Zhang H et al. (2010) Dev Biol "Cell autonomous specification of temporal identity by Caenorhabditis elegans microRNA lin-4."

Fire AZ, Zhang H

[Dev Biol, 2010]

MicroRNAs provide developing systems with substantial flexibility in posttranscriptional gene regulation. Despite advances made in understanding microRNA structure and function, the relationships between their site-of-synthesis and site-of-action ("autonomy" versus "non-autonomy") remain an open question. Given the well-defined role of microRNA lin-4 in a reproducible series of time-specific developmental switches, lin-4 is an excellent candidate for understanding whether microRNAs and the resulting heterochronic regulatory pathway have the potential to act cell autonomously. By monitoring temporal development and reporter activity in animals where lin-4 is modulated, we have demonstrated that lin-4 acts cell autonomously to specify temporal identity. This work (i) provides an example of cell autonomy in microRNA functions, and (ii) reveals a cell autonomous component of temporal regulation in C. elegans.

Arribere JA et al. (2018) Elife "Nonsense mRNA suppression via nonstop decay."

Arribere JA, Fire AZ

[Elife, 2018]

Nonsense-mediated mRNA decay is the process by which mRNAs bearing premature stop codons are recognized and cleared from the cell. While considerable information has accumulated regarding recognition of the premature stop codon, less is known about the ensuing mRNA suppression. During the characterization of a second, distinct translational surveillance pathway (nonstop mRNA decay), we trapped intermediates in nonsense mRNA degradation. We present data in support of a model wherein nonsense-mediated decay funnels into the nonstop decay pathway in C. elegans. Specifically, our results point to SKI-exosome decay and pelota-based ribosome removal as key steps facilitating suppression and clearance of prematurely-terminated translation complexes. These results suggest a model in which premature stop codons elicit nucleolytic cleavage, with the nonstop pathway disengaging ribosomes and degrading the resultant RNA fragments to suppress ongoing expression.

Maniar JM et al. (2011) Curr Biol "EGO-1, a C. elegans RdRP, modulates gene expression via production of mRNA-templated short antisense RNAs."

Maniar JM, Fire AZ

[Curr Biol, 2011]

BACKGROUND: The development of the germline in Caenorhabditis elegans is a complex process involving the regulation of thousands of genes in a coordinated manner. Several genes required for small RNA biogenesis and function are among those required for the proper organization of the germline. EGO-1 is a putative RNA-directed RNA polymerase (RdRP) that is required for multiple aspects of C. elegans germline development and efficient RNA interference (RNAi) of germline-expressed genes. RdRPs have been proposed to act through a variety of mechanisms, including the posttranscriptional targeting of specific mRNAs, as well as through a direct interaction with chromatin. Despite extensive investigation, the molecular role of EGO-1 has remained enigmatic. RESULTS: Here we use high-throughput small RNA and messenger RNA sequencing to investigate EGO-1 function. We found that EGO-1 is required to produce a distinct pool of small RNAs antisense to a number of germline-expressed mRNAs through several developmental stages. These potential mRNA targets fall into distinct classes, including genes required for kinetochore and nuclear pore assembly, histone-modifying activities, and centromeric proteins. We also found several RNAi-related genes to be targets of EGO-1. Finally, we show a strong association between the loss of small RNAs and the rise of mRNA levels in ego-1(-) animals. CONCLUSIONS: Our data support the conclusion that EGO-1 produces triphosphorylated small RNAs derived from mRNA templates and that these small RNAs modulate gene expression through the targeting of their cognate mRNAs.

Braukmann F et al. (2017) RNA Biol "Artificial and natural RNA interactions between bacteria and C. elegans."

Jordan D, Braukmann F, Miska E

[RNA Biol, 2017]

19years after Lisa Timmons and Andy Fire first described RNA transfer from bacteria to C. elegans in an experimental setting [Timmons and Fire, 1998 ] the biological role of this trans-kingdom RNA-based communication remains unknown. Here we summarize our current understanding on the mechanism and potential role of such social RNA.

Stigloher C et al. (2011) J Neurosci "The presynaptic dense projection of the Caenorhabditis elegans cholinergic neuromuscular junction localizes synaptic vesicles at the active zone through SYD-2/liprin and UNC-10/RIM-dependent interactions."

Richmond J, Bessereau JL, Zhen M, Zhan H, Stigloher C

[J Neurosci, 2011]

The active zone (AZ) of chemical synapses is a specialized area of the presynaptic bouton in which vesicles fuse with the plasma membrane and release neurotransmitters. Efficient signaling requires synaptic vesicles (SVs) to be recruited, primed, and retained at the AZ, in close proximity to voltage-dependent calcium channels that are activated during presynaptic depolarization. The electron-dense specializations at the AZ might provide a molecular platform for the spatial coordination of these different processes. To investigate this hypothesis, we examined high-resolution three-dimensional models of Caenorhabditis elegans cholinergic neuromuscular junctions generated by electron tomography. First, we found that SVs are interconnected within the bouton by filaments similar to those described in vertebrates. Second, we resolved the three-dimensional structure of the dense projection centered in the AZ. The dense projection is a more complex structure than previously anticipated, with filaments radiating from a core structure that directly contact SVs in the interior of the bouton as well as SVs docked at the plasma membrane. Third, we investigated the functional correlate of these contacts by analyzing mutants disrupting two key AZ proteins: UNC-10/RIM and SYD-2/liprin. In both mutants, the number of contacts between SVs and the dense projection was significantly reduced. Similar to unc-10 mutants, the dependence of SV fusion on extracellular calcium concentration was exacerbated in syd-2 mutants when compared with the wild type. Hence, we propose that the dense projection ensures proper coupling of primed vesicles with calcium signaling by retaining them at the AZ via UNC-10/RIM and SYD-2/liprin-dependent mechanisms.

Gabdank I et al. (2014) G3 (Bethesda) "Gamete-type dependent crossover interference levels in a defined region of Caenorhabditis elegans chromosome V."

Gabdank I, Fire AZ

[G3 (Bethesda), 2014]

In certain organisms, numbers of crossover events for any single chromosome are limited ("crossover interference") so that double crossover events are obtained at much lower frequencies than would be expected from the simple product of independent single-crossover events. We present a number of observations during which we examined interference over a large region of Caenorhabditis elegans chromosome V. Examining this region for multiple crossover events in heteroallelic configurations with limited dimorphism, we observed high levels of crossover interference in oocytes with only partial interference in spermatocytes.

Lipton DM et al. (2018) Cell Rep "Rapid Assembly of Presynaptic Materials behind the Growth Cone in Dopaminergic Neurons Is Mediated by Precise Regulation of Axonal Transport."

Maeder CI, Lipton DM, Shen K

[Cell Rep, 2018]

The proper assembly of neural circuits depends on the process of synaptogenesis, or the formation of synapses between partner neurons. Using the dopaminergic PDE neurons in C.elegans, we developed an invivo system to study the earliest steps of the formation of en passant presynaptic specializations behind an extending growth cone. We find that presynaptic materials coalesce into puncta in as littleas a few minutes and that both synaptic vesicle (SV) and active zone (AZ) proteins arrive nearly simultaneously at the nascent sites of synapse formation. We show that precise regulation of UNC-104/Kinesin-3 determines the distribution of SV proteins along the axon. The localization of AZ proteins to en passant puncta, however, is largely independent of the major axonal kinesins: UNC-104/Kinesin-3 and UNC-116/Kinesin-1. Moreover, AZ proteins play a crucial role in recruiting and tethering SV precursors (SVPs).

Wu YE et al. (2013) Neuron "The balance between capture and dissociation of presynaptic proteins controls the spatial distribution of synapses."

Wu YE, Maeder CI, Huo L, Feng W, Shen K

[Neuron, 2013]

The location, size, and number of synapses critically influence the specificity and strength of neural connections. In axons, synaptic vesicle (SV) and active zone (AZ) proteins are transported by molecular motors and accumulate at discrete presynaptic loci. Little is known about the mechanisms coordinating presynaptic protein transport and deposition to achieve proper distribution of synaptic material. Here we show that SV and AZ proteins exhibit extensive cotransport and undergo frequent pauses. At the axonal and synaptic pause sites, the balance between the capture and dissociation of mobile transport packets determines the extent of presynaptic assembly. The small G protein ARL-8 inhibits assembly by promoting dissociation, while a JNK kinase pathway and AZ assembly proteins inhibit dissociation. Furthermore, ARL-8 directly binds to the UNC-104/KIF1A motor to limit the capture efficiency. Together, molecular regulation of the dichotomy between axonal trafficking and local assembly controls vital aspects of synapse formation and maintenance.

Zhang H et al. (2015) RNA "Functional relevance of "seed" and "non-seed" sequences in microRNA-mediated promotion of C. elegans developmental progression."

Artiles KL, Zhang H, Fire AZ

[RNA, 2015]

The founding heterochronic microRNAs, lin-4 and let-7, together with their validated targets and well-characterized phenotypes in C. elegans, offer an opportunity to test functionality of microRNAs in a developmental context. In this study, we defined sequence requirements at the microRNA level for these two microRNAs, evaluating lin-4 and let-7 mutant microRNAs for their ability to support temporal development under conditions where the wild-type lin-4 and let-7 gene products are absent. For lin-4, we found a strong requirement for seed sequences, with function drastically affected by several central mutations in the seed sequence, while rescue was retained by a set of mutations peripheral to the seed. let-7 rescuing activity was retained to a surprising degree by a variety of central seed mutations, while several non-seed mutant effects support potential noncanonical contributions to let-7 function. Taken together, this work illustrates both the functional partnership between seed and non-seed sequences in mediating C. elegans temporal development and a diversity among microRNA effectors in the contributions of seed and non-seed regions to activity.

Alcazar RM et al. (2008) Genetics "Transmission dynamics of heritable silencing induced by double-stranded RNA in Caenorhabditis elegans."

Fire AZ, Alcazar RM, Lin R

[Genetics, 2008]

Heritable silencing effects are gene suppression phenomena that can persist for generations after induction. In the majority of RNAi experiments conducted in C. elegans, the silencing response results in a hypomorphic phenotype where the effects recede after the F1 generation. F2 and subsequent generations revert to the original phenotype. Specific examples of trans-generational RNAi have been described in which effects persist to the F2 generation and beyond. In this study, we describe a systematic pedigree-based analysis of heritable silencing processes resulting from initiation of interference targeted at the C. elegans oocyte maturation factor oma-1. Heritable silencing of oma-1 is a dose-dependent process where the inheritance of the silencing factor is unequally distributed among the population. Heritability is not constant over generational time, with silenced populations appearing to undergo a bottleneck 3-4 generations following microinjection of RNA. Transmission of silencing through these generations can be through either maternal or paternal gamete lines, and is surprisingly more effective through the male gametic line. Genetic linkage tests reveal that silencing in the early generations is transmitted independently of the original targeted locus, in a manner indicative of a diffusible epigenetic element.