Biology subjects

Kurzynska-Kokorniak, A.

Publications and source records attributed to Kurzynska-Kokorniak, A..

3 recordsLinked to original sources

bioRxiv2023

Short 2'-O-methyl/LNA oligomers as highly-selective inhibitors of miRNA production in vitro and in vivo

MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that members of the same miRNA family with high sequence similarity might have different roles and that this functional divergence might be rooted in their precursors sequence. Current knock-down strategies such as antisense oligonucleotides (ASOs) or miRNA sponges cannot distinguish between identical or near identical miRNAs originating from different precursors to allow exploring unique functions of these miRNAs. We now develop a method based on short 2'-OMe/LNA-modified oligonucleotides to selectively target specific precursor molecules and ablate the production of individual members of miRNA families in vitro and in vivo. Using the highly conserved Xenopus miR-181a family as a proof-of-concept, we demonstrate that 2'-OMe/LNA-ASOs targeting pre-miRNA apical region elicit a precursor-selective inhibition of mature miRNA-5p production. The levels of miRNAs released from the 3'-arm of these precursors are not reduced, suggesting that our approach is also arm-selective. Overall, we show that this strategy can be successfully applied in vivo to achieve high target selectivity to study identical or highly similar miRNAs stemming from different precursors.

molecular biology↗

bioRxiv2023

New insights into the helicase domain of human Dicer and its biochemical properties: ATPase and RNA rearrangement activities

Vertebrates have one Dicer ortholog that generates both microRNAs (miRNAs) and small interfering RNAs (siRNAs), in contrast to the multiple Dicer-like proteins found in flies and plants. Here, we focus on the functions of the human Dicer (hDicer) helicase domain. The helicase domain of hDicer is known to recognize pre-miRNA substrates through interactions with their apical loop regions. Besides interacting with canonical substrates, the hDicer helicase domain has also been suggested to bind many different cellular RNAs; however, a comprehensive study of the biochemical activities and substrate specificity of the hDicer helicase domain towards different nucleic acids has yet to be undertaken. Here, we conduct such an analysis and reveal that full-length hDicer, through its helicase domain, hydrolyzes ATP. We also show that the hDicer helicase domain binds single-but not double-stranded RNAs and DNAs and that a structural rearrangement of the substrate accompanies the binding of single-stranded RNAs. This RNA rearrangement activity is ATP-independent. Our findings open new avenues for future studies aimed at defining the cellular activities of hDicer that may be associated with these newly described biochemical properties.

molecular biology↗

bioRxiv2020

RNA and DNA G-quadruplexes bind to human Dicer and inhibit its activity

Guanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform-PAZ-Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation.

biochemistry↗