C. elegans contains numerous small RNAs of~21-24 nt in length. The microRNAs (miRNAs) are small noncoding RNAs produced by DCR-1-and ALG-dependent processing of self-complementary hairpin transcripts. Endogenous small interfering RNAs (endosiRNAs), associated with ongoing silencing of protein-coding genes in normal worms, are produced by mechanisms that involve DCR-1 but, unlike miRNAs, also involve RDE-2, RDE-3, RDE-4, RRF-1, and RRF-3. The tiny noncoding (tncRNAs) are similar to endo-siRNAs in their biogenesis except that they are derived from noncoding sequences. These endo-siRNA-and tncRNA-based endogenous RNAi pathways involve some components, including DCR-1 and RDE-4, that are shared with exogenous RNAi, and some components, including RRF-3 and ERI-1, that are specific to endogenous RNAi. rrf-3 and eri-1 mutants are enhanced for some silencing processes and defective for others, suggesting cross-regulatory interactions between RNAi pathways in C. elegans. Microarray expression profiling of RNAi-defective mutant worms further suggests diverse endogenous RNAi pathways for silencing different sets of genes.

The C. elegans TRIM-NHL protein NHL-2 functions as a co-factor for the microRNA Induced Silencing Complex (miRISC) and thereby enhances the post-transcriptional repression of several genetically verified microRNA targets, including hbl-1 and let-60/Ras (by the let-7-family of microRNAs) and cog-1 (by the lsy-6 microRNA). NHL-2 is localized to cytoplasmic processing bodies (P-bodies) and physically associates with the P-body protein CGH-1 and the core miRISC components ALG-1/2 and AIN-1. nhl-2 and cgh-1 mutations compromise the repression of microRNA targets in vivo, but do not affect microRNA biogenesis, indicating a role for a NHL-2-CGH-1 complex in the effector phase of miRISC activity. We propose that the NHL-2-CGH-1 complex functions in association with mature miRISC to modulate the efficacy of microRNA:target interactions in response to physiological and developmental signals, and thereby helps ensure the robustness of genetic regulatory pathways regulated by microRNAs.

Animal development is remarkably robust; cell fates are specified with spatial and temporal precision despite physiological and environmental contingencies. Favorable conditions cause Caenorhabditis elegans to develop rapidly through four larval stages (L1-L4) to the reproductive adult. In unfavorable conditions, L2 larvae can enter the developmentally quiescent, stress-resistant dauer larva stage, enabling them to survive for prolonged periods before completing development. A specific progression of cell division and differentiation events occurs with fidelity during the larval stages, regardless of whether an animal undergoes continuous or dauer-interrupted development. The temporal patterning of developmental events is controlled by the heterochronic genes, whose products include microRNAs (miRNAs) and regulatory proteins. One of these proteins, the DAF-12 nuclear hormone receptor, modulates the transcription of certain let-7-family miRNAs, and also mediates the choice between the continuous vs. dauer-interrupted life history. Here, we report a complex feedback loop between DAF-12 and the let-7-family miRNAs involving both the repression of DAF-12 by let-7-family miRNAs and the ligandmodulated transcriptional activation and repression of the let-7-Fam miRNAs by DAF-12. We propose that this feedback loop functions to ensure robustness of cell fate decisions and to coordinate cell fate with developmental arrest.gene regulation ͉ microRNA ͉ nuclear hormone receptor T he complexity of animal development requires the coordination of temporal, spatial, and physiological cues. Successful development is necessary to achieve optimal fitness, and natural selection has favored developmental programs that robustly produce a desired outcome in a range of physiological and environmental conditions. Forward genetics has revealed much of the internal programming of several developmental processes in standardized laboratory conditions. A fundamental question emerging from these studies is how these genetic circuits are affected by conditions that are more similar to natural, variable environments. Caenorhabditis elegans has been an excellent system to uncover developmental mechanisms because of its well-defined cell lineage and tractability to genetic analysis. C. elegans development is robust in a wide range of environmental conditions, providing an excellent opportunity to determine how genetic pathways are modulated in these diverse environments (1).There are two distinct life histories a C. elegans larva may follow: a rapid, continuous life history that occurs in favorable environments and an extended, interrupted life history (2) that occurs in response to increased population density, decreasing food supplies, and elevated temperature (Fig. 1) (3). Within the continuous life history, animals progress through four larval stages (L1-L4) to a reproductively competent adult within Ϸ40-50 h after hatching (4). By contrast, unfavorable environments sensed during L1 stage cause larvae to enter the predauer L2d stage, which is ...

We reported previously that heat or ethanol shock in Saccharomyces cerevisiae leads to nuclear retention of most poly(A) + RNA but heat shock mRNAs (encoding Hsp70 proteins Ssa1p and Ssa4p) are efficiently exported in a process that is independent of the small GTPase Ran/Gsp1p, which is essential for most nucleocytoplasmic transport. To gain further insights into proteins essential or nonessential for export of heat shock mRNAs, in situ hybridization analyses to detect mRNA and pulse-labeling of proteins were used to examine several yeast mutant strains for their ability to export heat shock mRNAs following stress. Rip1p is a 42-kD protein associated with nuclear pore complexes and contains nucleoporin-like repeat sequences. It is dispensable for growth of yeast cells under normal conditions, but we report that it is essential for the export of heat shock mRNAs following stress. When SSA4 mRNA was induced from a GAL promoter in the absence of stress, it was efficiently exported in a strain lacking RIP1, indicating that Rip1p is required for export of heat shock mRNAs only following stress. Npl3p, a key mediator of export of poly(A) + RNA, was not required for heat shock mRNA export, whereas Rss1p/Gle1p, a NES-containing factor essential for poly(A) + RNA export, was also required for export of heat shock mRNAs after stress. High-level expression of the HIV-1 Rev protein, but not of Rev mutants, led to a partial block in export of heat shock mRNAs following stress. The data suggest a model wherein the requirement for Npl3p defines the mRNA export pathway, the requirement for Rip1p defines a pathway used for export of heat shock mRNAs after stress, and additional factors, including Rss1p/Gle1p and several nucleoporins (Rat7p/Nup159p, Rat2p/Nup120p, and Nup145p/Rat10p), are required in both pathways.[Key Words: RNA export; heat shock; RIP1; hnRNP; RSS1/GLE1; Rev]Received July 1, 1997; revised version accepted August 28, 1997.A distinguishing feature of eukaryotic cells is the nucleus, a distinct subcellular compartment separated from the cytoplasm by the double-membraned nuclear envelope. Embedded within the nuclear envelope are nuclear pore complexes (NPCs) that serve as the only known channels for transport between the nucleus and the cytoplasm (for review, see Davis 1995;Panté and Aebi 1996). Transport of macromolecules through NPCs is signal-mediated, saturable, and energy dependent. Considerable progress has been made in recent years in identifying (1) receptor molecules that recognize nuclear localization signals (NLSs) within karyophilic proteins and mediate interactions between these proteins and NPCs, (2) a small Ras-like GTPase (Ran in metazoan cells and Gsp1p/Gsp2p in Saccharomyces cerevisiae) and its accessory proteins, which play a central role in nuclear protein import, and (3) distinct components of NPCs required for nuclear protein import (for review, see Gö rlich and

In a screen to identify genes required for mRNA export in Saccharomyces cerevisiae, we isolated an allele of poly(A) polymerase (PAP1) and novel alleles encoding several other 3 processing factors. Many newly isolated and some previously described mutants (rna14-48, rna14-49, rna14-64, rna15-58, and pcf11-1 strains) are defective in polymerase II (Pol II) termination but, interestingly, retain the ability to polyadenylate these improperly processed transcripts at the nonpermissive temperature. Deletion of the cis-acting sequences required to couple 3 processing and termination also produces transcripts that fail to exit the nucleus, suggesting that all of these processes (cleavage, termination, and export) are coupled. We also find that several but not all mRNA export mutants produce improperly 3 processed transcripts at the nonpermissive temperature. 3 maturation defects in mRNA export mutants include improper Pol II termination and/or the previously characterized hyperpolyadenylation of transcripts. Importantly, not all mRNA export mutants have defects in 3 processing. The similarity of the phenotypes of some mRNA export mutants and 3 processing mutants indicates that some factors from each process may mechanistically interact to couple mRNA processing and export. Consistent with this assumption, we present evidence that Xpo1p interacts in vivo with several 3 processing factors and that the addition of recombinant Xpo1p to in vitro processing reaction mixtures stimulates 3 maturation. Of the core 3 processing factors tested (Rna14p, Rna15p, Pcf11p, Hrp1p, Fip1p, and Cft1p), only Hrp1p shuttles. Overexpression of Rat8p/Dbp5p suppresses both 3 processing and mRNA export defects found in xpo1-1 cells.One of the defining features of eukaryotic cells is the physical separation of the nucleus, where mRNAs are synthesized, from the cytoplasm, where protein synthesis occurs. Gene expression and cell function require the efficient transport of macromolecules between these two compartments. All exchange between the nucleus and cytoplasm takes place through nuclear pore complexes (NPCs) that perforate the nuclear envelope and permit selective passage in both directions of molecules and complexes containing transport signals. Interactions between the import substrate or complex and NPC are mediated by import or export receptors that bind directly to specific transport signals on the substrate and NPCs. Directionality of this process is imparted through the activity of a small GTPase (Gsp1p in Saccharomyces cerevisiae and Ran in metazoans) that modulates the receptor's affinity for the substrate on opposite sides of the nuclear envelope.Export of mRNA to the cytoplasm appears to be more complex than transport of proteins. mRNAs are exported as messenger ribonucleoprotein complexes (mRNPs) whose assembly begins during transcription (27,55). mRNA biogenesis requires multiple processing steps including 5Ј capping, splicing, and 3Ј cleavage or polyadenylation. Most of these reactions are accomplished during transcription by se...