title: How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing.
abstract: Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzyme's activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.
authors.affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland.
authors.forename: Natalia
authors.initials: N
authors.lastname: Koralewska
authors.affiliation: Intercollegiate Faculty of Biotechnology of the University of Gdansk and Medical University of Gdańsk, Gdansk, Poland.
authors.forename: Weronika
authors.initials: W
authors.lastname: Hoffmann
authors.affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland.
authors.forename: Maria
authors.initials: M
authors.lastname: Pokornowska
authors.affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland.
authors.forename: Marek
authors.initials: M
authors.lastname: Milewski
authors.affiliation: Intercollegiate Faculty of Biotechnology of the University of Gdansk and Medical University of Gdańsk, Gdansk, Poland.
authors.forename: Andrea
authors.initials: A
authors.lastname: Lipinska
authors.affiliation: Intercollegiate Faculty of Biotechnology of the University of Gdansk and Medical University of Gdańsk, Gdansk, Poland.
authors.forename: Krystyna
authors.initials: K
authors.lastname: Bienkowska-Szewczyk
authors.affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland; Institute of Computing Science, Poznan University of Technology, Poznań, Poland.
authors.forename: Marek
authors.initials: M
authors.lastname: Figlerowicz
authors.affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Kurzynska-Kokorniak
mesh_terms: D001483:Base Sequence; D053487:DEAD-box RNA Helicases / Q000737:chemistry*; D025461:Feedback, Physiological; D006801:Humans; D055029:Inverted Repeat Sequences; D007700:Kinetics; D035683:MicroRNAs / Q000737:chemistry*; D011485:Protein Binding; D059367:RNA Cleavage; D012323:RNA Processing, Post-Transcriptional; D043244:Ribonuclease III / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D035683:MicroRNAs; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases