A fitness assay for comparing RNAi effects across multiple C. elegans genotypes

<p>Abstract</p> <p>Background</p> <p>RNAi technology by feeding of <it>E. coli </it>containing dsRNA in <it>C. elegans </it>has significantly contributed to further our understanding of many different fields, including genetics, molecular biology, developmental biology and functional genomics. Most of this research has been carried out in a single genotype or genetic background. However, RNAi effects in one genotype do not reveal the allelic effects that segregate in natural populations and contribute to phenotypic variation.</p> <p>Results</p> <p>Here we present a method that allows for rapidly comparing RNAi effects among diverse genotypes at an improved high throughput rate. It is based on assessing the fitness of a population of worms by measuring the rate at which <it>E. coli </it>is consumed. Critically, we demonstrate the analytical power of this method by QTL mapping the loss of RNAi sensitivity (in the germline) in a recombinant inbred population derived from a cross between Bristol and a natural isolate from Hawaii. Hawaii has lost RNAi sensitivity in the germline. We found that polymorphisms in <it>ppw-1 </it>contribute to this loss of RNAi sensitivity, but that other loci are also likely to be important.</p> <p>Conclusions</p> <p>In summary, we have established a fast method that improves the throughput of RNAi in liquid, that generates quantitative data, that is easy to implement in most laboratories, and importantly that enables QTL mapping using RNAi.</p

An RNAi-based dimorphic genetic screen identified the double bromodomain protein BET-1 as a sumo-dependent attenuator of RAS-mediated signalling

Attenuation of RAS/RAF/MAPK signalling is essential to prevent hyperactivation of this oncogenic pathway. In C. elegans, the sumoylation pathway and a combination of histone tail modifications regulate gene expression to attenuate the LET-60 (RAS) signalling pathway. We hypothesised that a number of chromatin regulators are likely to depend on sumoylation to attenuate the pathway. To reveal these, we designed an RNAi-based dimorphic genetic screen that selects candidates based on their ability to act as enhancers of a sumo mutant phenotype, such interactions would suggest that the candidates may be physically associated with sumoylation. We found 16 enhancers, one of which BET-1, is a conserved double bromodomain containing protein. We further characterised BET-1 and showed that it can physically associate with SMO-1 and UBC-9, and that it can be sumoylated in vitro within the second bromodomain at lysine 252. Previous work has shown that BET-1 can bind acetyl-lysines on histone tails to influence gene expression. In conclusion, our screening approach has identified BET-1 as a Sumo-dependent attenuator of LET-60-mediated signalling and our characterisation suggests that BET-1 can be sumoylated

A targeted RNAi screen for SUMO interactors that attenuate LET-60-mediated signalling.

<p>A) Activation (green arrow) of LET-60-mediated signalling in the vulva by the LIN-3 (EGF), LET-23 (RTK), LET-60 (RAS), LIN-45 (RAF), and MPK-1 (MAPK) signalling cascade. Negative feedback loop is produced by chromatin regulators and the sumoylation pathway to attenuate LET-60-mediated signalling (in red). B) Diagram depicting the RNAi screening approach in a SUMO-balanced background using the MVP (multiple ventral protrusions) phenotype as readout for hyperactivation of the LET-60 signalling pathway during vulval development. The green pharynx indicates GFP balanced animals lacking one copy of <i>smo-1</i>, the non-green animals are <i>smo-1</i>_<i>lf</i><i> /smo-1</i>_<i>lf</i> homozygotes. The percentage indicates the approximate ratio of adult animals for each genotype observed. <i>smo-1</i>_<i>lf</i><i> /smo-1</i>_<i>lf</i> are 100% sterile presenting a <i>p</i>rotruding <i>v</i>u<i>l</i>va (PVL) and about 10% of these Pvl animals will display the <i>mu</i>lti<i>v</i>ulvae (MUV) phenotype. This was taken into account during the screen (see Materials and Methods).</p

Comparative analysis between the double <i>smo-1</i>_<i>lf</i> bet<i>-</i>1_<i>lf</i> mutant and the respective single mutants.

<p>A) Two alternative models (linear and parallel) for attenuation of the LET-60(RAS) signalling pathwayby SMO-1 and BET-1. B) The percentage of VPCs expressing <i>egl-17::cfp</i> is indicated on the y axis. Each bar represents the descendants of the indicated parental VPC. The genotype analysed is indicated in the inset legend. We analysed a minimum of 40 animals for each genotype. B) Western blots performed in triplicate, and prepared from larval stage 4 (L4) against phosphorylated MPK-1 (top panel), MPK-1 (middle panel) and tubulin (bottom panel). Quantification of pMPK-1 is relative to tubulin levels. </p

BET-1 can be sumoylated <i>in</i><i>vitro</i>.

<p>A) A schematic map depicting BET-1 full length and the different fragments used in yeast two-hybrid and for the <i>in </i><i>vitro</i> sumoylation assay. B) The yeast two-hybrid assay shows that BET-1 and SMO-1 interact. The interaction requires lysine 252 within the C-terminal part of the bromodomain. The fragment from amino acid 614 to 853 auto activates and could not be tested for interactions. The photographs show the results from six serial (1 in 5) dilutions to allow semi-quantitative comparisons between conditions. Omitted amino acids are indicated on top left hand side. C) The <i>in </i><i>vitro</i> sumoylation assay analysed by Western blot against MBP shows that lysine 252 is sumoylated. The expected size of MBP fused to the bromodomain is roughly 80kDa and its sumoylated form corresponding to an added SUMO-1, branching out of the bromodomain, produces a shift of ~20kDa. The bands detected below are likely to be degradation products and/or incomplete translation products.</p

Analysis of <i>egl-17::cfp</i> expression.

<p>A) DIC and fluorescence photographs showing <i>egl-17::cfp</i> expression (in cyan blue) in wild type (N2) <i>versus</i> bet<i>-</i>1_<i>lf</i>. Arrowheads indicate VPCs. B) Absence of <i>smo-1</i> or bet<i>-</i>1 causes <i>egl-17::cfp</i> ectopic expression. A gap-<i>1</i> (GTPase Activating Protein, a known negative modulator of the pathway [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083659#B44" target="_blank">44</a>]) loss of function mutant (gap-1_ga133) is also shown. C and D) Detailed analysis of the VPCs expressing <i>egl-17::cfp</i>. Each bar represents the descendants of the indicated parental VPC with the 50% ectopic expression mark indicated. We analysed a minimum of 30 animals for each genotype. The genotype analysed is indicated in the top left hand corner.</p

Structure and biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes

Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts