title: siRNA release from pri-miRNA scaffolds is controlled by the sequence and structure of RNA.
abstract: shmiRs are pri-miRNA-based RNA interference triggers from which exogenous siRNAs are expressed in cells to silence target genes. These reagents are very promising tools in RNAi in vivo applications due to their good activity profile and lower toxicity than observed for other vector-based reagents such as shRNAs. In this study, using high-resolution northern blotting and small RNA sequencing, we investigated the precision with which RNases Drosha and Dicer process shmiRs. The fidelity of siRNA release from the commonly used pri-miRNA shuttles was found to depend on both the siRNA insert and the pri-miR scaffold. Then, we searched for specific factors that may affect the precision of siRNA release and found that both the structural features of shmiR hairpins and the nucleotide sequence at Drosha and Dicer processing sites contribute to cleavage site selection and cleavage precision. An analysis of multiple shRNA intermediates generated from several reagents revealed the complexity of shmiR processing by Drosha and demonstrated that Dicer selects substrates for further processing. Aside from providing new basic knowledge regarding the specificity of nucleases involved in miRNA biogenesis, our results facilitate the rational design of more efficient genetic reagents for RNAi technology.
authors.affiliation: Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.
authors.forename: Paulina
authors.initials: P
authors.lastname: Galka-Marciniak
authors.affiliation: Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Olejniczak
authors.affiliation: Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.
authors.forename: Julia
authors.initials: J
authors.lastname: Starega-Roslan
authors.affiliation: Department of Bioinformatics, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Umultowska 89, 61-614 Poznan, Poland.
authors.forename: Michal W
authors.initials: MW
authors.lastname: Szczesniak
authors.affiliation: Department of Bioinformatics, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Umultowska 89, 61-614 Poznan, Poland.
authors.forename: Izabela
authors.initials: I
authors.lastname: Makalowska
authors.affiliation: Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland. Electronic address: wlodkrzy@ibch.poznan.pl.
authors.forename: Wlodzimierz J
authors.initials: WJ
authors.lastname: Krzyzosiak
mesh_terms: D001483:Base Sequence / Q000235:genetics; D053487:DEAD-box RNA Helicases / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics*; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D043244:Ribonuclease III / Q000235:genetics* / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D034741:RNA, Small Interfering; C408087:DICER1 protein, human; C517918:DROSHA protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases
keywords: Artificial miRNA; Dicer; Drosha; RNAi; miRNA biogenesis; pri-miRNA-based shRNA