The Period protein homolog LIN-42 regulates germline development in C. elegans.

Germline stem cells are maintained in the distal region of the C. elegans gonad. These cells undergo mitotic divisions, and GLP-1/Notch signaling dictates whether they remain in this state. The somatic distal tip cell (DTC) caps the end of the distal gonad and is essential for maintenance of the germline mitotic zone. As germ cells move away from the DTC they exit mitosis and enter early meiotic prophase. Here we identify the Period protein homolog LIN-42 as a new regulator of germline development in C. elegans. LIN-42 is expressed in almost all somatic cells including the DTC, and LIN-42 functions as a transcription factor in the heterochronic pathway and to regulate molting. We found that the mitotic proliferative zone size in the distal gonad was significantly reduced by ~25% in lin-42 mutants compared to WT N2 worms. A lin-42 mutation also reduced the mitotic proliferative zone size caused by glp-1 partial loss-of-function and gain-of-function alleles. LIN-42 mediates this effect, at least in part, by regulating expression of the GLP-1/Notch ligand LAG-2. We further show that lin-42 expression itself is regulated by ATX-2, which promotes germline proliferation and is the homolog of the RNA binding protein ataxin-2 that is implicated in human neurodegenerative diseases. Altogether our results establish a new role for the conserved, important Period protein homolog LIN-42 in regulating early germline development. These results also suggest that in addition to regulating behavioral rhythms, the circadian clock plays an important role in communicating environmental signals to essential reproductive pathways.

The Doubletime Homolog KIN-20 Mainly Regulates let-7 Independently of Its Effects on the Period Homolog LIN-42 in Caenorhabditis elegans.

The Caenorhabditis elegans (C. elegans) heterochronic pathway, which regulates developmental timing, is thought to be an ancestral form of the circadian clock in other organisms. An essential member of this clock is the Period protein whose homolog, lin-42, in C. elegans is an important heterochronic gene. LIN-42 functions as a transcriptional repressor of multiple genes including the conserved lin-4 and let-7 microRNAs. Like other Period proteins, levels of LIN-42 oscillate throughout development. In other organisms this cycling is controlled in part by phosphorylation. KIN-20 is the C. elegans homolog of the Drosophila Period protein kinase Doubletime. Worms containing a large deletion in kin-20 have a significantly smaller brood size and develop slower than wild type C. elegans Here we analyze the effect of kin-20 on lin-42 phenotypes and microRNA expression. We find that kin-20 RNAi enhances loss-of-function lin-42 mutant phenotypes and that kin-20 mutant worms express lower levels of LIN-42 We also show that kin-20 is important for post-transcriptional regulation of mature let-7 and lin-4 microRNA expression. In addition, the increased level of let-7 found in lin-42(n1089) mutant worms is not maintained after kin-20 RNAi treatment. Instead, let-7 is further repressed when levels of kin-20 and lin-42 are both decreased. Altogether these results suggest that though kin-20 regulates lin-42 and let-7 microRNA, it mainly affects let-7 microRNA expression independently of lin-42 These findings further our understanding of the mechanisms by which these conserved circadian rhythmic genes interact to ultimately regulate rhythmic processes, developmental timing and microRNA biogenesis in C. elegans.

The Period protein homolog LIN-42 negatively regulates microRNA biogenesis in C. elegans.

MicroRNAs (miRNAs) are small RNAs that post-transcriptionally regulate gene expression in many multicellular organisms. They are encoded in the genome and transcribed into primary (pri-) miRNAs before two processing steps that ultimately produce the mature miRNA. In order to generate the appropriate amount of a particular miRNA in the correct location at the correct time, proper regulation of miRNA biogenesis is essential. Here we identify the Period protein homolog LIN-42 as a new regulator of miRNA biogenesis in Caenorhabditis elegans. We mapped a spontaneous suppressor of the normally lethal let-7(n2853) allele to the lin-42 gene. Mutations in this allele (ap201) or a second lin-42 allele (n1089) caused increased mature let-7 miRNA levels at most time points when mature let-7 miRNA is normally expressed. Levels of pri-let-7 and a let-7 transcriptional reporter were also increased in lin-42(n1089) worms. These results indicate that LIN-42 normally represses pri-let-7 transcription and thus the accumulation of let-7 miRNA. This inhibition is not specific to let-7, as pri- and mature levels of lin-4 and miR-35 were also increased in lin-42 mutants. Furthermore, small RNA-seq analysis showed widespread increases in the levels of mature miRNAs in lin-42 mutants. Thus, we propose that the period protein homolog LIN-42 is a global regulator of miRNA biogenesis.

Period homolog LIN-42 regulates miRNA transcription to impact developmental timing.

Two recent studies by Van Wynsberghe et al. and Perales et al. in the nematode C. elegans have demonstrated a new function of the Period protein homolog LIN-42 in negatively regulating microRNA (miRNA) biogenesis at the transcriptional level. LIN-42 is a complex gene with 4 isoforms and multiple functions including the regulation of molting, developmental timing and entry into dauer. These recent studies uncover an additional function of LIN-42 as a negative regulator of miRNA transcription. Approximately 95% of miRNAs present in eggs and 33% of miRNAs present in L4 stage worms were upregulated in lin-42 mutant worms relative to wild type (WT) worms, suggesting that LIN-42 globally regulates miRNA biogenesis. Expression from both a let-7 miRNA and a lin-4 miRNA transcriptional reporter were enhanced in the absence of lin-42. Additionally, chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) of late larval stage worms showed that LIN-42 bound the let-7 promoter, suggesting that LIN-42 affects mature miRNA levels by inhibiting their transcription. In addition to miRNAs, LIN-42 also predominantly bound to the promoters of many diverse protein-coding genes. These findings support the action of LIN-42 at multiple points within the heterochronic and other regulatory pathways to impact a multitude of functions including developmental timing.

The cellular decapping activators LSm1, Pat1, and Dhh1 control the ratio of subgenomic to genomic Flock House virus RNAs.

Positive-strand RNA viruses depend on recruited host factors to control critical replication steps. Previously, it was shown that replication of evolutionarily diverse positive-strand RNA viruses, such as hepatitis C virus and brome mosaic virus, depends on host decapping activators LSm1-7, Pat1, and Dhh1 (J. Diez et al., Proc. Natl. Acad. Sci. U. S. A. 97:3913-3918, 2000; A. Mas et al., J. Virol. 80:246 -251, 2006; N. Scheller et al., Proc. Natl. Acad. Sci. U. S. A. 106:13517-13522, 2009). By using a system that allows the replication of the insect Flock House virus (FHV) in yeast, here we show that LSm1-7, Pat1, and Dhh1 control the ratio of subgenomic RNA3 to genomic RNA1 production, a key feature in the FHV life cycle mediated by a long-distance base pairing within RNA1. Depletion of LSM1, PAT1, or DHH1 dramatically increased RNA3 accumulation during replication. This was not caused by differences between RNA1 and RNA3 steady-state levels in the absence of replication. Importantly, coimmunoprecipitation assays indicated that LSm1-7, Pat1, and Dhh1 interact with the FHV RNA genome and the viral polymerase. By using a strategy that allows dissecting different stages of the replication process, we found that LSm1-7, Pat1, and Dhh1 did not affect the early replication steps of RNA1 recruitment to the replication complex or RNA1 synthesis. Furthermore, their function on RNA3/RNA1 ratios was independent of the membrane compartment, where replication occurs and requires ATPase activity of the Dhh1 helicase. Together, these results support that LSm1-7, Pat1, and Dhh1 control RNA3 synthesis. Their described function in mediating cellular mRNP rearrangements suggests a parallel role in mediating key viral RNP transitions, such as the one required to maintain the balance between the alternative FHV RNA1 conformations that control RNA3 synthesis.

Epigenetic control of germline development.

Dynamic regulation of histone modifications and small noncoding RNAs is observed throughout the development of the C. elegans germ line. Histone modifications are differentially regulated in the mitotic vs meiotic germ line, on X chromosomes vs autosomes and on paired chromosomes vs unpaired chromosomes. Small RNAs function in transposon silencing and developmental gene regulation. Histone modifications and small RNAs produced in the germ line can be inherited and impact embryonic development. Disruption of histone-modifying enzymes or small RNA machinery in the germ line can result in sterility due to degeneration of the germ line and/or an inability to produce functional gametes.

Analysis of microRNA expression and function.

Originally discovered in C. elegans, microRNAs (miRNAs) are small RNAs that regulate fundamental cellular processes in diverse organisms. MiRNAs are encoded within the genome and are initially transcribed as primary transcripts that can be several kilobases in length. Primary transcripts are successively cleaved by two RNase III enzymes, Drosha in the nucleus and Dicer in the cytoplasm, to produce ∼70 nucleotide (nt) long precursor miRNAs and 22 nt long mature miRNAs, respectively. Mature miRNAs regulate gene expression post-transcriptionally by imperfectly binding target mRNAs in association with the multiprotein RNA induced silencing complex (RISC). The conserved sequence, expression pattern, and function of some miRNAs across distinct species as well as the importance of specific miRNAs in many biological pathways have led to an explosion in the study of miRNA biogenesis, miRNA target identification, and miRNA target regulation. Many advances in our understanding of miRNA biology have come from studies in the powerful model organism C. elegans. This chapter reviews the current methods used in C. elegans to study miRNA biogenesis, small RNA populations, miRNA-protein complexes, and miRNA target regulation.

LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in Caenorhabditis elegans.

The highly conserved let-7 microRNA (miRNA) regulates developmental pathways across animal phyla. Mis-expression of let-7 causes lethality in C. elegans and has been associated with several human diseases. We show that timing of let-7 expression in developing worms is under complex transcriptional and post-transcriptional control. Expression of let-7 primary transcripts oscillates during each larval stage, but precursor and mature let-7 miRNAs do not accumulate until later in development after LIN-28 protein has diminished. We demonstrate that LIN-28 binds endogenous primary let-7 transcripts co-transcriptionally. We further show that LIN-28 binds endogenous primary let-7 transcripts in the nuclear compartment of human ES cells, suggesting that this LIN-28 activity is conserved across species. We conclude that co-transcriptional interaction of LIN-28 with let-7 primary transcripts blocks Drosha processing and, thus, precocious expression of mature let-7 during early development.

Regulation of lin-4 miRNA expression, organismal growth and development by a conserved RNA binding protein in C. elegans.

Transcription and multiple processing steps are required to produce specific 22 nucleotide microRNAs (miRNAs) that can regulate the expression of target genes. In C. elegans, mature lin-4 miRNA accumulates at the end of the first larval stage to repress its direct targets lin-14 and lin-28, allowing the progression of several somatic cell types to later larval fates. In this study, we characterized the expression of endogenous lin-4 and found that temporally regulated independent transcripts, but not constitutive lin-4 containing RNAs derived from an overlapping gene, are processed to mature lin-4 miRNA. Through an RNAi screen, we identified a conserved RNA binding protein gene rbm-28 (R05H10.2), homologous to the human RBM28 and yeast Nop4p proteins, that is important for lin-4 expression in C. elegans. We also demonstrate that rbm-28 genetically interacts with the lin-4 developmental timing pathway and uncover a previously unrecognized role for lin-14 and lin-28 in coordinating organismal growth.

5' cis elements direct nodavirus RNA1 recruitment to mitochondrial sites of replication complex formation.

Positive-strand RNA viruses replicate their genomes on intracellular membranes, usually in conjunction with virus-induced membrane rearrangements. For the nodavirus flock house virus (FHV), we recently showed that multifunctional FHV replicase protein A induces viral RNA template recruitment to a membrane-associated state, but the site(s) and function of this recruitment were not determined. By tagging viral RNA with green fluorescent protein, we show here in Drosophila cells that protein A recruits FHV RNA specifically to the outer mitochondrial membrane sites of RNA replication complex formation. Using Drosophila cells and yeast cells, which also support FHV replication, we also defined the cis-acting regions that direct replication and template recruitment for FHV genomic RNA1. RNA1 nucleotides 68 to 205 were required for RNA replication and directed efficient protein A-mediated RNA recruitment in both cell types. RNA secondary structure prediction, structure probing, and phylogenetic comparisons in this region identified two stable, conserved stem-loops with nearly identical loop sequences. Further mutational analysis showed that both stem-loops and certain flanking sequences were required for RNA1 recruitment, negative-strand synthesis, and subsequent positive-strand amplification in yeast and Drosophila cells. Thus, we have shown that protein A recruits RNA1 templates to mitochondria, as expected for RNA replication, and identified a new RNA1 cis element that is necessary and sufficient for RNA1 template recognition and recruitment to these mitochondrial membranes for negative-strand RNA1 synthesis. These results establish RNA recruitment to the sites of replication complex formation as an essential, distinct, and selective early step in nodavirus replication.

Nodavirus RNA replication protein a induces membrane association of genomic RNA.

Positive-strand RNA virus genome replication occurs in membrane-associated RNA replication complexes, whose assembly remains poorly understood. Here we show that prior to RNA replication, the multifunctional, transmembrane RNA replication protein A of the nodavirus flock house virus (FHV) recruits FHV genomic RNA1 to a membrane-associated state in both Drosophila melanogaster and Saccharomyces cerevisiae cells. Protein A has mitochondrial membrane-targeting, self-interaction, RNA-dependent RNA polymerase (RdRp), and RNA capping domains. In the absence of RdRp activity due to an active site mutation (A(D692E)), protein A stimulated RNA1 accumulation by increasing RNA1 stability. Protein A(D692E) stimulated RNA1 accumulation in wild-type cells and in xrn1(-) yeast defective in decapped RNA decay, showing that increased RNA1 stability was not due to protein A-mediated RNA1 recapping. Increased RNA1 stability was closely linked with protein A-induced membrane association of the stabilized RNA and was highly selective for RNA1. Substantial N- and C-proximal regions of protein A were dispensable for these activities. However, increased RNA1 accumulation was eliminated by deleting protein A amino acids (aa) 1 to 370 but was restored completely by adding back the transmembrane domain (aa 1 to 35) and partially by adding back peripheral membrane association sequences in aa 36 to 370. Moreover, although RNA polymerase activity was not required, even small deletions in or around the RdRp domain abolished increased RNA1 accumulation. These and other results show that prior to negative-strand RNA synthesis, multiple domains of mitochondrially targeted protein A cooperate to selectively recruit FHV genomic RNA to membranes where RNA replication complexes form.