The axoneme, a microtubule-based array at the center of every cilium, has been the subject of structural investigations for decades, but only recent advances in cryo-EM and cryo-ET have allowed a molecular-level interpretation of the entire complex to be achieved. The unique properties of the nine doublet microtubules and central pair of singlet microtubules that form the axoneme, including the highly decorated tubulin lattice and the docking of massive axonemal complexes, provide opportunities and challenges for sample preparation, 3D reconstruction and atomic modeling. Here, the approaches used for cryo-EM and cryo-ET of axonemes are reviewed, while highlighting the unique opportunities provided by the latest generation of AI-guided tools that are transforming structural biology.

Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections 1 . Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes 2 . The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.

Axonemal dyneins are tethered to doublet microtubules inside cilia to drive ciliary beating, a process critical for cellular motility and extracellular fluid flow. Axonemal dyneins are evolutionarily and biochemically distinct from cytoplasmic dyneins that transport cargo, and the mechanisms regulating their localization and function are poorly understood. Here, we report a single-particle cryo-EM reconstruction of a three-headed axonemal dynein natively bound to doublet microtubules isolated from cilia. The slanted conformation of the axonemal dynein causes interaction of its motor domains with the neighboring dynein complex. Our structure shows how a heterotrimeric docking complex specifically localizes the linear array of axonemal dyneins to the doublet microtubule by directly interacting with the heavy chains. Our structural analysis establishes the arrangement of conserved heavy, intermediate and light chain subunits, and provides a framework to understand the roles of individual subunits and the interactions between dyneins during ciliary waveform generation. Axonemal dyneins are tethered to doublet microtubules inside cilia to drive ciliary beating but the mechanisms regulating their localization and function are poorly understood. Here authors report a cryo-EM reconstruction of a three-headed axonemal dynein natively bound to doublet microtubules isolated from cilia which provides a framework to understand the roles of individual subunits.

Nonenzymatic copying of RNA templates with activated nucleotides is a useful model for studying the emergence of heredity at the origin of life. Previous experiments with defined-sequence templates have pointed to the poor fidelity of primer extension as a major problem. Here we examine the origin of mismatches during primer extension on random templates in the simultaneous presence of all four 2-aminoimidazole-activated nucleotides. Using a deep sequencing approach that reports on millions of individual template-product pairs, we are able to examine correct and incorrect polymerization as a function of sequence context. We have previously shown that the predominant pathway for primer extension involves reaction with imidazolium-bridged dinucleotides, which form spontaneously by the reaction of two mononucleotides with each other. We now show that the sequences of correctly paired products reveal patterns that are expected from the bridged dinucleotide mechanism, whereas those associated with mismatches are consistent with direct reaction of the primer with activated mononucleotides. Increasing the ratio of bridged dinucleotides to activated mononucleotides, either by using purified components or by using isocyanide-based activation chemistry, reduces the error frequency. Our results point to testable strategies for the accurate nonenzymatic copying of arbitrary RNA sequences.

The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozyme-catalyzed polymerization using more stable substrates such as biological 5'-triphosphates. Despite significant progress in both approaches in recent years, the assembly and copying of functional RNA sequences under prebiotic conditions remains a challenge. Here, we explore an alternative approach to RNA-templated RNA copying that combines ribozyme catalysis with RNA substrates activated with a prebiotically plausible leaving group, 2-aminoimidazole (2AI). We applied in vitro selection to identify ligase ribozymes that catalyze phosphodiester bond formation between a template-bound primer and a phosphor-imidazolide-activated oligomer. Sequencing revealed the progressive enrichment of 10 abundant sequences from a random sequence pool. Ligase activity was detected in all 10 RNA sequences; all required activation of the ligator with 2AI and generated a 3'-5' phosphodiester bond. We propose that ribozyme catalysis of phosphodiester bond formation using intrinsically reactive RNA substrates, such as imidazolides, could have been an evolutionary step connecting purely nonenzymatic to ribozyme-catalyzed RNA template copying during the origin of life.

The emergence of the replication of RNA oligonucleotides was a critical step in the origin of life. An important model for the study of nonenzymatic template copying, which would be a key part of any such pathway, involves the reaction of ribonucleoside-5 '-phosphorimidazolides with an RNA primer/template complex. The mechanism by which the primer becomes extended by one nucleotide was assumed to be a classical in-line nucleophilic-substitution reaction in which the 3 '-hydroxyl of the primer attacks the phosphate of the incoming activated monomer with displacement of the imidazole leaving group. Surprisingly, this simple model has turned out to be incorrect, and the dominant pathway has now been shown to involve the reaction of two activated nucleotides with each other to form a 5 '-5 '-imidazolium bridged dinucleotide intermediate. Here we review the discovery of this unexpected intermediate, and the chemical, kinetic, and structural evidence for its role in template copying chemistry.

We report the synthesis of guanosine 5′-(4-methylimidazolyl)phosphonate (ICG), the third member of a series of nonhydrolyzable nucleoside 5′-phosphoro-2-methylimidazolide (2-MeImpN) analogues designed for mechanistic studies of nonenzymatic RNA primer extension. The addition of a 2-MeImpN monomer to a primer is catalyzed by the presence of a downstream activated monomer, yet the three nonhydrolyzable analogues do not show catalytic effects under standard mildly basic primer extension conditions. Surprisingly, ICG, which has a pKa similar to that of 2-MeImpG, is a modest catalyst of nonenzymatic primer extension at acidic pH. Here we show that ICG reacts with 2-MeImpC to form a stable 5′–5′-imidazole-bridged guanosine-cytosine dinucleotide, with both a labile nitrogen–phosphorus and a stable carbon–phosphorus linkage flanking the central imidazole bridge. Cognate RNA primer–template complexes react with this GC-dinucleotide by attack of the primer 3′-hydroxyl on the activated N–P side of the 5′-5′-imidazole bridge. These observations support the hypothesis that 5′–5′-imidazole-bridged dinucleotides can bind to cognate RNA primer–template duplexes and adopt appropriate conformations for subsequent phosphodiester bond formation, consistent with our recent mechanistic proposal that the formation of activated 5′–5′-imidazolium-bridged dinucleotides is responsible for 2-MeImpN-driven primer extension.

The importance of genome replication has inspired detailed crystallographic studies of enzymatic DNA/RNA polymerization. In contrast, the mechanism of nonenzymatic polymerization is less well understood, despite its critical role in the origin of life. Here we report the direct observation of nonenzymatic RNA primer extension through time-resolved crystallography. We soaked crystals of an RNA primer-template-dGMP complex with guanosine-5′-phosphoro-2-aminoimidazolide for increasing times. At early times we see the activated ribonucleotides bound to the template, followed by formation of the imidazolium-bridged dinucleotide intermediate. At later times, we see a new phosphodiester bond forming between the primer and the incoming nucleotide. The intermediate is pre-organized because of the constraints of base-pairing with the template and hydrogen bonding between the imidazole amino group and both flanking phosphates. Our results provide atomic-resolution insight into the mechanism of nonenzymatic primer extension, and set the stage for further structural dissection and optimization of the RNA copying process.

Before the advent of polymerase enzymes, the copying of genetic material during the origin of life may have involved the nonenzymatic polymerization of RNA monomers that are more reactive than the biological nucleoside triphosphates. Activated RNA monomers such as nucleotide 5'-phosphoro-2-aminoimidazolides spontaneously form an imidazolium-bridged dinucleotide intermediate that undergoes rapid nonenzymatic template-directed primer extension. However, it is unknown whether the intermediate can form on the template or only in solution and whether the intermediate is prone to hydrolysis when bound to the template or reacts preferentially with the primer. Here we show that an activated monomer can first bind the template and then form an imidazolium-bridged intermediate by reacting with a 2-aminoimidazole-activated downstream oligonucleotide. We have also characterized the partition of the template-bound intermediate between hydrolysis and primer extension. In the presence of the catalytic metal ion Mg2+, >90% of the template-bound intermediate reacts with the adjacent primer to generate the primer extension product while less than 10% reacts with competing water. Our results indicate that an RNA template can catalyze a multistep phosphodiester bond formation pathway while minimizing hydrolysis with a specificity reminiscent of an enzyme-catalyzed reaction.

The nonenzymatic copying of RNA templates with imidazole-activated nucleotides is a well-studied model for the emergence of RNA self-replication during the origin of life. We have recently discovered that this reaction can proceed through the formation of an imidazolium-bridged dinucleotide intermediate that reacts rapidly with the primer. To gain insight into the relationship between the structure of this intermediate and its reactivity, we cocrystallized an RNA primer-template complex with a close analog of the intermediate, the triphosphate-bridged guanosine dinucleotide GpppG, and solved a high-resolution X-ray structure of the complex. The structure shows that GpppG binds the RNA template through two Watson-Crick base pairs, with the primer 3'-hydroxyl oriented to attack the 5'-phosphate of the adjacent G residue. Thus, the GpppG structure suggests that the bound imidazolium-bridged dinucleotide intermediate would be preorganized to react with the primer by in-line S(N)2 substitution. The structures of bound GppG and GppppG suggest that the length and flexibility of the 5'-5' linkage are important for optimal preorganization of the complex, whereas the position of the 5'-phosphate of bound pGpG explains the slow rate of oligonucleotide ligation reactions. Our studies provide a structural interpretation for the observed reactivity of the imidazolium-bridged dinucleotide intermediate in nonenzymatic RNA primer extension.

The nonenzymatic polymerization of RNA may have enabled copying of functional sequences during the origin of life. Recent progress utilizing 5'-phosphoro-2-aminoimidazole activation has reinvigorated the possibility of using nonenzymatic RNA polymerization for copying arbitrary sequences. However, the reasons why 2-aminoimidazole (AI) is a superior activation group remain unclear. Here we report that the predominant mechanism of polymerization using cytidine-5'-phosphoro-2-aminoimidazolide (Cp*) involves a 2-aminoimidazolium-bridged dinucleotide (Cp*pC) intermediate. To explore the role of this intermediate, we first identify and quantify four reactions involving the synthesis and breakdown of Cp*pC that occur in the absence of the primer-template duplex. We then analyze the dependence of the rate of polymerization on the concentration of the Cp*pC intermediate in the presence and absence of the competitive inhibitor Cp. We also show that the contribution of the monomer Cp* to the polymerization rate is negligible under our primer extension conditions. Finally, we use the experimentally determined rate constants of these reactions to develop a kinetic model that helps explain the changing rate of nonenzymatic RNA polymerization over time. Our model accounts for the concentration of Cp*pC formed by Cp* under primer extension conditions. The model does not completely account for the decline in polymerization rate observed over long times, which indicates that additional important inhibitory processes have not yet been identified. Our results suggest that the superiority of 2-aminoimidazole over the traditional 2-methylimidazole activation is mostly due to the higher level of accumulation of the imidazolium-bridged intermediate under primer extension conditions.

Reads per million for each gene. (XLSX 6277Â kb)

Because of its importance for the origin of life, the nonenzymatic copying of RNA templates has been the subject of intense study for several decades. Previous characterizations of template-directed primer extension using 5′-phosphoryl-2-methylimidazole-activated nucleotides (2-MeImpNs) as substrates have assumed a classical in-line nucleophilic substitution mechanism, in which the 3′-hydroxyl of the primer attacks the phosphate of the incoming monomer, displacing the 2-methylimidazole leaving group. However, we have found that the initial rate of primer extension depends on the pH and concentration at which the activated monomer is maintained prior to the primer extension reaction. These and other results suggest an alternative mechanism, in which two monomers react with each other to form an imidazolium-bridged dinucleotide intermediate, which then binds to the template. Subsequent attack of the 3′-hydroxyl of the primer displaces an activated nucleotide as the leaving group and results in extension of the primer by one nucleotide. Analysis of monomer solutions by NMR indicates formation of the proposed imidazolium-bridged dinucleotide in the expected pH-dependent manner. We have used synthetic methods to prepare material that is enriched in this proposed intermediate and show that it is a highly reactive substrate for primer extension. The formation of an imidazolium-bridged dinucleotide intermediate provides a mechanistic interpretation of previously observed catalysis by an activated nucleotide located downstream from the site of primer extension.

Number of motifs found significant using different numbers of clusters, at different cutoffs. (XLSX 5Â kb)

Background Understanding gene expression across the diverse metazoan cell types during development is critical to understanding their function and regulation. However, most cell types have not been assayed for expression genome-wide. Results We applied a novel approach we term “Profiling of Overlapping Populations of cells (POP-Seq)” to assay differential expression across all embryonic cells in the nematode Caenorhabditis elegans . In this approach, we use RNA-seq to define the transcriptome of diverse partially overlapping FACS-sorted cell populations. This identified thousands of transcripts differentially expressed across embryonic cells. Hierarchical clustering analysis identified over 100 sets of coexpressed genes corresponding to distinct patterns of cell type specific expression. We identified thousands of candidate regulators of these clusters based on enrichment of transcription factor motifs and experimentally determined binding sites. Conclusions Our analysis provides new insight into embryonic gene regulation, and provides a resource for improving our knowledge of tissue-specific expression and its regulation throughout C. elegans development.

While many transcriptional regulators of pluripotent and terminally differentiated states have been identified, regulation of intermediate progenitor states is less well understood. Previous high throughput cellular resolution expression studies identified dozens of transcription factors with lineage-specific expression patterns in C. elegans embryos that could regulate progenitor identity. In this study we identified a broad embryonic role for the C. elegans OTX transcription factor ceh-36, which was previously shown to be required for the terminal specification of four neurons. ceh-36 is expressed in progenitors of over 30% of embryonic cells, yet is not required for embryonic viability. Quantitative phenotyping by computational analysis of time-lapse movies of ceh-36 mutant embryos identified cell cycle or cell migration defects in over 100 of these cells, but most defects were low-penetrance, suggesting redundancy. Expression of ceh-36 partially overlaps with that of the PITX transcription factor unc-30. unc-30 single mutants are viable but loss of both ceh-36 and unc-30 causes 100% lethality, and double mutants have significantly higher frequencies of cellular developmental defects in the cells where their expression normally overlaps. These factors are also required for robust expression of the downstream developmental regulator mls-2/HMX. This work provides the first example of genetic redundancy between the related yet evolutionarily distant OTX and PITX families of bicoid class homeodomain factors and demonstrates the power of quantitative developmental phenotyping in C. elegans to identify developmental regulators acting in progenitor cells.

The Wnt signaling pathway plays a conserved role during animal development in transcriptional regulation of distinct targets in different developmental contexts but it remains unclear whether quantitative differences in the nuclear localization of effector proteins TCF and β-catenin contribute to context-specific regulation. We investigated this question in Caenorhabditis elegans embryos by quantifying nuclear localization of fluorescently tagged SYS-1/β-catenin and POP-1/TCF and expression of Wnt ligands at cellular resolution by time-lapse microscopy and automated lineage tracing. We identified reproducible, quantitative differences that generate a subset of Wnt-signaled cells with a significantly higher nuclear concentration of the TCF/β-catenin activating complex. Specifically, β-catenin and TCF are preferentially enriched in nuclei of daughter cells whose parents also had high nuclear levels of that protein, a pattern that could influence developmental gene expression. Consistent with this, we found that expression of synthetic reporters of POP-1-dependent activation is biased towards cells that had high nuclear SYS-1 in consecutive divisions. We identified new genes whose embryonic expression patterns depend on pop-1. Most of these require POP-1 for either transcriptional activation or repression, and targets requiring POP-1 for activation are more likely to be expressed in the cells with high nuclear SYS-1 in consecutive divisions than those requiring POP-1 for repression. Taken together, these results indicate that SYS-1 and POP-1 levels are influenced by the parent cell’s SYS-1/POP-1 levels and this may provide an additional mechanism by which POP-1 regulates distinct targets in different developmental contexts.