Luisa Cochella

A Toolkit and Robust Pipeline for the Generation of Fosmid-Based Reporter Genes in C. elegans

Engineering fluorescent proteins into large genomic clones, contained within BACs or fosmid vectors, is a tool to visualize and study spatiotemporal gene expression patterns in transgenic animals. Because these reporters cover large genomic regions, they most likely capture all cis-regulatory information and can therefore be expected to recapitulate all aspects of endogenous gene expression. Inserting tags at the target gene locus contained within genomic clones by homologous recombination (“recombineering”) represents the most straightforward method to generate these reporters. In this methodology paper, we describe a simple and robust pipeline for recombineering of fosmids, which we apply to generate reporter constructs in the nematode C. elegans, whose genome is almost entirely covered in an available fosmid library. We have generated a toolkit that allows for insertion of fluorescent proteins (GFP, YFP, CFP, VENUS, mCherry) and affinity tags at specific target sites within fosmid clones in a virtually seamless manner. Our new pipeline is less complex and, in our hands, works more robustly than previously described recombineering strategies to generate reporter fusions for C. elegans expression studies. Furthermore, our toolkit provides a novel recombineering cassette which inserts a SL2-spliced intercistronic region between the gene of interest and the fluorescent protein, thus creating a reporter controlled by all 5′ and 3′ cis-acting regulatory elements of the examined gene without the direct translational fusion between the two. With this configuration, the onset of expression and tissue specificity of secreted, sub-cellular compartmentalized or short-lived gene products can be easily detected. We describe other applications of fosmid recombineering as well. The simplicity, speed and robustness of the recombineering pipeline described here should prompt the routine use of this strategy for expression studies in C. elegans.

Mutational analysis reveals two independent molecular requirements during transfer RNA selection on the ribosome

Accurate discrimination between cognate and near-cognate aminoacyl-tRNAs during translation relies on the specific acceleration of forward rate constants for cognate tRNAs. Such specific rate enhancement correlates with conformational changes in the tRNA and small ribosomal subunit that depend on an RNA-specific type of interaction, the A-minor motif, between universally conserved 16S ribosomal RNA nucleotides and the cognate codon-anticodon helix. We show that perturbations of these two components of the A-minor motif, the conserved rRNA bases and the codon-anticodon helix, result in distinct outcomes. Although both cause decreases in the rates of tRNA selection that are rescued by aminoglycoside antibiotics, only disruption of the codon-anticodon helix is overcome by a miscoding tRNA variant. On this basis, we propose that two independent molecular requirements must be met to allow tRNAs to proceed through the selection pathway, providing a mechanism for exquisite control of fidelity during this step in gene expression.

Fidelity in protein synthesis.

Given that tRNAs are aminoacylated with such great accuracy by aminoacyl-tRNA synthetases (10−4–10−5), high fidelity translation then depends on selection of the cognate aminoacyl-tRNA corresponding to the codon presented by the mRNA in the ribosome. Here, the substrates, aminoacyl-tRNAs, are discriminated primarily on the basis of their anticodon sequences. The difference in free energy of binding between the cognate and a non-cognate aminoacyl-tRNA (with two or three mismatches to the codon in the mRNA) is easily large enough to exclude the latter from the ribosome. Discrimination of near-cognate aminoacyl-tRNAs, with only one mismatch between codon and anticodon, with high accuracy, however, is not a trivial problem. There are generally sufficient binding energy differences to allow discrimination between cognate and near-cognate pairings (especially if these differences are sampled several times). But because tRNA selection has the additional constraint of needing to be fast, as translation is rapid and processive, these differences cannot be adequately exploited. Indeed, the rapid rate of translation apparently precludes the establishment of equilibrium between the various tRNAs and the ribosome–mRNA complex, thus calling into action kinetic discrimination mechanisms.The first strategy shown to operate during aminoacyl-tRNA selection has been termed kinetic proofreading. It was long ago realized that, if substrate selection were separated into two distinct phases by an irreversible step (in this case GTP hydrolysis), there would be two opportunities to examine and discard an incorrect aminoacyl-tRNA (Figure 3Figure 3). This means that the binding energy between the ribosome and the ternary complex can be sampled twice and the specificity thus increased. While the idea of having consecutive selective steps is similar to the ‘double-sieve editing’ mechanism discussed above, it is distinguished by the fact that kinetic proofreading applies the same basic selective step twice, whereas editing generally relies on a second distinct site or activity that monitors different properties than the first selective step.Figure 3Detailed kinetic scheme for tRNA selection highlighting the two stages of the process, initial selection and proofreading.The selectivity of the initial selection stage is determined by the difference in rate of GTPase activation (k3) between the cognate and a near-cognate tRNA. The selectivity of the proofreading stage is determined primarily by the difference in rate of accommodation (k5) between the cognate and a near-cognate tRNA. EF-Tu (green) is shown in two different conformations before and after GTP hydrolysis.View Large Image | View Hi-Res Image | Download PowerPoint SlideKinetic proofreading during tRNA selection is made possible by the fact that aminoacyl-tRNAs are delivered to the ribosome in a ternary complex with the GTPase elongation factor Tu (EF-Tu in bacteria, EF1A in eukaryotes) and GTP. In an encounter between ternary complex and the ribosome (initial selection), a cognate ternary complex is more likely to trigger GTP hydrolysis than to dissociate, whereas a near-cognate ternary complex is more likely to dissociate. Simply put, the cognate species partitions forward in the stepwise scheme whereas the near-cognate partitions backward. This initial selection step is followed by the proofreading step where inherent binding differences between codon and anticodon are again sampled. As before, the cognate aminoacyl-tRNA species is more likely to partition forward (and ‘accommodate’ into the A site and participate in peptide bond formation), while the near-cognate aminoacyl-tRNAs are more likely to partition backward (and be rejected from the ribosome).The relative contribution of each of these selective steps, initial selection and proofreading, has been measured in vitro in multiple ways, where overall error rates of ~1 in 450 to 1 in 1600 approach the overall fidelity measured in vivo. In these systems, essentially all non-cognate aminoacyl-tRNAs are rejected during initial selection. Near-cognate aminoacyl-tRNAs, however, can pass through initial selection and trigger GTP hydrolysis with a frequency of ~1 in 30. These sneaky aminoacyl-tRNAs are generally rejected during the second stage, thus increasing selectivity by ~15–45-fold.Interestingly, the maximal theoretical selectivity of kinetic proofreading is not realized here because of the processive nature of translation and associated requirement for speed. To maximize each selective step, the forward rates should be slow enough that differences in dissociation rates can be exploited. Indeed, experimental evidence shows that, when GTP hydrolysis is made extremely slow, the selectivity observed in this initial selection step is substantially increased. It was long ago suggested that ribosome mutations which affect the fidelity of tRNA selection act similarly by increasing or decreasing the rates of individual steps in the selection process.While separating the process into two steps — kinetic proofreading — does provide an advantage during tRNA selection, it is not because differences in dissociation rates between cognate and near-cognate aminoacyl-tRNAs are exploited twice, as previously thought. Rather, during each stage of tRNA selection a second strategy comes into play that introduces a large difference in the rates of two critical forward steps. During initial selection, the rate of GTPase activation (k3) is significantly faster for the cognate than for near-cognate aminoacyl-tRNAs, and during proofreading, there are similar differential rates of accommodation (k5) (Figure 3Figure 3). These differences in forward rates have been attributed to a mechanism historically termed induced fit, which is used by the translation machinery, polymerases and a number of other enzymes. Induced fit refers to the ability of a correct substrate, but not an incorrect one, to cause conformational changes in the enzyme and/or the substrate which have downstream effects on catalysis. During tRNA selection on the ribosome a series of conformational changes induced by binding of the cognate aminoacyl-tRNA, but not a near-cognate one, result in a number of rearrangements in the ribosome and the tRNA itself that result in the kinetic effects discussed above.It appears then that the combination of kinetic proofreading and induced fit in tRNA selection provides a suitable balance between fidelity and rapid elongation rates. If we take the simplest case of kinetic proofreading, where there are no differences in forward rate constants introduced by induced fit — where k3 and k5 are equivalent for both cognate and near-cognate tRNAs — significant discrimination between cognate and near-cognate tRNAs will only be observed when k3 and k5 are very, very slow relative to k–2 and k7. In other words, if tRNA selection were an equilibrium process where the full discrimination potential was extracted from the binding energy, there would be no need for other discriminatory mechanisms. But such a slow step in translation is apparently not compatible with the overall rapid rate of elongation.The addition of induced fit to the process of tRNA selection boosts selectivity when the reaction is constrained to be fast by accelerating the rate of passage of cognate species relative to near-cognate ones. Because forward rates are fast relative to reverse ones, the selectivity of each step is lower than the theoretical maximum allowed by intrinsic energetic differences between cognate and near-cognate tRNAs in the complex. In this case, the energetic cost of inducing conformational changes has little impact on cognate tRNA selection but has substantial detrimental effects on near-cognate tRNA selection thus conferring increased specificity. Effects on forward rates that result from an induced fit mechanism have been shown to be a dominant determinant of fidelity in tRNA selection. A body of experimental data supports this idea by showing that miscoding increases when the differences in GTPase activation and accommodation rates are decreased either by introduction of a mutation in the tRNA body or by addition of antibiotics like paromomycin and streptomycin.We have discussed two general mechanisms used to maintain the high fidelity of protein synthesis (as well as of DNA replication and transcription). The first mechanism is comprised of editing and kinetic proofreading. Although different in detail, both strategies amplify the available discrimination power, determined by differences in free energy of binding, by having more than one selective step. Of these two strategies, editing has an advantage arising from the use of two distinct sites that scrutinize different properties of the substrate. The second mechanism, induced fit, depends on substrate-specific conformational changes that result in selective modulation of forward rate constants, permitting high fidelity discrimination when rapid rates are essential. Such distinct solutions for different enzymes ultimately result from the evolutionary constraints imposed by the diverse requirements for fidelity, speed and efficiency on each molecular problem.

Arginine kinase of the flagellate protozoa Trypanosoma cruzi: Regulation of its expression and catalytic activity

In epimastigotes of Trypanosoma cruzi, the etiological agent of Chagas’ disease, arginine kinase activity increased continuously during the exponential phase of growth. A correlation between growth rate, enzyme‐specific activity and enzyme protein was observed. Arginine kinase‐specific activity, expressed as a function of enzyme protein, remains roughly constant up to 18 days of culture. In the whole range of the culture time mRNA levels showed minor changes indicating that the enzyme activity is post‐transcriptionally regulated. Arginine kinase could be proposed as a modulator of energetic reserves under starvation stress condition.

Temporal and spatial regulation of microRNA activity with photoactivatable cantimirs.

MicroRNAs (miRNAs) are small non-coding RNAs that play numerous important roles in physiology and human diseases. During animal development, many miRNAs are expressed continuously from early embryos throughout adults, yet it is unclear whether these miRNAs are actually required at all the stages of development. Current techniques of manipulating microRNA function lack the required spatial and temporal resolution to adequately address the functionality of a given microRNA at a specific time or at single-cell resolution. To examine stage- or cell-specific function of miRNA during development and to achieve precise control of miRNA activity, we have developed photoactivatable antisense oligonucleotides against miRNAs. These caged oligonucleotides can be activated with 365 nm light with extraordinarily high efficiency to release potent antisense reagents to inhibit miRNAs. Initial application of these caged antimirs in a model organism (C. elegans) revealed that the activity of a miRNA (lsy-6) is required specifically around the comma stage during embryonic development to control a left/right asymmetric differentiation program in the C. elegans nervous system. This suggests that a transient input of lsy-6 during development is sufficient to specify the neuronal cell fate.

Diverse functions of microRNAs in nervous system development.

MicroRNAs (miRNAs) are integral parts of the gene regulatory networks that control most developmental processes. Through their regulatory action, miRNAs introduce an additional layer of genetic complexity that can translate into increased cellular diversity, something that is extremely relevant to nervous system structure. In addition, miRNAs sharpen the spatial and temporal boundaries between different cellular states during development. Here, we illustrate these roles with a number of specific miRNAs that act during distinct steps of neural development. We further discuss specific aspects of miRNA function that make these regulators particularly suited to provide the robustness and complexity that are essential for the dynamic nature of both the development and activity of the nervous system.

A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans

One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome), we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1) the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2) the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single-cell embryo.

Wobble during decoding: more than third-position promiscuity.

Structural studies by the Ramakrishnan and Agris groups allow us to directly observe how the ribosomes decoding site accommodates non-Watson-Crick base pairs in the third position of the codon-anticodon triplet while maintaining the one-amino-acid-per-codon framework that is central to life.

Cell-type specific sequencing of microRNAs from complex animal tissues

MicroRNAs (miRNAs) play an essential role in the post-transcriptional regulation of animal development and physiology. However, in vivo studies aimed at linking miRNA function to the biology of distinct cell types within complex tissues remain challenging, partly because in vivo miRNA-profiling methods lack cellular resolution. We report microRNome by methylation-dependent sequencing (mime-seq), an in vivo enzymatic small-RNA-tagging approach that enables high-throughput sequencing of tissue- and cell-type-specific miRNAs in animals. The method combines cell-type-specific 3′-terminal 2′-O-methylation of animal miRNAs by a genetically encoded, plant-specific methyltransferase (HEN1), with chemoselective small-RNA cloning and high-throughput sequencing. We show that mime-seq uncovers the miRNomes of specific cells within Caenorhabditis elegans and Drosophila at unprecedented specificity and sensitivity, enabling miRNA profiling with single-cell resolution in whole animals. Mime-seq overcomes current challenges in cell-type-specific small-RNA profiling and provides novel entry points for understanding the function of miRNAs in spatially restricted physiological settings.

Olfaction regulates organismal proteostasis and longevity via microRNA-dependent signaling

The maintenance of proteostasis is crucial for any organism to survive and reproduce in an ever changing environment, but its efficiency declines with age. Posttranscriptional regulators such as microRNAs control protein translation of target mRNAs with major consequences for development, physiology, and longevity. However, the precise function of lifespan-determining microRNAs remains poorly understood. Here we show that the microRNA mir-71 controls organismal proteostasis and aging in Caenorhabditis elegans by regulating its conserved target tir-1 in AWC olfactory neurons. We screened a collection of microRNAs that control aging4 to identify regulators of organismal proteostasis and discovered that the lifespan promoting mir-71 affects ubiquitin-dependent protein turnover, particularly in the intestine. We show that mir-71 directly inhibits the toll receptor domain protein TIR-1 in AWC olfactory neurons. Neuronal signaling is required for mir-71/tir-1-dependent and diet-dependent regulation of organismal proteostasis. Disruption of mir-71/tir-1 or loss of AWC olfactory neurons eliminates the influence of food source on proteostasis. Mir-71-mediated regulation of TIR-1 controls chemotactic behavior and is regulated by odor. Our findings support a model whereby odor promotes mir-71-mediated inhibition of TIR-1 in AWC neurons to stimulate organismal protein turnover. Thus, odor perception influences cell-type specific miRNA-target interaction to regulate organismal proteostasis and longevity. We anticipate that the proposed mechanism of food perception will stimulate further research on neuroendocrine brain-to-gut communication and may open the possibility for therapeutic interventions to improve proteostasis and organismal health via the sense of smell, with potential implication for obesity, diabetes and aging.