Critical role of backbone coordination in the mRNA recognition by RNA induced silencing complex.

Despite its functional importance, the molecular mechanism underlying target mRNA recognition by Argonaute (Ago) remains largely elusive. Based on extensive all-atom molecular dynamics simulations, we constructed quasi-Markov State Model (qMSM) to reveal the dynamics during recognition at position 6-7 in the seed region of human Argonaute 2 (hAgo2). Interestingly, we found that the slowest mode of motion therein is not the gRNA-target base-pairing, but the coordination of the target phosphate groups with a set of positively charged residues of hAgo2. Moreover, the ability of Helix-7 to approach the PIWI and MID domains was found to reduce the effective volume accessible to the target mRNA and therefore facilitate both the backbone coordination and base-pair formation. Further mutant simulations revealed that alanine mutation of the D358 residue on Helix-7 enhanced a trap state to slow down the loading of target mRNA. Similar trap state was also observed when wobble pairs were introduced in g6 and g7, indicating the role of Helix-7 in suppressing non-canonical base-paring. Our study pointed to a general mechanism for mRNA recognition by eukaryotic Agos and demonstrated the promise of qMSM in investigating complex conformational changes of biomolecular systems.

Rapid and cost-effective screening of CRISPR/Cas9-induced mutants by DNA-guided Argonaute nuclease.

OBJECTIVE: With the widespread application of CRISPR/Cas9 gene editing technology, new methods are needed to screen mutants quickly and effectively. Here, we aimed to develop a simple and cost-effective method to screen CRISPR/Cas9-induced mutants. RESULT: We report a novel method to identify CRISPR/Cas9-induced mutants through a DNA-guided Argonaute nuclease derived from the archaeon Pyrococcus furiosus. We demonstrated that the Pyrococcus furiosus Argonaute (PfAgo)-based method could distinguish among biallelic mutants, monoallelic mutants and wild type (WT). Furthermore, this method was able to identify 1 bp indel mutations. CONCLUSION: The PfAgo-based method is simple to implement and can be applied to screen biallelic mutants and mosaic mutants generated by CRISPR-Cas9 or other kinds of gene editing tools.

Assessment of the function of gonad-specific PmAgo4 in viral replication and spermatogenesis in Penaeus monodon.

Argonaute family is phylogenetically subdivided into Ago and Piwi subfamilies that operate either transcriptional or post-transcriptional regulation in association with particular types of small RNAs. Among the four members of Ago subfamily (PmAgo1-4) found in black tiger shrimp Penaeus monodon, PmAgo4 exhibits gonad-restricted expression and takes part in transposon repression as the Piwi subfamily. While PmAgo1-3 participate in RNA interference (RNAi)-based mechanism, the role of PmAgo4 in RNAi is still mysterious, and was therefore investigated in this study. The results showed that knockdown of PmAgo4 in shrimp testis did not have a significant effect on the potency of PmRab7 silencing by dsPmRab7. In addition, replication of YHV as well as YHV-induced cumulative mortality in PmAgo4-knockdown shrimp are comparable to the control shrimp, suggesting the irrelevant association of PmAgo4 with RNAi-mediated gene silencing and antiviral immunity. Since PmAgo4 did not function in common with the Ago subfamily, its potential function in gametogenesis of male shrimp was further investigated. The reduction of PmAgo4 transcript levels in male shrimp revealed significant defect in testicular maturity as measured by Testicular Index (TI). Moreover, the numbers of mature sperm in spermatophore of PmAgo4-knockdown shrimp were significantly decreased comparing with the control shrimp. Our studies thus suggest a distinctive role of PmAgo4 that is not consistent with a dsRNA-mediate gene regulation and virus replication, but has a key function in controlling spermatogenesis in P. monodon.

New opportunities for designing effective small interfering RNAs.

Small interfering RNAs (siRNAs) that silence genes of infectious diseases are potentially potent drugs. A continuing obstacle for siRNA-based drugs is how to improve their efficacy for adequate dosage. To overcome this obstacle, the interactions of antiviral siRNAs, tested in vivo, were computationally examined within the RNA-induced silencing complex (RISC). Thermodynamics data show that a persistent RISC cofactor is significantly more exothermic for effective antiviral siRNAs than their ineffective counterparts. Detailed inspection of viral RNA secondary structures reveals that effective antiviral siRNAs target hairpin or pseudoknot loops. These structures are critical for initial RISC interactions since they partially lack intramolecular complementary base pairing. Importing two temporary RISC cofactors from magnesium-rich hairpins and/or pseudoknots then kickstarts full RNA hybridization and hydrolysis. Current siRNA design guidelines are based on RNA primary sequence data. Herein, the thermodynamics of RISC cofactors and targeting magnesium-rich RNA secondary structures provide additional guidelines for improving siRNA design.

A Rapid and Versatile Assay for Ago2-Mediated Cleavage by Using Branched Rolling Circle Amplification.

Micro RNA (miRNA) research has evolved into an essential part of investigating gene regulation in which deregulation of numerous miRNAs is associated with various cellular dysfunction and diseases. Here, we describe a rapid and homogenous assay for Ago2-mediated target RNA cleavage, based on branched rolling circle amplification (BRCA). In particular, the ability to investigate small molecule binders for inhibition of miRNA function is within the potential of our assay. This method uses no artificial fluorescence labeling of RNA components, which can be an advantage in screening of potential inhibitors. To visualize cleavage of RNA substrate by Ago2, we developed a two-step assay composed of Ago2-mediated cleavage and BRCA-based detection. The assay is cost-effective and practicable and can be performed in 96-well format by using a standard qPCR machine.