Unknown Areas of Activity of Human Ribonuclease Dicer: A Putative Deoxyribonuclease Activity.

Marta Wojnicka,Anna Kurzynska-Kokorniak,Agnieszka Szczepanska

doi:10.3390/molecules25061414

Marta Wojnicka, Anna Kurzynska-Kokorniak + Show 1 more

Open Access

https://doi.org/10.3390/molecules25061414

Copy DOI

Abstract

The Dicer ribonuclease plays a crucial role in the biogenesis of small regulatory RNAs (srRNAs) by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. Dicer-generated srRNAs can control gene expression by targeting complementary transcripts and repressing their translation or inducing their cleavage. Human Dicer (hDicer) is a multidomain enzyme comprising a putative helicase domain, a DUF283 domain, platform, a PAZ domain, a connector helix, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Specific, ~20-base pair siRNA or miRNA duplexes with 2 nucleotide (nt) 3’-overhangs are generated by Dicer when an RNA substrate is anchored within the platform-PAZ-connector helix (PPC) region. However, increasing number of reports indicate that in the absence of the PAZ domain, binding of RNA substrates can occur by other Dicer domains. Interestingly, truncated variants of Dicer, lacking the PPC region, have been found to display a DNase activity. Inspired by these findings, we investigated how the lack of the PAZ domain, or the entire PPC region, would influence the cleavage activity of hDicer. Using immunopurified 3xFlag-hDicer produced in human cells and its two variants: one lacking the PAZ domain, and the other lacking the entire PPC region, we show that the PAZ domain deletion variants of hDicer are not able to process a pre-miRNA substrate, a dsRNA with 2-nt 3ʹ-overhangs, and a blunt-ended dsRNA. However, the PAZ deletion variants exhibit both RNase and DNase activity on short single-stranded RNA and DNAs, respectively. Collectively, our results indicate that when the PAZ domain is absent, other hDicer domains may contribute to substrate binding and in this case, non-canonical products can be generated.

Highlights

Ribonuclease Dicer is one of the key enzymes involved in the biogenesis of small regulatory
The Dicer-produced miRNA and small interfering RNAs (siRNAs) duplexes are loaded into a multi-protein complex referred to as the RNA-induced silencing complex (RISC) [2]
Transcript variant 2 of DICER1 (NM_030621) as a template

Summary

Introduction

RNAs (srRNAs), such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) [1]. The Dicer-produced miRNA and siRNA duplexes are loaded into a multi-protein complex referred to as the RNA-induced silencing complex (RISC) [2]. During RISC activation, one strand of the RNA duplex is discarded (designated as “passenger”) while the mature miRNA or siRNA strand (designated as “guide”) remains in the RISC. The activated RISC targets the cellular mRNA complementary to the guide strand [2,3,4]. Depending on the degree of complementarity between the small RNA and the target molecule, RISC binding results in either mRNA cleavage and degradation or translational repression [5]. Depending on the degree of complementarity between the small RNA and the target molecule, RISC binding results in either mRNA cleavage and degradation or translational repression [5]. srRNAs play significant roles in many biological processes, including developmental timing, growth control, Molecules 2020, 25, 1414; doi:10.3390/molecules25061414 www.mdpi.com/journal/molecules

Methods

Results

Conclusion