Biology subjects

MacRae, I. J.

Publications and source records attributed to MacRae, I. J..

10 recordsLinked to original sources

bioRxiv2026

Organyl 5'-Phosphates in siRNA Guide Strands: Structure-Function Relationships Governing Anchoring in Argonaute 2 and Metabolic Stability

Efficient siRNA loading into Argonaute2 (AGO2) requires a 5'-phosphate (5'-P) on the guide strand, yet this group is vulnerable to metabolic degradation in vivo. Although numerous chemical mimics of 5'-P have been reported, structural principles governing AGO2 interactions with organyl substituents on the 5'-P remain unclear. Moreover, structural determinants of 5'-P mimic recognition by known degradative enzymes (principally phosphatases and 5'-exonucleases) are also poorly understood. The 5'-P binding site of the AGO2 MID domain contains a stack of aromatic residues (Y527/F811/Y815), presenting a structural basis for augmenting canonical anchoring interactions. Herein, we systematically synthesized and characterized a diverse panel of organyl 5'-phosphates (5'-POR; R = 35 variable substituents) as guide strand 5'-P mimics designed to engage this unique hydrophobic pocket. Among the compounds evaluated, 5'-POR guide strands bearing methyl (Me) or phenylpropargyl (PhPrp) substituents are well-tolerated by AGO2 in cells. Previously uncharacterized 5'-P mimics, including 5'-phosphorothioate (5'-PS), phenylpropargyl 5'-phosphorothioate (5'-PS-PhPrp), and 5'-mesylphosphoramidate (5'-MsPA), maintain comparable AGO2 compatibility. All examined 5'-P mimics are markedly resistant to phosphatase, while 5'-POR variants and 5'-PS-PhPrp are also resistant to 5'-exonuclease degradation due to masking a negative charge of 5'-P. A crystal structure of a 5'-PO-PhPrp guide strand loaded into AGO2 reveals an unexpected network of {pi}-{pi} interactions between the rigid phenylpropargyl group and the targeted hydrophobic pocket of the MID domain. Collectively, these findings expand the functional chemical space of 5'-P mimics and define new modes for metabolically stabilizing the guide strand 5'-end while augmenting AGO2 MID anchoring. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/705631v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@e6f88forg.highwire.dtl.DTLVardef@1c87dfaorg.highwire.dtl.DTLVardef@1c6e68corg.highwire.dtl.DTLVardef@14a1f60_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Structural basis for gene silencing by siRNAs in humans

Small interfering RNAs (siRNAs) guide mRNA cleavage by human Argonaute2 (hAgo2), leading to targeted gene silencing. Despite their laboratory and clinical impact, structural insights into human siRNA catalytic activity remain elusive. Here, we show that disrupting siRNA 3-end binding by hAgo2 accelerates target cleavage and stabilizes its catalytic conformation, enabling detailed structural analysis. A 3.16 [A] global resolution cryo-EM reconstruction reveals that distortion of the siRNA-target duplex at position 6 allows target RNA entry into the catalytic cleft and shifts Lysine-709, a previously unrecognized catalytic residue, into the active site. A pyrimidine at target nucleotide t10 positions another unrecognized catalytic residue, Arginine-710, for optimal cleavage. Expansion of the guide-target duplex major groove docks the scissile phosphate for hydrolysis and subsequent groove compression after position 16 permits target RNAs to exit the catalytic cleft. These findings reveal how hAgo2 catalyzes siRNA target hydrolysis, providing a high-resolution model for therapeutic design.

molecular biology↗

bioRxiv2023

RNA-guided RNA silencing by an Asgard archaeal Argonaute

Eukaryotic Argonaute proteins achieve gene repression and defense against viruses and transposons by RNA-guided RNA silencing. By contrast, known prokaryotic Argonautes adopt single-stranded DNA as guides and/or targets, leaving the evolutionary origin of RNA-guided RNA silencing elusive. Here, we show an evolutionary expansion of Asgard archaeal Argonautes (asAgos), including the discovery of HrAgo1 from the Lokiarchaeon Candidatus Harpocratesius repetitus that shares a common origin with eukaryotic PIWI proteins. HrAgo1 exhibits RNA-guided RNA cleavage in vitro and RNA silencing in human cells. The cryo-EM structure of HrAgo1 combined with quantitative single-molecule experiments reveals that HrAgo1 possesses hybrid structural features and target binding modes bridging those of the eukaryotic AGO and PIWI clades. Finally, genomic evidence suggests that eukaryotic Dicer-like processing of double-stranded RNA likely emerged as a mechanism of generating guide RNA for asAgos prior to eukaryogenesis. Our study provides new insights into the evolutionary origin and plasticity of Argonaute-based RNA silencing.

microbiology↗

bioRxiv2023

MicroRNAs are necessary for the emergence of Purkinje cell identity

Brain computations are dictated by the unique morphology and connectivity of neuronal subtypes, features established by closely timed developmental events. MicroRNAs (miRNAs) are critical for brain development, but current technologies lack the spatiotemporal resolution to determine how miRNAs instruct the steps leading to subtype identity. Here, we developed new tools to tackle this major gap. Fast and reversible miRNA loss-of-function revealed that miRNAs are necessary for cerebellar Purkinje cell (PC) differentiation, which previously appeared miRNA-independent, and resolved distinct miRNA critical windows in PC dendritogenesis and climbing fiber synaptogenesis, key determinants of PC identity. To identify underlying mechanisms, we generated a mouse model, which enables precise mapping of miRNAs and their targets in rare cell types. With PC-specific maps, we found that the PC-enriched miR-206 drives exuberant dendritogenesis and modulates synaptogenesis. Our results showcase vastly improved approaches for dissecting miRNA function and reveal that many critical miRNA mechanisms remain largely unexplored. HighlightsO_LIFast miRNA loss-of-function with T6B impairs postnatal Purkinje cell development C_LIO_LIReversible T6B reveals critical miRNA windows for dendritogenesis and synaptogenesis C_LIO_LIConditional Spy3-Ago2 mouse line enables miRNA-target network mapping in rare cells C_LIO_LIPurkinje cell-enriched miR-206 regulates its unique dendritic and synaptic morphology C_LI

neuroscience↗

bioRxiv2022

Structural basis for differential targeting properties of small RNAs in plants and animals

microRNAs (miRNAs) regulate gene expression in plants and animals. Animals use miRNAs to sculpt the transcriptome, with each miRNA modestly repressing hundreds of otherwise unrelated targets. By contrast, each plant miRNA potently silences a small number of physiologically related targets. Here, we show that this major functional distinction depends on a minor structural difference between plant and animal Argonaute (AGO) proteins. A 7-amino-acid loop in the PIWI domain of Arabidopsis Argonaute10 (AtAGO10) reduces the affinity of the miRNA seed region for target RNAs. Swapping the PIWI-loop from human Argonaute2 (HsAGO2) into AtAGO10 increases seed strength and target-binding promiscuity, resembling animal miRNA targeting. Conversely, swapping the plant PIWI-loop into HsAGO2 increases targeting stringency and elevates target cleavage rates. The loop-swapped HsAGO2-siRNA complex silences targets more potently, with reduced off-targeting, than wild-type HsAGO2 and small interfering RNA (siRNA) duplexes in mammalian cells. Thus, tiny structural differences can tune the targeting properties of AGO proteins for distinct biological roles and HsAGO2 can be engineered for potential applications in siRNA therapeutics.

molecular biology↗

bioRxiv2022

Structural basis for RNA slicing by a plant Argonaute

Argonaute (AGO) proteins use small RNAs to recognize transcripts targeted for silencing in plants and animals. Many AGOs possess an endoribonuclease activity termed RNA slicing, which catalyzes rapid turnover of target RNAs. The nuclease activity of Thermus thermophilus AGO (TtAgo)1,2 is often used as a model for RNA slicing, but how well DNA-guided slicing by this bacterial thermophile resembles RNA-guided slicing by eukaryotic AGOs is not known. We present cryo-EM structures of the Arabidopsis thaliana Argonaute10 (AtAgo10)-guide RNA complex with and without a target RNA representing a slicing substrate. Like TtAgo, AtAgo10 conformation expands in response to target binding. However, the AtAgo10-guide-target complex adopts slicing-competent and -incompetent conformations that are distinct from structures of TtAgo. AtAgo10 slicing activity is licensed by docking target (t) nucleotides t9-t13 into a surface channel containing the AGO endoribonuclease active site. A {beta}-hairpin, conserved in the L1 domain of eukaryotic AGOs, secures the t9-t13 segment and coordinates t9-t13 docking with extended guide-target pairing, preventing the complex from becoming trapped in slicing-incompetent conformations. Results show the mechanism for achieving RNA slicing in eukaryotes is distinct from that of bacteria and provide insights for controlling small interfering RNA (siRNA) potency.

molecular biology↗

bioRxiv2020

Structural basis for piRNA-targeting

Piwi proteins use PIWI-interacting RNAs (piRNAs) to identify and silence the transposable elements (TEs) pervasively found in animal genomes. The Piwi targeting mechanism is proposed to be similar to targeting by Argonaute proteins, which employ microRNA (miRNA) guides to repress cellular mRNAs, but has not been characterized in detail. We present cryo-EM structures of a Piwi-piRNA complex with and without target RNAs and analysis of target recognition. Resembling Argonaute, Piwi identifies targets using the piRNA seed-region. However, Piwi creates a much weaker seed so that prolonged target association requires further piRNA-target pairing. Beyond the seed, Piwi creates wide central cleft wide for unencumbered piRNA-target pairing, enabling long-lived Piwi-piRNA-target interactions that are tolerant of mismatches. Piwi ensures targeting fidelity by blocking propagation of the piRNA-target duplex in the absence of faithful seed pairing, and by requiring extended piRNA-target pairing to reach an endonucleolytically active conformation. This mechanism allows Piwi to minimize off-targeting cellular mRNAs and adapt piRNA sequences to evolving genomic threats.

molecular biology↗

bioRxiv2020

Skipping and sliding to optimize target search on protein-bound DNA and RNA

Rapidly finding a specific nucleic-acid sequences in a large pool of competing off-targets is a fundamental challenge overcome by all living systems. To optimize the search and beat the diffusion limit, it is known that searchers should spend time sliding along the nucleic-acid substrate. Still, such sliding generally has to contend with high levels of molecular crowding on the substrate, and it remains unclear what effect this has on optimal search strategies. Using mechanistic modelling informed by single-molecule data, we show how sliding combined with correlated short-ranged skips allow searchers to maintain search speed on densely crowded substrates. We determine the conditions of optimal search, which show that an optimized searchers always spend more than half its time skipping and sliding along the substrate. Applying our theory to single-molecule data, we determine that both human and bacterial Argonaute proteins alternate between sliding 10 nt and skipping 30 nt along the substrate. We show that this combination of skipping and sliding lengths allows the searcher to maintain search speeds largely unaffected by molecular roadblocks covering up to 70% of the substrate. Our novel combination of experimental and theoretical approach could also help elucidate how other systems ensure rapid search in crowded environments.

biophysics↗

bioRxiv2019

mRNA structural dynamics shape Argonaute-target interactions

Small RNAs (such as miRNAs, siRNAs and piRNAs) regulate protein expression in a wide variety of biological processes and play an important role in cellular function, development and disease. Association of small RNAs with Argonaute (AGO) family proteins guide AGO to target RNAs, generally resulting in target silencing through transcriptional silencing, translational repression or mRNA degradation. Here we develop a live-cell single-molecule imaging assay to simultaneously visualize translation of individual mRNA molecules and their silencing by human AGO2-siRNA complexes. We find that siRNA target sites are commonly masked in vivo by RNA secondary structures, which inhibit AGO2-target interactions. Translating ribosomes unmask AGO2 binding sites, stimulating AGO2-target interactions and promoting mRNA degradation. Using a combination of mathematical modeling and experiments, we find that mRNA structures are highly heterogeneous and continuously refolding. We show that structural dynamics of mRNAs shape AGO2-target recognition, which may be a common feature controlling mRNA-protein interactions.

molecular biology↗

bioRxiv2018

Beyond the seed: structural basis for supplementary mircoRNA targeting

microRNAs (miRNA) guide Argonaute proteins to mRNAs targeted for repression. Target recognition occurs primarily through the miRNA seed region, composed of guide (g) nucleotides g2-g8. However, nucleotides beyond the seed are also important for some known miRNA-target interactions. Here, we report the structure of human Argonaute2 (Ago2) engaged with a target RNA recognized through both miRNA seed and supplementary (g13-g16) regions. Ago2 creates a \"supplementary chamber\" that accommodates up to 5 miRNA-target base pairs. Seed and supplementary chambers are adjacent to each other, and can be bridged by an unstructured target loop of 1-15 nucleotides. Opening of the supplementary chamber may be constrained by tension in the miRNA 3' tail as increases in miRNA length stabilize supplementary interactions. Contrary to previous reports, we demonstrate optimal supplementary interactions can increase target affinity >20-fold. These results provide a mechanism for extended miRNA-targeting, suggest a function for 3' isomiRs in tuning miRNA targeting specificity, and indicate that supplementary interactions may contribute more to target recognition than is widely appreciated.

biochemistry↗