The RNase III called Dicer generates small RNAs that post-transcriptionally
silence the expression of certain genes, and this regulation is essential for
development and normal physiology. Models for small interfering RNA (siRNA) and
microRNA (miRNA) processing by vertebrate Dicer have emerged from structural
and biochemical studies, yet the active dicing state in Dicer-RNA structures
has not been observed and characterized. We used cryo-electron microscopy to
determine the structures of mouse Dicer and DicerO, an oocyte
isoform lacking the HEL1 module, alone and in complex with pre-miR-15a RNA. The
apo-structure of Dicer showed that HEL1 stabilizes a closed conformation, and
its absence promotes helicase opening for substrate engagement into a dicing
state. Consequently, DicerO with pre-miR-15a exclusively forms an
active dicing-competent conformation, whereas the structure of
Dicer–pre-miR-15a complex captures Dicer in a pre-dicing state. We show that
the absence of HEL1 not only activates Dicer but also alters substrate
selectivity by facilitating direct substrate loading into the catalytic site
and curtailing pre-dicing state, which serves as a selectivity filter for the
microRNA precursor architecture.
This work was supported by the Czech Science Foundation (GA22-19896S to RS). At the initial stage, it also received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 649030 to R.S.), Operational Programme Research, Development and Education-project „Internal Grant Agency of Masaryk University“ (No.CZ.02.2.69/0.0/0.0/19_073/0016943) to D.Z.. Institutional funding was provided by the Ministry of Education, Youth and Sports (MEYS) of the Czech Republic (CEITEC 2020 project (LQ1601)). Research in the lab of P.S. was supported by the Czech Science Foundation EXPRO grant (20-03950X). We gratefully acknowledge the cryo-EM Core Facility and Proteomics Core Facility supported by the CIISB research infrastructure, an Instruct-CZ Centre of Instruct-ERIC EU consortium (funded by MEYS CR infrastructure project LM2018127) for their support with obtaining the scientific data presented in this paper.