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RNA granules are protein/RNA condensates. How specific mRNAs are recruited to cytoplasmic RNA granules is not known. Here, we characterize the transcriptome and assembly of P granules, RNA granules in the C. elegans germ plasm. We find that P granules recruit mRNAs by condensation with the disordered protein MEG-3. MEG-3 traps mRNAs into non-dynamic condensates in vitro and binds to ~500 mRNAs in vivo in a sequence-independent manner that favors embryonic mRNAs with low ribosome coverage. Translational stress causes additional mRNAs to localize to P granules and translational activation correlates with P granule exit for two mRNAs coding for germ cell fate regulators. Localization to P granules is not required for translational repression but is required to enrich mRNAs in the germ lineage for robust germline development. Our observations reveal similarities between P granules and stress granules and identify intrinsically-disordered proteins as drivers of RNA condensation during P granule assembly.

Animals with germ plasm assemble cytoplasmic RNA granules (germ granules) that segregate with the embryonic germ lineage. How germ granules assemble and recruit RNA is not well understood. Here we characterize the assembly and RNA composition of the germ (P) granules ofC. elegans. ∼500 maternal mRNAs are recruited into P granules by a sequence independent mechanism that favors mRNAs with low ribosome coverage. Translational activation correlates temporally with P granule exit for two mRNAs that code for germ cell fate regulators. mRNAs are recruited into the granules by MEG-3, an intrinsically disordered protein that condenses with RNA to form nanoscale gels. Our observations reveal parallels between germ granules and stress granules and suggest that cytoplasmic RNA granules are reversible super-assemblies of nanoscale RNA-protein gel condensates.

The Nanos RNA-binding protein has been implicated in the specification of primordial germ cells (PGCs) in metazoans, but the underlying mechanisms remain poorly understood. We have profiled the transcriptome of PGCs lacking thenanoshomologuesnos-1andnos-2iC. elegans.nos-1nos-2PGCs fail to silence hundreds of genes normally expressed in oocytes and somatic cells, a phenotype reminiscent of PGCs lacking the repressive PRC2 complex. Thenos-1nos-2phenotype depends on LIN-15B, a broadly expressed synMuvB class transcription factor known to antagonize PRC2 activity in somatic cells. LIN-15B is maternally-inherited by all embryonic cells and is down-regulated specifically in PGCs in anos-1nos-2-dependent manner. Consistent with LIN-15B being a critical target of Nanos regulation, inactivation of maternal LIN-15B restores fertility tonos-1nos-2mutants. These studies demonstrate a central role for Nanos in reprogramming the transcriptome of PGCs away from an oocyte/somatic fate by down-regulating an antagonist of PRC2 activity.

Stressed eukaryotic cells store mRNAs in protein-rich condensates called stress granules. Using single-molecule tracking techniques to examine how mRNAs enter stress granules, a new study shows that mRNAs make transient contacts with the granule surface before stable association, and become largely immobile after entry.

Homology-directed repair (HDR) of double-strand DNA breaks is a promising method for genome editing, but is thought to be less efficient than error-prone nonhomologous end joining in most cell types. We have investigated HDR of double-strand breaks induced by CRISPR-associated protein 9 (Cas9) in Caenorhabditis elegans. We find that HDR is very robust in the C. elegans germline. Linear repair templates with short (∼30–60 bases) homology arms support the integration of base and gene-sized edits with high efficiency, bypassing the need for selection. Based on these findings, we developed a systematic method to mutate, tag, or delete any gene in the C. elegans genome without the use of co-integrated markers or long homology arms. We generated 23 unique edits at 11 genes, including premature stops, whole-gene deletions, and protein fusions to antigenic peptides and GFP. Whole-genome sequencing of five edited strains revealed the presence of passenger variants, but no mutations at predicted off-target sites. The method is scalable for multi-gene editing projects and could be applied to other animals with an accessible germline.

SUMMARYP granules are perinuclear condensates in C. elegans germ cells proposed to serve as hubs for self/non-self RNA discrimination by Argonautes. We report that a mutant (meg-3 meg-4) that does not assemble P granules in primordial germ cells loses competence for RNA-interference over several generations and accumulates silencing small RNAs against hundreds of endogenous genes, including the RNA-interference genes rde-11 and sid-1. In wild-type, rde-11 and sid-1 transcripts are heavily targeted by piRNAs, accumulate in P granules, but maintain expression. In the primordial germ cells of meg-3 meg-4 mutants, rde-11 and sid-1 transcripts disperse in the cytoplasm with the small RNA biogenesis machinery, become hyper-targeted by secondary sRNAs, and are eventually silenced. Silencing requires the PIWI-class Argonaute PRG-1 and the nuclear Argonaute HRDE-1 that maintains trans-generational silencing of piRNA targets. These observations support a “safe harbor” model for P granules in protecting germline transcripts from piRNA-initiated silencing.

The prolyl hydroxylase Ofd1 enables yeast cells to adapt to hypoxia by regulating the DNA binding and degradation of the hypoxic transcription factor Sre1N. These two regulatory functions are inseparable in vivo. A mathematical model of the Ofd1 system is used to show that both functions are necessary for Ofd1 to work as observed., Cells adapt to changes in ambient oxygen by changing their gene expression patterns. In fission yeast, the sterol regulatory element–binding protein Sre1 is proteolytically cleaved under low oxygen, and its N-terminal segment (Sre1N) serves as a hypoxic transcription factor. When oxygen is present, the prolyl hydroxylase Ofd1 down-regulates Sre1N activity in two ways: first, by inhibiting its binding to DNA, and second, by accelerating its degradation. Here we use a mathematical model to assess what each of these two regulatory functions contributes to the hypoxic response of the cell. By disabling individual regulatory functions in the model, which would be difficult in vivo, we found that the Ofd1 function of inhibiting Sre1N binding to DNA is essential for oxygen-dependent Sre1N regulation. The other Ofd1 function of accelerating Sre1N degradation is necessary for the yeast to quickly turn off its hypoxic response when oxygen is restored. In addition, the model predicts that increased Ofd1 production at low oxygen plays an important role in the hypoxic response, and the model indicates that the Ofd1 binding partner Nro1 tunes the response to oxygen. This model quantifies our understanding of a novel oxygen-sensing mechanism that is widely conserved.

Regulation of gene expression plays an integral role in adaptation of cells to hypoxic stress. In mammals, prolyl hydroxylases control levels of the central transcription factor hypoxia inducible factor (HIF) through regulation of HIFα subunit stability. Here, we report that the hydroxylase Ofd1 regulates the Sre1 hypoxic transcription factor in fission yeast by controlling DNA binding. Prolyl hydroxylases require oxygen as a substrate, and the activity of Ofd1 regulates Sre1-dependent transcription. In the presence of oxygen, Ofd1 binds the Sre1 N-terminal transcription factor domain (Sre1N) and inhibits Sre1-dependent transcription by blocking DNA binding. In the absence of oxygen, the inhibitor Nro1 binds Ofd1, thereby releasing Sre1N and leading to activation of genes required for hypoxic growth. In contrast to the HIF system, where proline hydroxylation is essential for regulation, Ofd1 inhibition of Sre1N does not require hydroxylation and, thus, defines a new mechanism for hypoxic gene regulation.

In fission yeast, orthologs of mammalian SREBP and Scap, called Sre1 and Scp1, monitor oxygen-dependent sterol synthesis as a measure of cellular oxygen supply. Under low oxygen conditions, sterol synthesis is inhibited, and Sre1 cleavage is activated. However, the sterol signal for Sre1 activation is unknown. In this study, we characterized the sterol signal for Sre1 activation using a combination of Sre1 cleavage assays and gas chromatography sterol analysis. We find that Sre1 activation is regulated by levels of the 4-methyl sterols 24-methylene lanosterol and 4,4-dimethylfecosterol under conditions of low oxygen and cell stress. Both increases and decreases in the level of these ergosterol pathway intermediates induce Sre1 proteolysis in a Scp1-dependent manner. The SREBP ortholog in the pathogenic fungus Cryptococcus neoformans is also activated by high levels of 4-methyl sterols, suggesting that this signal for SREBP activation is conserved among unicellular eukaryotes. Finally, we provide evidence that the sterol-sensing domain of Scp1 is important for regulating Sre1 proteolysis. The conserved mutations Y247C, L264F, and D392N in Scp1 that render Scap insensitive to sterols cause constitutive Sre1 activation. These findings indicate that unlike Scap, fission yeast Scp1 responds to 4-methyl sterols and thus shares properties with mammalian HMG-CoA reductase, a sterol-sensing domain protein whose degradation is regulated by the 4-methyl sterol lanosterol.

This protocols is from: Alexandre Paix, et al. (2014) Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in Caenorhabditis elegans.Genetics198:1347-1356;doi:10.1534/genetics.114.170423 Please see the full manuscript or additional details

Escherichia coli MutY is an adenine and weak guanine DNA glycosylase involved in reducing the mutagenic effects of 7,8-dihydro-8-oxoguanine (GO). MutY contains three structural domains: an iron-sulfur module, a six-helix barrel module with the helix-hairpin-helix motif, and a C-terminal domain. Here, we demonstrate that the mutant MutY(Δ26–134), which lacks the six-helix barrel domain, cannot complement the mutator phenotype of a mutY mutant in vivo. However, the mutant can still bind DNA and has weak catalytic activity at high enzyme concentrations. The mutant is a dimer in solution and assembled into two and multiple (up to five) complexes with 20- and 44-bp DNA fragments, respectively, in a concentration-dependent manner. Higher order complexes with DNA substrates containing A/GO mismatches were formed at lower protein concentrations than with the A/G mismatch and homoduplex DNA. Measurement of equilibrium binding using fluorescence anisotropy showed that the mutant protein retains some specificity for A/GO-containing DNA substrates and that the binding event is highly cooperative. This is consistent with the MutY structure determined, which indicates that GO specificity is contributed by both the six-helix barrel and C-terminal domains. The nonspecific binding of MutY(Δ26–134) to DNA suggests a model in which the specific binding of mismatched DNA by MutY involves sequential interactions, in which one MutY molecule scans the DNA and enhances binding of another MutY molecule to the A/GO mismatch.

Sterol homeostasis is tightly controlled by the sterol regulatory element-binding protein (SREBP) transcription factor that is highly conserved from fungi to mammals. In fission yeast, SREBP functions in an oxygen-sensing pathway to promote adaptation to decreased oxygen supply that limits oxygen-dependent sterol synthesis. Low oxygen stimulates proteolytic cleavage of the SREBP homolog Sre1, generating the active transcription factor Sre1N that drives expression of sterol biosynthetic enzymes. In addition, low oxygen increases the stability and DNA binding activity of Sre1N. To identify additional signals controlling Sre1 activity, we conducted a genetic overexpression screen. Here, we describe our isolation and characterization of the casein kinase 1 family member Hhp2 as a novel regulator of Sre1N. Deletion of Hhp2 increases Sre1N protein stability and ergosterol levels in the presence of oxygen. Hhp2-dependent Sre1N degradation by the proteasome requires Hhp2 kinase activity, and Hhp2 binds and phosphorylates Sre1N at specific residues. Our results describe a role for casein kinase 1 as a direct regulator of sterol homeostasis. Given the role of mammalian Hhp2 homologs, casein kinase 1δ and 1ϵ, in regulation of the circadian clock, these findings may provide a mechanism for coordinating circadian rhythm and lipid metabolism.

Recent progress in L1 biology highlights its role as a major driving force in the evolution of mammalian genome structure and function. This coincides with direct confirmation of the preponderance of long interspersed elements in mammalian genomes at the nucleotide level by large scale sequencing efforts. Two assay systems have been prominently featured in L1 studies over the past decade, which are used to assess L1 activities in cultured cells and transgenic mice respectively. However, constructing retrotransposon assay vectors and subsequent mapping of integration sites remain technically challenging aspects of the field. Synthetic biology approaches have changed the playing field with regard to the strategic design of retrotransposons. To streamline the construction and optimization of synthetic retrotransposons, we have implemented a highly efficient modular design for L1 vectors allowing “plug and play” swapping of individual modules as new knowledge is gained and optimization of constructs proceeds. Seven functional modules are divided by strategically placed unique restriction sites. These are utilized to facilitate module exchange and construction of L1 vectors for gene targeting, transgenesis and cell culture assays. A “double SfiI” strategy utilizing two non-complementary overhangs allows insert swapping to be carried out with a single, robust restriction/ligation cycle. The double-SfiI strategy is generic and can be applied to many other problems in synthetic biology or genetic engineering. To facilitate genomic mapping of L1 insertions, we have developed an optimized inverse PCR protocol using 4-base cutters and step-down cycling conditions. Using this protocol, de novo L1 insertions can be efficiently recovered after a single round of PCR. The proposed modular design also incorporates features allowing streamlined insertion mapping without repeated optimization. Furthermore, we have presented evidence that efficient L1 retrotransposition is not dependent on pCEP4 conferred autonomous replication capabilities when a shortened puromycin selection protocol is used, providing a great opportunity for further optimization of L1 cell culture assay vectors by using alternative vector backbones.

Sre1, the fission yeast sterol regulatory element-binding protein, is an ER membrane-bound transcription factor that controls adaptation to low oxygen growth. Under low oxygen, Sre1 is proteolytically cleaved and the N-terminal transcription factor domain (Sre1N) is released from the membrane and enters the nucleus to activate hypoxic gene expression. Ofd1, a prolyl 4-hydroxylase-like 2-oxoglutarate dioxygenase, controls the oxygen-dependent stability of Sre1N. In the presence of oxygen, Ofd1 accelerates the degradation of Sre1N, but under low oxygen Ofd1 is inhibited and Sre1N accumulates. To identify the regulators of Sre1N, we performed a plasmid-based screen for genes that increased Sre1N transcriptional activity. Here, we identify Nro1 (SPCC4B3.07) as a positive regulator of Sre1N stability and a direct inhibitor of Ofd1. In the absence of oxygen, Nro1 binds to the Ofd1 C-terminal degradation domain and inhibits Sre1N degradation. In the presence of oxygen, Nro1 binding to Ofd1 is disrupted, leading to rapid degradation of Sre1N. We conclude that the Ofd1 dioxygenase domain functions as an oxygen sensor that regulates binding of Nro1 to Ofd1 to control oxygen-dependent Sre1N stability.

Both GO (7,8-dihydro-8-oxoguanine) and hoU (5-hydroxyuracil) are highly mutagenic because DNA polymerase frequently misincorporates adenine opposite these damaged bases. In Escherichia coli, MutY DNA glycosylase can remove misincorporated adenine opposite G or GO on the template strand during DNA replication. MutY remains bound to the product that contains an AP (apurinic/apyrimidinic) site. Endo VIII (endonuclease VIII) can remove oxidized pyrimidine and weakly remove GO by its DNA glycosylase and β/δ-elimination activities. In the present paper, we demonstrate that Endo VIII can promote MutY dissociation from AP/G, but not from AP/GO, and can promote β/δ-elimination on the products of MutY. MutY interacts physically with Endo VIII through its C-terminal domain. MutY has a moderate affinity for DNA containing a hoU/A mismatch, which is a substrate of Endo VIII. MutY competes with Endo VIII and inhibits Endo VIII activity on DNA that contains a hoU/A mismatch. Moreover, MutY has a weak adenine glycosylase activity on hoU/A mismatches. These results suggest that MutY may have some role in reducing the mutagenic effects of hoU.

Sre1, the fission yeast homologue of the mammalian sterol regulatory element binding protein, SREBP, is a hypoxic transcription factor required for sterol homeostasis and low oxygen growth. The level of the N-terminal transcription factor domain of Sre1, Sre1N, is regulated by Ofd1-Nro1 in an oxygen-dependent manner. Ofd1 is a putative prolyl hydroxylase of the 2-oxyoglutarate-Fe(II) dioxygenase protein superfamily. Unlike the prolyl hydroxylase PHD which hydroxylates proline residues in the hypoxia-inducible factor HIF, Ofd1 utilizes its N-terminal dioxygenase domain to control the interaction between itself and its inhibitor Nro1: in hypoxia, Nro1 binds to Ofd1 and this inhibits its C-terminal degradation domain (CTDD) function on Sre1N destabilization. As shown previously, Ofd1CTDD by itself is sufficient to promote Sre1N degradation and there's direct interaction between Nro1 and Ofd1CTDD. However, the molecular mechanism by which Ofd1CTDD accelerates Sre1N degradation remains unclear. Here we report the crystal structure of Ofd1CTDD at 2.0 A resolution. Interestingly, Ofd1CTDD has a double-stranded beta helix (DBSH) fold like that of the 2-oxyoglutarate-Fe(II) dioxygenase protein superfamily but lacks the iron-binding motif characteristic of the superfamily. This structure may help elucidate how Ofd1CTDD promotes Sre1N turnover and how Ofd1-Nro1 as a whole functions as an oxygen-sensor.

SummaryFission yeast protein Sre1, the homolog of the mammalian sterol regulatory element-binding protein (SREBP), is a hypoxic transcription factor required for sterol homeostasis and low-oxygen growth. Nro1 regulates the stability of the N-terminal transcription factor domain of Sre1 (Sre1N) by inhibiting the action of the prolyl 4-hydroxylase-like Ofd1 in an oxygen-dependent manner. The crystal structure of Nro1 determined at 2.2 Å resolution shows an all-α-helical fold that can be divided into two domains: a small N-terminal domain, and a larger C-terminal HEAT-repeat domain. Follow-up studies showed that Nro1 defines a new class of nuclear import adaptor that functions both in Ofd1 nuclear localization and in the oxygen-dependent inhibition of Ofd1 to control the hypoxic response.