Guide-directed DNA cleavage by a prokaryotic Argonaute protein induces chromosome recombination in Escherichia coli

Emerging evidence supports the argument that some prokaryotic argonautes (pAgos) serve as a defensive system against invasion of viruses and plasmids through guide DNAs (gDNAs) directed DNA cleavage. This DNA-guided DNA interference motivates research to induce genomic mutations via pAgo mediated cleavage. Here we demonstrate that CbAgo, a pAgo from Clostridium butyricum, is able to induce chromosomal recombination between direct repeat sequences via its gDNA-directed cleavage in Escherichia coli chromosome. We also show that CbAgo targeting can assist Lambda-Red recombineering in RecA-deficient strain. Our study reveals that cleavage by CbAgo in E. coli chromosome can be mutagenic and suggests its broader application in genetic manipulation.

An siRNA-guided ARGONAUTE protein directs RNA polymerase V to initiate DNA methylation

In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without pre-existing DNA methylation. We find that Pol V follows and is dependent on the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of pre-existing DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.

microRNA-mediated regulation of microRNA machinery controls cell fate decisions

microRNAs associate with Argonaute proteins, forming the microRNA-induced silencing complex (miRISC), to repress target gene expression post-transcriptionally. Although microRNAs are critical regulators in mammalian cell differentiation, our understanding of how microRNA machinery, such as the miRISC, are regulated during development is still limited. We previously showed that repressing the production of one Argonaute protein, Ago2, by Trim71 is important for mouse embryonic stem cells (mESC) self-renewal (Liu et al., 2021). Here we show that among the four Argonaute proteins in mammals, Ago2 is the major developmentally regulated Argonaute protein in mESCs. Moreover, in pluripotency, besides the Trim71-mediated regulation of Ago2 (Liu et al., 2021), Mir182/Mir183 also repress Ago2. Specific inhibition of this microRNA-mediated repression results in stemness defects and accelerated differentiation through the let-7 microRNA pathway. These results reveal a microRNA-mediated regulatory circuit on microRNA machinery that is critical to maintaining pluripotency.

An siRNA-guided Argonaute protein directs RNA Polymerase V to initiate DNA methylation

In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA Polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without preexisting DNA methylation. We find that Pol V follows and is dependent upon the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of preexisting DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.

microRNA-mediated regulation of microRNA machinery controls cell fate decisions

microRNAs associate with Argonaute proteins, forming the microRNA-induced silencing complex (miRISC), to repress target gene expression post-transcriptionally. Although microRNAs are critical regulators in mammalian cell differentiation, our understanding of how microRNA machinery, such as the miRISC, are regulated during development is still limited. We previously showed that repressing the production of one Argonaute protein, Ago2, by Trim71 is important for mouse embryonic stem cells (mESC) self-renewal (Liu et al., 2021). Here we show that among the four Argonaute proteins in mammals, Ago2 is the major developmentally regulated Argonaute protein in mESCs. Moreover, in pluripotency, besides the Trim71-mediated regulation of Ago2 (Liu et al., 2021), microRNA-182/microRNA-183 also repress Ago2. Specific inhibition of this microRNA-mediated repression results in stemness defects and accelerated differentiation through the let-7 microRNA pathway. These results reveal a microRNA-mediated regulatory circuit on microRNA machinery that is critical to maintaining pluripotency.

Arginine methylation promotes siRNA-binding specificity for a spermatogenesis-specific isoform of the Argonaute protein CSR-1

CSR-1 is an essential Argonaute protein that binds to a subclass of 22G-RNAs targeting most germline-expressed genes. Here we show that the two isoforms of CSR-1 have distinct expression patterns; CSR-1B is ubiquitously expressed throughout the germline and during all stages of development while CSR-1A expression is restricted to germ cells undergoing spermatogenesis. Furthermore, CSR-1A associates preferentially with 22G-RNAs mapping to spermatogenesis-specific genes whereas CSR-1B-bound small RNAs map predominantly to oogenesis-specific genes. Interestingly, the exon unique to CSR-1A contains multiple dimethylarginine modifications, which are necessary for the preferential binding of CSR-1A to spermatogenesis-specific 22G-RNAs. Thus, we have discovered a regulatory mechanism for C. elegans Argonaute proteins that allows for specificity of small RNA binding between similar Argonaute proteins with overlapping temporal and spatial localization.

Profiling Thermus thermophilus Argonaute Guide DNA Sequence Preferences by Functional Screening

Prokaryotic Argonautes (pAgo) are an increasingly well-studied class of guided endonucleases, and the underlying mechanisms by which pAgo generate nucleic acid guides in vivo remains an important topic of investigation. Recent insights into these mechanisms for the Argonaute protein from Thermus thermophilus has drawn attention to global sequence and structural feature preferences involved in oligonucleotide guide selection. In this work, we approach the study of guide sequence preferences in T. thermophilus Argonaute from a functional perspective. Screening a library of 1,968 guides against randomized single- and double-stranded DNA substrates, endonuclease activity associated with each guide was quantified using high-throughput capillary electrophoresis, and localized sequence preferences were identified which can be used to improve guide design for molecular applications. The most notable preferences include: a strong cleavage enhancement from a first position dT independent of target sequence; a significant decrease in activity with dA at position 12; and an impact of GC dinucleotides at positions 10 and 11. While this method has been useful in characterizing unique preferences of T. thermophilus Argonaute and criteria for creating efficient guides, it could be expanded further to rapidly characterize more recent mesophilic variants reported in the literature and drive their utility toward molecular tools in biology and genome editing applications.

siRNA Specificity: RNAi Mechanisms and Strategies to Reduce Off-Target Effects

Short interfering RNAs (siRNAs) are processed from long double-stranded RNA (dsRNA), and a guide strand is selected and incorporated into the RNA-induced silencing complex (RISC). Within RISC, a member of the Argonaute protein family directly binds the guide strand and the siRNA guides RISC to fully complementary sites on-target RNAs, which are then sequence-specifically cleaved by the Argonaute protein—a process commonly referred to as RNA interference (RNAi). In animals, endogenous microRNAs (miRNAs) function similarly but do not lead to direct cleavage of the target RNA but to translational inhibition followed by exonucleolytic decay. This is due to only partial complementarity between the miRNA and the target RNA. SiRNAs, however, can function as miRNAs, and partial complementarity can lead to miRNA-like off-target effects in RNAi applications. Since siRNAs are widely used not only for screening but also for therapeutics as well as crop protection purposes, such miRNA-like off-target effects need to be minimized. Strategies such as RNA modifications or pooling of siRNAs have been developed and are used to reduce off-target effects.