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AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading

RNA interference (RNAi) is mediated by small, 20-24-nt-long, non-coding regulatory (s)RNAs such as micro (mi) and small interfering (si) RNAs via the action of ARGONAUTE (AGO) proteins. High-throughput sequencing of size-separated sRNA pools of plant crude extracts revealed that the majority of the...

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Autores principales: Dalmadi, Ágnes, Gyula, Péter, Bálint, Jeannette, Szittya, György, Havelda, Zoltán
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765109/
https://www.ncbi.nlm.nih.gov/pubmed/31392979
http://dx.doi.org/10.1093/nar/gkz690
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author Dalmadi, Ágnes
Gyula, Péter
Bálint, Jeannette
Szittya, György
Havelda, Zoltán
author_facet Dalmadi, Ágnes
Gyula, Péter
Bálint, Jeannette
Szittya, György
Havelda, Zoltán
author_sort Dalmadi, Ágnes
collection PubMed
description RNA interference (RNAi) is mediated by small, 20-24-nt-long, non-coding regulatory (s)RNAs such as micro (mi) and small interfering (si) RNAs via the action of ARGONAUTE (AGO) proteins. High-throughput sequencing of size-separated sRNA pools of plant crude extracts revealed that the majority of the canonical miRNAs were associated with high molecular weight RNA-induced silencing complexes co-migrating with AGO1 (HMW RISC). In contrast, the majority of 24-nt-long siRNAs were found in association with low molecular weight complexes co-migrating with AGO4 (LMW RISC). Intriguingly, we identified a large set of cytoplasmic sRNAs, including mature miRNA sequences, in the low molecular size range corresponding to protein-unbound sRNAs. By comparing the RISC-loaded and protein-unbound pools of miRNAs, we identified miRNAs with highly different loading efficiencies. Expression of selected miRNAs in transient and transgenic systems validated their altered loading abilities implying that this process is controlled by information associated with the diverse miRNA precursors. We also showed that the availability of AGO proteins is a limiting factor determining the loading efficiency of miRNAs. Our data reveal the existence of a regulatory checkpoint determining the RISC-loading efficiencies of various miRNAs by sorting only a subset of the produced miRNAs into the biologically active RISCs.
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spelling pubmed-67651092019-10-02 AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading Dalmadi, Ágnes Gyula, Péter Bálint, Jeannette Szittya, György Havelda, Zoltán Nucleic Acids Res RNA and RNA-protein complexes RNA interference (RNAi) is mediated by small, 20-24-nt-long, non-coding regulatory (s)RNAs such as micro (mi) and small interfering (si) RNAs via the action of ARGONAUTE (AGO) proteins. High-throughput sequencing of size-separated sRNA pools of plant crude extracts revealed that the majority of the canonical miRNAs were associated with high molecular weight RNA-induced silencing complexes co-migrating with AGO1 (HMW RISC). In contrast, the majority of 24-nt-long siRNAs were found in association with low molecular weight complexes co-migrating with AGO4 (LMW RISC). Intriguingly, we identified a large set of cytoplasmic sRNAs, including mature miRNA sequences, in the low molecular size range corresponding to protein-unbound sRNAs. By comparing the RISC-loaded and protein-unbound pools of miRNAs, we identified miRNAs with highly different loading efficiencies. Expression of selected miRNAs in transient and transgenic systems validated their altered loading abilities implying that this process is controlled by information associated with the diverse miRNA precursors. We also showed that the availability of AGO proteins is a limiting factor determining the loading efficiency of miRNAs. Our data reveal the existence of a regulatory checkpoint determining the RISC-loading efficiencies of various miRNAs by sorting only a subset of the produced miRNAs into the biologically active RISCs. Oxford University Press 2019-10-10 2019-08-08 /pmc/articles/PMC6765109/ /pubmed/31392979 http://dx.doi.org/10.1093/nar/gkz690 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle RNA and RNA-protein complexes
Dalmadi, Ágnes
Gyula, Péter
Bálint, Jeannette
Szittya, György
Havelda, Zoltán
AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading
title AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading
title_full AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading
title_fullStr AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading
title_full_unstemmed AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading
title_short AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading
title_sort ago-unbound cytosolic pool of mature mirnas in plant cells reveals a novel regulatory step at ago1 loading
topic RNA and RNA-protein complexes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765109/
https://www.ncbi.nlm.nih.gov/pubmed/31392979
http://dx.doi.org/10.1093/nar/gkz690
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