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

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.

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

“…Accordingly, we performed cleavage assays with a 21-nt RNA substrate, called "RNA21". We found that ∆DUF(625-752) generated 15-nt, 6-nt and 2-nt RNA products (Figure 4), as it was earlier observed for hDcr and the hDicer variants lacking the PAZ domain [25]. With incubation time, we observed disappearance of 15-nt and 6-nt RNA products and accumulation of 2-nt RNAs.…”

“…This 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNA or DNA molecules in vitro [17]. 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 (called "hDcr") that was earlier obtained in our laboratory [25]) (Figure 1a,b) (for details please see Materials and Methods). The expression plasmid was used for the transfection of 293T NoDice cells (human cells that do not produce hDicer) [26].…”

“…hDicer, apart from the canonical substrates, can bind and cut miRNA-size RNA molecules [24,25,[28][29][30]; therefore, we assume that Dicer proteins might be involved in the miRNA turnover [25]. Accordingly, we performed cleavage assays with a 21-nt RNA substrate, called "RNA21".…”

Computational search of hybrid human/SARS-CoV-2 dsRNA reveals unique viral sequences that diverge from those of other coronavirus strains

“…Some paired 19-mers with a dTdT overhang at the 3′ end bind Dicer with high affinity; these sequences are not cleaved by Dicer, are fully transferred to Ago and trigger high-efficiency gene-silencing activity ( Snead and Rossi, 2012 ; Snead et al, 2013 ). Dicer also binds single RNAs at a specific site with high affinity, the function of which is to accelerate hybridization with a partner from the milieu of RNA metabolism ( Kurzynska-Kokorniak et al, 2016 ; Wojnicka et al, 2020 ). This leads to the paradigm that hDicer acts as an annealer and presents a RNA molecule for searching a complementary segment in the RNA degradome, thus facilitating pairing between a small RNA and a complementary longer RNA in asymmetric mode.…”

Genetic Insight into the Domain Structure and Functions of Dicer-Type Ribonucleases

“…It was demonstrated that, in contrast to the PAZ-containing Dicers, which can bind the substrate termini, noncanonical Dicers initiate processing in the interior and work outward [ 100 ]. Moreover, in vitro studies revealed that the PAZ domain deletion variants of hDicer do not process pre-miRNAs and pre-siRNAs; however, they can cleave single-stranded RNA and DNA substrates, and the resulting products differ in size from typical hDicer products [ 104 ]. These results suggest that when the PAZ domain is absent (or not available), other hDicer domains may contribute to substrate binding, and in such cases, noncanonical products can be generated.…”