MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally via the 3′ UTR of target mRNAs and were first identified in the Caenorhabditis elegans heterochronic pathway. miRNAs have since been found in many organisms and have broad functions, including control of differentiation and pluripotency in humans. lin-4 and let-7-family miRNAs regulate developmental timing in C. elegans, and their proper temporal expression ensures cell lineage patterns are correctly timed and sequentially executed. Although much is known about miRNA biogenesis, less is understood about how miRNA expression is timed and regulated. lin-42, the worm homolog of the circadian rhythm gene period of flies and mammals, is another core component of the heterochronic gene pathway. lin-42 mutants have a precocious phenotype, in which later-stage programs are executed too early, but the placement of lin-42 in the timing pathway is unclear. Here, we demonstrate that lin-42 negatively regulates heterochronic miRNA transcription. let-7 and the related miRNA miR-48 accumulate precociously in lin-42 mutants. This defect reflects transcriptional misregulation because enhanced expression of both primary miRNA transcripts (pri-miRNAs) and a let-7 promoter::gfp fusion are observed. The pri-miRNA levels oscillate during larval development, in a pattern reminiscent of lin-42 expression. Importantly, we show that lin-42 is not required for this cycling; instead, peak amplitude is increased. Genetic analyses further confirm that lin-42 acts through let-7 family miRNAs. Taken together, these data show that a key function of lin-42 in developmental timing is to dampen pri-miRNAs levels, preventing their premature expression as mature miRNAs.M etazoan development uses both positional and temporal information to pattern life stages from the single-cell embryo to the reproductive adult. In Caenorhabditis elegans, the temporal component of postembryonic patterning is conveyed through the heterochronic gene regulatory circuit (see ref. 1 for review). A core theme of the heterochronic circuit is that a series of microRNA (miRNA) switches promote transitions from one stage to the next by negatively regulating key factors that direct stage-specific programs, thereby allowing successive temporal fates to be executed.Five conserved miRNAs, lin-4 and the let-7 family members, let-7, miR-48, miR-84, and miR-241, play prominent roles in the circuit (2-6). lin-4 appears first, midway through the L1 larval stage and guides the transition from the L1 to L2 stage (7). miR-48, miR-84, and miR-241 amass in the L2 and control the transition to the L3 stage, when let-7 levels rise dramatically and govern the switch to the L4. Mutations in these miRNAs cause the relevant transition to fail to advance. This process results in, for example, repetition of the L1-stage pattern in lin-4 mutants or L2-stage patterns in mir-48/84/241 mutants, consistent with the sequential expression of these miRNAs. Deciphering this temporal control mechanism then simplifies, to a...

Alteration in the timing of particular developmental events can lead to major morphological changes that have profound effects on the life history of an organism. Insights into developmental timing mechanisms have been revealed in the model organism C. elegans, in which a regulatory network of heterochronic genes times events during larval development, ensuring that stage-specific programs occur in the appropriate sequence and on schedule. Developmental timing studies in C. elegans led to the landmark discovery of miRNAs and continue to enhance our understanding of the regulation and activity of these small regulatory molecules. Current views of the heterochronic gene pathway are summarized here, with a focus on the ways in which miRNAs contribute to temporal control and how miRNAs themselves are regulated. Finally, the conservation of heterochronic genes and their functions in timing, as well as their related roles in stem cells and cancer are highlighted.

The Caenorhabditis elegans heterochronic gene pathway regulates the relative timing of events during postembryonic development. lin-42, the worm homolog of the circadian clock gene, period, is a critical element of this pathway. lin-42 function has been defined by a set of hypomorphic alleles that cause precocious phenotypes, in which later developmental events, such as the terminal differentiation of hypodermal cells, occur too early. A subset of alleles also reveals a significant role for lin-42 in molting; larval stages are lengthened and ecdysis often fails in these mutant animals. lin-42 is a complex locus, encoding overlapping and nonoverlapping isoforms. Although existing alleles that affect subsets of isoforms have illuminated important and distinct roles for this gene in developmental timing, molting, and the decision to enter the alternative dauer state, it is essential to have a null allele to understand all of the roles of lin-42 and its individual isoforms. To remedy this problem and discover the null phenotype, we engineered an allele that deletes the entire lin-42 protein-coding region. lin-42 null mutants are homozygously viable, but have more severe phenotypes than observed in previously characterized hypomorphic alleles. We also provide additional evidence for this conclusion by using the null allele as a base for reintroducing different isoforms, showing that each isoform can provide heterochronic and molting pathway activities. Transcript levels of the nonoverlapping isoforms appear to be under coordinate temporal regulation, despite being driven by independent promoters. The lin-42 null allele will continue to be an important tool for dissecting the functions of lin-42 in molting and developmental timing.

The translation initiation complex eIF3 imparts specialized functions to regulate protein expression. However, understanding of eIF3 activities in neurons remains limited despite widespread dysregulation of eIF3 subunits in neurological disorders. Here, we report a selective role of the C. elegans RNA-binding subunit EIF-3.G in shaping the neuronal protein landscape. We identify a missense mutation in the conserved Zinc-Finger (ZF) of EIF-3.G that acts in a gain-of-function manner to dampen neuronal hyperexcitation. Using neuron type-specific seCLIP, we systematically mapped EIF-3.G-mRNA interactions and identified EIF-3.G occupancy on GC-rich 5′UTRs of a select set of mRNAs enriched in activity-dependent functions. We demonstrate that the ZF mutation in EIF-3.G alters translation in a 5′UTR dependent manner. Our study reveals an in vivo mechanism for eIF3 in governing neuronal protein levels to control neuronal activity states and offers insights into how eIF3 dysregulation contributes to neuronal disorders.

Acetylcholine (ACh) receptors (AChR) regulate neural circuit activity in multiple contexts. In humans, mutations in ionotropic acetylcholine receptor (iAChR) genes can cause neurological disorders, including myasthenia gravis and epilepsy. In Caenorhabditis elegans, iAChRs play multiple roles in the locomotor circuit. The cholinergic motor neurons express an ACR-2-containing pentameric AChR (ACR-2R) comprised of ACR-2, ACR-3, ACR-12, UNC-38, and UNC-63 subunits. A gain-of-function mutation in the non-α subunit gene acr-2 [acr-2(gf)] causes defective locomotion as well as spontaneous convulsions. Previous studies of genetic suppressors of acr-2(gf) have provided insights into ACR-2R composition and assembly. Here, to further understand how the ACR-2R regulates neuronal activity, we expanded the suppressor screen for acr-2(gf)-induced convulsions. The majority of these suppressor mutations affect genes that play critical roles in synaptic transmission, including two novel mutations in the vesicular ACh transporter unc-17. In addition, we identified a role for a conserved major facilitator superfamily domain (MFSD) protein, mfsd-6, in regulating neural circuit activity. We further defined a role for the sphingosine (SPH) kinase (Sphk) sphk-1 in cholinergic neuron activity, independent of previously known signaling pathways. Overall, the genes identified in our study suggest that optimal modulation of synaptic activity is balanced by the differential activities of multiple pathways, and the novel alleles provide valuable reagents to further dissect neuronal mechanisms regulating the locomotor circuit.