Structural determinants in the miRNA/miRNA* duplex and the DCL1 PAZ domain for precise and efficient plant miRNA processing.

The accessory proteins Hyponastic-like 1 (HYL1) and Serrated (SE) enhance the precise and efficient processing of miRNAs by Dicer-like 1 (DCL1), which is important for proper miRNA function. However, other factors determining the precision and efficiency of miRNA biogenesis are not well-known. Here, we found that an asymmetric bulge (AB) at the 3' end of miR-5p (produced from the 5' arm of the pre-miRNA) reduced the precision of the second cleavage, whereas an AB at other sites of miR-5p mainly affected the accumulation level of miR-5p in transient expression in Nicotiana benthamiana. In contrast, many ABs in miR-3p (produced from the 3' arm of the pre-miRNA) impose strong negative impact on the processing precision and the accumulation level of miR-5p in N. benthamiana. Arabidopsis DCL1/SE/HYL1 complex-mediated miRNA processing was reconstituted in Saccharomyces cerevisiae to further investigate AB-mediated interference with DCL1 processing. With this system, the positional effect of AB on miRNA processing was tested. The results showed that ABs on the middle of miR-5p have less of an impact on DCL1 cleavage efficiency and precision, whereas those on miR-3p or near the ends of miR-5p strongly reduce DCL1 cleavage activity, precision or both. Studies using the yeast miRNA processing system and transgenic Arabidopsis also revealed the importance of the interaction between the 2-nt 3' overhang of pre-miRNA and the 3' overhang binding pocket (3'BP) on the precision of the second cleavage reaction for many endogenous miRNAs. These findings provide new insights into the mechanism of miRNA biogenesis.

PAZ domain is critical for spermatogenesis in chicken.

Piwi-like protein 1 (PIWIL1) plays a crucial role in stem cell proliferation, embryogenesis, growth, and development. We aimed to unravel the function of PIWIL1 and its Piwi/Argonaute/Zwille (PAZ) domain in chicken embryogenesis. The expression of PIWI1 at different stages of spermatogenesis was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and the PAZ domain was mutated based on its 3D structure model using the clustered regularly interspaced short palindromic repeats Cas9 (CRISPR/Cas9) technology. The results indicated that PIWIL1 mRNA was specifically expressed in spermatogonium cells undergoing meiosis. After targeting the PAZ domain (300-370 amino acid residues), we obtained two mutant DF-1 cell clones with 23-bp and 8-bp deletions. Injection of the pCMV-Cas9-puro-sgRNA-2 construct into 2.5-day embryos resulted in generation of 19 different PAZ mutants (13 males and 6 females), which showed delayed hatching, reduced quality of semen, and decreased expression of PIWIL1 and SOX2 at embryonic days 5 and 18. However, we could not obtain PAZ double knockout (KO) chickens by crossing of the F0 generation, suggesting that PAZ double KO may halt embryonic development. Our results indicate that PIWIL1 plays an important role in meiosis and that PAZ mutations can lead to decreased sperm quality, whereas its double KO may arrest embryogenesis in chicken.

A Glimpse of "Dicer Biology" Through the Structural and Functional Perspective.

The RNA interference pathway (RNAi) is executed by two core enzymes, Dicer and Argonaute, for accomplishing a tailored transcriptional and post-transcriptional gene regulation. Dicer, an RNase III enzyme, initiates the RNAi pathway, plays a pivotal role in fighting infection against pathogens, and acts as a housekeeping enzyme for cellular homeostasis. Here, we review structure-based functional insights of Dicer and its domains present in a diverse group of organisms. Although Dicer and its domains are evolutionarily conserved from microsporidian parasites to humans, recent cryo-electron microscopy structures of Homo sapiens Dicer and Drosophila melanogaster Dicer-2 suggest characteristic variations in the mechanism of the dsRNA substrate recognition. Interestingly, the necessity for more than one functionally distinct Dicer paralogs in insects and plants compared with a single Dicer in other eukaryotic life forms implies Dicer's role in the interplay of RNAi and other defense mechanisms. Based on the structural and mechanistic information obtained during the last decade, we aim to highlight the significance of key Dicer domains that are crucial to Dicer specific recognition and precise cleavage of dsRNA substrates. Further, the role of Dicer in the formation of Argonaute-based RNA-induced silencing complex (RISC) assembly formation, Dicer's ability to regulate a complex protein interaction network, and its role in other cellular processes, as well as its therapeutic potentials, are emphasized.

Structural basis for RNA 3'-end recognition by the PIWIL2 PAZ domain.

PIWI family proteins are important members of Argonaute family that play an essential role in spermatogenesis and development when loaded with piRNAs. Here we solved the crystal structure of the human PIWIL2 PAZ domain and found its PAZ domain adopts a canonical PAZ fold. We furhter built a homology model of PIWIL2 bound to 2 nt 3' overhangs. We found that PIWIL2 utilizes a deep hydrophobic concave to accommodate the 2 nt at 3'-end of RNAs. The recognition of 2 nt 3' overhangs by PIWIL2 is conserved in other human PIWIL proteins, implicating the evolutionarily conserved role of PAZ domain in binding to target RNAs.

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.

Sulfonamide antibiotics inhibit RNAi by binding to human Argonaute protein 2 PAZ.

We found that sulfisomidine, a sulfonamide antibiotic, potently binds to the Piwi/Argonaute/Zwille (PAZ) domain of human Argonaute protein 2 and inhibits RNA interference (RNAi). To elucidate the effect on RNAi of strong affinity of the 3'-ends in small interfering RNA (siRNA) to the PAZ domain, chemically modified siRNAs bearing sulfisomidine at the 3'-end were synthesized.

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

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 3'-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.

Synthesis and characterization of small interfering RNAs with haloalkyl groups at their 3'-dangling ends.

With the aim to create a small interfering RNA (siRNA) with enhanced activity and resistance to nuclease degradation, we synthesized and evaluated the properties of the following siRNAs containing haloalkyl ß-d-ribofuranosides at their 3'-dangling ends: 2,2,2-trifluoroethyl ß-d-ribofuranoside, 2,2,2-trichloroethyl ß-d-ribofuranoside and 2,2,2-tribromoethyl ß-d-ribofuranoside. The gene silencing activities of the modified siRNAs were investigated through a dual luciferase reporter assay using HeLa cells. The highest silencing activity was observed for the trichloroethyl analog modified siRNA, which was closely followed by the trifluoroethyl and tribromoethyl analogs. The modified siRNAs were found to show increased binding affinity towards the Piwi-Argonaute-Zwille (PAZ) domain protein based on computational analysis and an experimental study. Furthermore, the RNAs modified with the analogs at their 3'-ends exhibited improved resistance to hydrolysis by a 3'-exonuclease.

Characterization of DCL4 missense alleles provides insights into its ability to process distinct classes of dsRNA substrates.

In the model plant Arabidopsis thaliana, four Dicer-like proteins (DCL1-4) mediate the production of various classes of small RNAs (sRNAs). Among these four proteins, DCL4 is by far the most versatile RNaseIII-like enzyme, and previously identified dcl4 missense alleles were shown to uncouple the production of the various classes of DCL4-dependent sRNAs. Yet little is known about the molecular mechanism behind this uncoupling. Here, by studying the subcellular localization, interactome and binding to the sRNA precursors of three distinct dcl4 missense alleles, we simultaneously highlight the absolute requirement of a specific residue in the helicase domain for the efficient production of all DCL4-dependent sRNAs, and identify, within the PAZ domain, an important determinant of DCL4 versatility that is mandatory for the efficient processing of intramolecular fold-back double-stranded RNA (dsRNA) precursors, but that is dispensable for the production of small interfering RNAs (siRNAs) from RDR-dependent dsRNA susbtrates. This study not only provides insights into the DCL4 mode of action, but also delineates interesting tools to further study the complexity of RNA silencing pathways in plants, and possibly other organisms.

Molecular dynamics and binding selectivity of nucleotides and polynucleotide substrates with EIF2C2/Ago2 PAZ domain.

RNA interference (RNAi) constitutes a major target in drug discovery. Recently, we reported that the Argonaute protein 2 (Ago2) PAZ domain selectively binds with all ribonucleotides except adenine and poorly recognizes deoxyribonucleotides. The binding properties of the PAZ domain with polynucleotides and the molecular mechanisms of substrates' selectivity remains unclear. In this study, the binding potencies of polynucleotides and the associated conformational and dynamic changes in PAZ domain are investigated. Coinciding with nucleotides' binding profile with the PAZ domain, polyuridylate (PolyU) and polycytidylate (PolyC) were potent binders. However, KdPolyU and KdPolyC were 15.8 and 9.3µM, respectively. In contrast, polyadenylate (PolyA) binding was not detectable. Molecular dynamics (MD) simulation revealed the highest change in root mean square deviation (RMSD) with ApoPAZ or PAZ domain bound with experimentally approved, low affinity substrates, whereas stronger binding substrates such as UMP or PolyU showed minimal RMSD changes. The loop between α3 and ß5 in the ß-hairpin subdomain showed the most responsive change in RMSD, being highly movable in the ApoPAZ and PAZ-AMP complex. Favorable substrate recognition was associate with moderate change in secondary structure content. In conclusion, the PAZ domain retains differential substrate selectivity associated with corresponding dynamic and structural changes upon binding.

Guide Strand 3'-End Modifications Regulate siRNA Specificity.

Short interfering RNA (siRNA)-triggered gene knockdown through the RNA interference (RNAi) pathway is widely used to study gene function, and siRNA-based therapeutics are in development. However, as the guide strand of an siRNA can function like a natural microRNA (miRNA), siRNAs often repress hundreds of off-target transcripts with complementarity only to the seed region (nucleotides 2-8) of the guide strand. Here, we describe novel guide strand 3'-end modifications derived from 1-ethynylribose (1-ER) and copper-catalyzed azide-alkyne cycloaddition reactions and evaluate their impact on target versus miRNA-like off-target knockdown. Surprisingly, when positioned at the guide strand 3'-end, the parent 1-ER modification substantially reduced off-target knockdown while having no measurable effect on on-target knockdown potency. In addition, these modifications were shown to modulate siRNA affinity for the hAgo2 PAZ domain. However, the change in PAZ domain binding affinity was not sufficient to predict the modification's effect on miRNA-like off targeting.

Synthesis of small interfering RNAs containing acetal-type nucleoside analogs at their 3'-ends and analysis of their silencing activity and their ability to bind to the Argonaute2 PAZ domain.

In this study, we aimed to create small interfering RNAs (siRNAs) with increased silencing activities and nuclease resistance properties. Therefore, we designed and synthesized five types of siRNA containing acetal-type nucleoside analogs at their 3'-dangling ends. We found that the siRNA containing 1-O-(2,2,2-trifluoroethyl)-ß-D-ribofuranose at the 3'-dangling end was the most potent among the synthesized siRNAs and showed more resistance to nucleolytic degradation by a 3' exonuclease than a natural RNA did. Thus, modification of siRNAs by addition of 1-O-(2,2,2-trifluoroethyl)-ß-D-ribofuranose may hold promise as a means of improving the silencing activity and nuclease resistance of siRNAs.