be transcribed as a single long consecutive transcript, however the maturation into functional forms of miRNAs is regulated individually. In seven of the miRNAs, subjected to editing, we analyze how editing relates to miRNA maturation. Of particular interest has been maturation of miR-381-3p and miR-376b-3p, both important for neuronal plasticity, dendrite outgrowth and neuronal homeostasis. Most of the edited miRNAs from the cluster, are highly edited in their unprocessed primary transcript, including miR-381-3p and miR-376b-3p. However, editing in miR-381-3p is almost entirely absent in the mature form, while editing is increased in the mature form of miR-376b-3p compared to the primary transcript. We propose that ADAR1 positively influences the maturation of pri-miR-381 in an editing independent manner. In pri-miR-376b we hypothesize that ADAR1 and ADAR2 competes for editing, and while ADAR2 inhibits miRNA maturation, ADAR1 editing is frequently present in the mature miR-376b-3p. We further show that miR-381-3p and miR-376b-3p regulate the dendritically expressed Pumilio 2 (Pum2) protein. By next generation RNA sequencing (NGS RNA-seq) on purified synaptoneurosomes, we show that miR-381-3p is highly expressed at the synapse, suggesting its functional role in locally regulating Pum2. Furthermore, we identify a set of highly expressed miRNAs at the synapse, which may act locally to target synaptic mRNAs.
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| 3. |
- Edelbroek, Bart, et al.
(författare)
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Evolution of microRNAs in Amoebozoa and implications for the origin of multicellularity
- 2024
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 52:6, s. 3121-3136
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Tidskriftsartikel (refereegranskat)abstract
- MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression in both plants and animals. They are thought to have evolved convergently in these lineages and hypothesized to have played a role in the evolution of multicellularity. In line with this hypothesis, miRNAs have so far only been described in few unicellular eukaryotes. Here, we investigate the presence and evolution of miRNAs in Amoebozoa, focusing on species belonging to Acanthamoeba, Physarum and dictyostelid taxonomic groups, representing a range of unicellular and multicellular lifestyles. miRNAs that adhere to both the stringent plant and animal miRNA criteria were identified in all examined amoebae, expanding the total number of protists harbouring miRNAs from 7 to 15. We found conserved miRNAs between closely related species, but the majority of species feature only unique miRNAs. This shows rapid gain and/or loss of miRNAs in Amoebozoa, further illustrated by a detailed comparison between two evolutionary closely related dictyostelids. Additionally, loss of miRNAs in the Dictyostelium discoideum drnB mutant did not seem to affect multicellular development and, hence, demonstrates that the presence of miRNAs does not appear to be a strict requirement for the transition from uni- to multicellular life.
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| 4. |
- Kalogeropoulos, Panagiotis, et al.
(författare)
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Imputation of miRNA activity in single cells from their transcriptome footprint
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- MicroRNAs (miRNAs) are major class of small RNAs that play important role in post-transcriptional gene regulation. There is an increasing interest in profiling miRNAs in single cells for studying their expression heterogeneity; however, all current methods to directly sequence miRNA in single cells have low sensitivity (<5%). Because of Poissonian (sampling) noise, this limits what type of quantitative questions can be addressed using direct sequencing. Here, we investigate if we can - as an alternative approach – indirectly infer activity of miRNAs from their footprint on the transcriptome. Specifically, we want to leverage the high sensitivity (60%) of single-cell RNA-seq data like SmartSeq3 to detect patterns of coordinated repression of the mRNA targets of specific miRNA - thus extracting orthogonal single-cell information on gene regulation from already existing resources. Using our new method micro-imp, we show that activity of miRNAs can be inferred from their footprint of the transcriptome with an accuracy that approaches that of directly sequencing the miRNAs.
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| 5. |
- Kang, Wenjing, 1988-, et al.
(författare)
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MapToCleave : High-throughput profiling of microRNA biogenesis in living cells
- 2021
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 37:7
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Tidskriftsartikel (refereegranskat)abstract
- Previous large-scale studies have uncovered many features that determine the processing of microRNA (miRNA) precursors; however, they have been conducted in vitro. Here, we introduce MapToCleave, a method to simultaneously profile processing of thousands of distinct RNA structures in living cells. We find that miRNA precursors with a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from 20 animal species. We systematically compare the importance of known and novel sequence and structural features and test biogenesis of miRNA precursors from 10 animal and plant species in human cells. Lastly, we provide evidence that the GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryogenic electron microscopy (cryo-EM) studies. In summary, we apply a screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression.
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| 6. |
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| 7. |
- Kang, Wenjing, et al.
(författare)
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miRTrace reveals the organismal origins of microRNA sequencing data
- 2018
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Ingår i: Genome Biology. - : Springer Science and Business Media LLC. - 1465-6906 .- 1474-760X. ; 19
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Tidskriftsartikel (refereegranskat)abstract
- We present here miRTrace, the first algorithm to trace microRNA sequencing data back to their taxonomic origins. This is a challenge with profound implications for forensics, parasitology, food control, and research settings where cross-contamination can compromise results. miRTrace accurately (> 99%) assigns real and simulated data to 14 important animal and plant groups, sensitively detects parasitic infection in mammals, and discovers the primate origin of single cells. Applying our algorithm to over 700 public datasets, we find evidence that over 7% are cross-contaminated and present a novel solution to clean these computationally, even after sequencing has occurred.
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| 8. |
- Mármol-Sánchez, Emilio, et al.
(författare)
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Historical RNA expression profiles from the extinct Tasmanian tiger
- 2023
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Ingår i: Genome Research. - 1088-9051 .- 1549-5469. ; 33:8, s. 1299-1316
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Tidskriftsartikel (refereegranskat)abstract
- Paleogenomics continues to yield valuable insights into the evolution, population dynamics, and ecology of our ancestors and other extinct species. However, DNA sequencing cannot reveal tissue-specific gene expression, cellular identity, or gene regulation, which are only attainable at the transcriptional level. Pioneering studies have shown that useful RNA can be extracted from ancient specimens preserved in permafrost and historical skins from extant canids, but no attempts have been made so far on extinct species. We extract, sequence, and analyze historical RNA from muscle and skin tissue of a ∼130-year-old Tasmanian tiger (Thylacinus cynocephalus) preserved in desiccation at room temperature in a museum collection. The transcriptional profiles closely resemble those of extant species, revealing specific anatomical features such as slow muscle fibers or blood infiltration. Metatranscriptomic analysis, RNA damage, tissue-specific RNA profiles, and expression hotspots genome-wide further confirm the thylacine origin of the sequences. RNA sequences are used to improve protein-coding and noncoding annotations, evidencing missing exonic loci and the location of ribosomal RNA genes while increasing the number of annotated thylacine microRNAs from 62 to 325. We discover a thylacine-specific microRNA isoform that could not have been confirmed without RNA evidence. Finally, we detect traces of RNA viruses, suggesting the possibility of profiling viral evolution. Our results represent the first successful attempt to obtain transcriptional profiles from an extinct animal species, providing thought-to-be-lost information on gene expression dynamics. These findings hold promising implications for the study of RNA molecules across the vast collections of natural history museums and from well-preserved permafrost remains.
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| 9. |
- Melnikova, Larisa, et al.
(författare)
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Long-distance interactions between regulatory elements are suppressed at the end of a terminally deficient chromosome in Drosophila melanogaster.
- 2008
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Ingår i: Chromosoma. - : Springer Science and Business Media LLC. - 0009-5915 .- 1432-0886. ; 117:1, s. 41-50
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Tidskriftsartikel (refereegranskat)abstract
- In Drosophila melanogaster, broken chromosome ends behave as real telomeres and are believed to be covered with telomere-specific chromatin. It has been shown previously that the telomeric chromatin represses normal activity of enhancers that regulate yellow expression in wings and body cuticle. In this paper, we have found that a modified yellow promoter is fully active in the wing and body cuticle when it is located at the chromosome end, which is evidence that the telomeric chromatin does not repress transcription. Substitution of the yellow core promoter region, including TATA and Inr, with the promoter regions of the eve, hsp70 (TATA-containing), and white (TATA-less) promoters does not affect the ability of the promoter to be cis- or trans-activated by the yellow enhancers if the heterologous promoter is located at a distance of about 6 kb from the chromosome end. The best characterized Drosophila insulator found in the gypsy retrotransposon can specifically repress the yellow promoter at a distance when one component of the insulator complex, Mod(mdg4)-67.2 protein, is inactive. We have also found that, in the mod(mdg4) mutant background, the gypsy insulator can repress the heterologous promoters, indicating that the core promoter elements are not critical for specificity of repression. However, long-distance functional enhancer-promoter and gypsy-promoter interactions were suppressed when the distance between the yellow promoter and the end of the deficient chromosome was less than 6 kb. These results suggest that Drosophila telomeric chromatin does not generally repress transcription but is somehow involved in suppression of some long-distance interactions between regulatory elements.
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| 10. |
- Mohammed, Mubasher, Msc, et al.
(författare)
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Single-Cell Transcriptomics To Define Plasmodium falciparum Stage Transition in the Mosquito Midgut
- 2023
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Ingår i: Microbiology Spectrum. - : American Society for Microbiology. - 2165-0497. ; 11:2
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Tidskriftsartikel (refereegranskat)abstract
- Malaria inflicts the highest rate of morbidity and mortality among the vector-borne diseases. The dramatic bottleneck of parasite numbers that occurs in the gut of the obligatory mosquito vector provides a promising target for novel control strategies. Using single-cell transcriptomics, we analyzed Plasmodium falciparum development in the mosquito gut, from unfertilized female gametes through the first 20 h after blood feeding, including the zygote and ookinete stages. This study revealed the temporal gene expression of the ApiAP2 family of transcription factors and of parasite stress genes in response to the harsh environment of the mosquito midgut. Further, employing structural protein prediction analyses, we found several upregulated genes predicted to encode intrinsically disordered proteins (IDPs), a category of proteins known for their importance in regulation of transcription, translation, and protein-protein interactions. IDPs are known for their antigenic properties and may serve as suitable targets for antibody- or peptide-based transmission suppression strategies. In total, this study uncovers the P. falciparum transcriptome from early to late parasite development in the mosquito midgut, inside its natural vector, which provides an important resource for future malaria transmission-blocking initiatives.
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| 11. |
- Mozūraitis, Raimondas, et al.
(författare)
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Male swarming aggregation pheromones increase female attraction and mating success among multiple African malaria vector mosquito species
- 2020
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Ingår i: Nature Ecology & Evolution. - : Nature Research. - 2397-334X. ; 4:10, s. 1395-1401
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Tidskriftsartikel (refereegranskat)abstract
- Accumulating behavioural data indicate that aggregation pheromones may mediate the formation and maintenance of mosquito swarms. However, chemical cues possibly luring mosquitoes to swarms have not been adequately investigated, and the likely molecular incitants of these complex reproductive behaviours remain unknown. Here we show that males of the important malaria vector species Anopheles arabiensis and An. gambiae produce and release aggregation pheromones that attract individuals to the swarm and enhance mating success. We found that males of both species released significantly higher amounts of 3-hydroxy-2-butanone (acetoin), 6-methyl-5-hepten-2-one (sulcatone), octanal, nonanal and decanal during swarming in the laboratory. Feeding males with stable-isotope-labelled glucose revealed that the males produced these five compounds. A blend composed of synthetic analogues to these swarming odours proved highly attractive to virgin males and females of both species under laboratory conditions and substantially increased mating in five African malaria vectors (An. gambiae,An. coluzzii,An. arabiensis,An. merus and An. funestus) in semi-field experiments. Our results not only narrow a conspicuous gap in understanding a vital aspect of the chemical ecology of male mosquitoes but also demonstrate fundamental roles of rhythmic and metabolic genes in the physiology and behavioural regulation of these vectors. These identified aggregation pheromones have great potential for exploitation against these highly dangerous insects. Manipulating such pheromones could increase the efficacy of malaria-vector control programmes.
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| 12. |
- Sekar, Vaishnovi, 1993-, et al.
(författare)
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Detection of transcriptome-wide microRNA-target interactions in single cells with agoTRIBE
- 2024
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Ingår i: Nature Biotechnology. - 1087-0156 .- 1546-1696. ; , s. 1296-1302
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Tidskriftsartikel (refereegranskat)abstract
- MicroRNAs (miRNAs) exert their gene regulatory effects on numerous biological processes based on their selection of target transcripts. Current experimental methods available to identify miRNA targets are laborious and require millions of cells. Here we have overcome these limitations by fusing the miRNA effector protein Argonaute2 to the RNA editing domain of ADAR2, allowing the detection of miRNA targets transcriptome-wide in single cells. miRNAs guide the fusion protein to their natural target transcripts, causing them to undergo A>I editing, which can be detected by sensitive single-cell RNA sequencing. We show that agoTRIBE identifies functional miRNA targets, which are supported by evolutionary sequence conservation. In one application of the method we study microRNA interactions in single cells and identify substantial differential targeting across the cell cycle. AgoTRIBE also provides transcriptome-wide measurements of RNA abundance and allows the deconvolution of miRNA targeting in complex tissues at the single-cell level.
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| 13. |
- Sekar, Vaishnovi, et al.
(författare)
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microRNA targeting variability in single cells from a homogenous cell population
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally. miRNAs have distinct expression profiles across tissues and cell types, and there is even evidence that miRNAs can vary in abundance between individual cells in otherwise homogenous cell populations. However, due to a paucity of single-cell methods to measure miRNA targeting, the functional implications of these variations have not been studied transcriptome-wide. In this study, we re-analyze our published agoTRIBE single-cell targeting data from HEK293T cells, to investigate if single cells from a homogenous population display differences in miRNA targeting. We find that two cell populations display distinct targeting patterns, with one population being targeted specifically by miR-10 and miR-16. Transcripts targeted in one population have functions in biosynthesis and chromatin organization, while transcripts in the other population have functions in various stress responses. In summary, we find that miRNAs appear to have overlapping but also distinct functions in two subpopulations of cells in an otherwise homogenous human cell culture.
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| 14. |
- Tarbier, Marcel, 1990-, et al.
(författare)
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Landscape of microRNA and target expression variation and covariation in single mouse embryonic stem cells
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- MicroRNAs are small RNA molecules that can repress the expression of protein coding genes post-transcriptionally. Previous studies have shown that microRNAs can also have alternative functions including target noise buffering and co-expression, but these observations have been limited to a few microRNAs. Here we systematically study microRNA alternative functions in mouse embryonic stem cells, by genetically deleting Drosha - leading to global loss of microRNAs. We apply complementary single-cell RNA-seq methods to study the variation of the targets and the microRNAs themselves, and transcriptional inhibition to measure target half-lives. We find that microRNAs form four distinct co-expression groups across single cells. In particular the mir-290 and the mir-182 clusters are abundantly, variably and inversely expressed. Intriguingly, some cells have global biases towards specific miRNAs originating from either end of the hairpin precursor, suggesting the presence of unknown regulatory cofactors. We find that miRNAs generally increase variation and covariation of their targets at the RNA level, but we also find miRNAs such as miR-182 that appear to have opposite functions. In particular, miRNAs that are themselves variable in expression, such as miR-291a, are more likely to induce covariations. In summary, we apply genetic perturbation and multi-omics to give the first global picture of microRNA dynamics at the single cell level.
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| 15. |
- Tarbier, Marcel, et al.
(författare)
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Single-cell sequencing reveals heterogeneity in miRNA biogenesis and function
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- MiRNAs are ~22 nucleotide small RNAs that repress protein coding genes post- transcriptionally. They have been studied extensively in bulk studies that pool hundreds of thousands of cells, but there is a paucity of studies of their biogenesis and function in single cells. Here we present single-cell small RNA sequencing (sc- sRNAseq) data from 192 individual mouse embryonic stem cells. We find that some miRNAs are stably expressed across cells, while others have variable expression. In particular, we find that the miR-290 cluster, which promotes progression through the cell cycle and proliferation, and the miR-182/183 cluster, which targets differentiation factors, are negatively correlated. We also find that some cells have global biases towards 5’ or 3’ miRNA, suggesting the presence of protein cofactors. Complementing our data with single-cell mRNA sequencing and protein data from other studies in embryonic stem cells, we find that miRNAs generally increase variation of their targets at the RNA level, but we find examples of miRNAs that buffer variation of their targets at the protein level. In summary, we integrate single- cell gene expression data of small RNA, mRNA and protein to give new insights into miRNA biogenesis, dynamics and functions at the cell level.
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| 16. |
- Widmark, Albin, 1991-, et al.
(författare)
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ADAR1-and ADAR2-mediated regulation of maturation and targeting of miR-376b to modulate GABA neurotransmitter catabolism
- 2022
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 298:3
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Tidskriftsartikel (refereegranskat)abstract
- miRNAs are short noncoding RNA molecules that regulate gene expression by inhibiting translation or inducing degradation of target mRNAs. miRNAs are often expressed as polycistronic transcripts, so-called miRNA clusters, containing several miRNA precursors. The largest mammalian miRNA cluster, the miR-379-410 cluster, is expressed primarily during embryonic development and in the adult brain; however, downstream regulation of this cluster is not well understood. Here, we investigated adenosine deamination to inosine (RNA editing) in the miR-379-410 cluster by adenosine deaminase acting on RNA (ADAR) enzymes as a possible mechanism modulating the expression and activity of these miRNAs in a brain-specific manner. We show that the levels of editing in the majority of mature miRNAs are lower than the editing levels of the corresponding site in primary miRNA precursors. However, for one miRNA, miR-376b-3p, editing was significantly higher in the mature form than in the primary precursor. We found miR-376b-3p maturation is negatively regulated by ADAR2 in an editing activity-independent manner, whereas ADAR1-mediated and ADAR2-mediated editing were observed to be competitive. In addition, the edited miR-376b-3p targets a different set of mRNAs than unedited miR-376b-3p, including 4-aminobutyrate aminotransferase, encoding the enzyme responsible for the catabolism of the neurotransmitter gamma aminobutyric acid (GABA). Expression of edited miR-376b-3p led to increased intracellular GABA levels as well as increased cell surface presentation of GABA type A receptors. Our results indicate that both editing and editing-independent effects modulate the expression of miR-376b-3p, with the potential to regulate GABAergic signaling in the brain.
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| 17. |
- Widmark, Albin, 1991-, et al.
(författare)
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ADAR1- and ADAR2-mediated regulation of miR-376b maturation and targeting modulates GABA catabolism
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate gene expression by inhibiting translation or inducing degradation of target mRNAs. miRNAs are often expressed as polycistronic transcripts, so-called miRNA clusters, where several miRNA precursors are present in the same transcript. The largest mammalian miRNA cluster, the miR-379-410 cluster, is expressed primarily during embryonic development and in the adult brain. We investigated adenosine-to-inosine (A-to-I) RNA editing by the adenosine deaminase acting on RNA (ADAR) enzymes of the miR-379-410 cluster as a possible mechanism of modulating expression and activity of the miRNAs in a brain-specific manner. We show that editing levels of the majority of mature miRNAs are lower than the editing level of the corresponding site in primary miRNA (pri-miRNA) precursors. However, for one miRNA, miR-376b-3p, editing was significantly higher in the mature form than in the primary precursor. miR-376b-3p maturation is negatively regulated by ADAR2 in an editing-independent manner, while ADAR1 and ADAR2 editing was observed to be competitive. The edited miR-376b-3p targets a different set of mRNAs than unedited miR-376b-3p, including 4-aminobutyrate aminotransferase (Abat), the enzyme responsible for the catabolism of the neurotransmitter GABA. Expression of edited miR-376b-3p led to increased intracellular GABA levels, as well as increased cell surface presentation of GABAA receptors, previously shown to be dependent on intracellular GABA levels. Our results indicate that editing and editing-independent effects modulates the expression of miR-376b-3p, with the potential to regulate GABAergic signaling in the brain.
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