The Significance of the DUF283 Domain for the Activity of Human Ribonuclease Dicer.

Agnieszka Szczepanska,Anna Kurzynska-Kokorniak,Marta Wojnicka

doi:10.3390/ijms22168690

Agnieszka Szczepanska, Anna Kurzynska-Kokorniak + Show 1 more

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https://doi.org/10.3390/ijms22168690

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Abstract

Dicers are multidomain proteins, usually comprising an amino-terminal putative helicase domain, a DUF283 domain (domain of unknown function), a PAZ domain, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Dicer homologs play an important role in the biogenesis of small regulatory RNAs by cleaving single-stranded precursors adopting stem-loop structures (pre-miRNAs) and double-strand RNAs into short RNA duplexes containing functional microRNAs or small interfering RNAs, respectively. Growing evidence shows that apart from the canonical role, Dicer proteins can serve a number of other functions. For example, results of our previous studies showed that human Dicer (hDicer), presumably through its DUF283 domain, can facilitate hybridization between two complementary RNAs, thus, acting as a nucleic acid annealer. Here, to test this assumption, we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain. The respective 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNAs in vitro. We show that the ΔDUF(625-752) hDicer variant loses the potential to facilitate RNA-RNA base pairing, which strongly proves our hypothesis about the importance of the DUF283 domain for the RNA-RNA annealing activity of hDicer. Interestingly, the in vitro biochemical characterization of the obtained deletion variant reveals that it displays different RNA cleavage properties depending on the pre-miRNA substrate.

Highlights

We hypothesize that the DUF283 domain is crucial for the RNA-RNA annealing activity of human Dicer (hDicer). To test this assumption, here we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain
To investigate whether the DUF283 domain is indispensable for hDicer RNARNA base pairing activity, we obtained the hDicer variant lacking the amino acid residues
The expression plasmid encoding the hDicer ∆DUF(625-752) variant was produced by a PCR approach using as a template the plasmid encoding the wild-type full-length hDicer fused with the 3xFlagtag that was earlier obtained in our laboratory [25]) (Figure 1a,b)

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Dicer belongs to the ribonuclease III (RNase III) family of double stranded RNA (dsRNA)-specific endoribonucleases [1]. Dicer proteins generate short RNA duplexes containing functional microRNAs (miRNAs) or small interfering RNAs (siRNAs) [2], which are loaded into a multi-protein complex referred to as the RNA-induced silencing complex (RISC) [3]. During RISC activation, one strand of the RNA duplex (called the “passenger” strand) is released and degraded and the other strand

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