MLPCN maternal gene expression-MEX-5 TCR-2 binding assay-Primary Screen
Keywords: Zinc finger, C. elegans, maternal gene expression, RNA-protein interaction, gene regulation, MEX-5, POS-1, embryonic development ..more
BioActive Compounds: 1137
Broad Institute: MLPCN maternal gene expression
Project ID: 2024
Keywords: Zinc finger, C. elegans, maternal gene expression, RNA-protein interaction, gene regulation, MEX-5, POS-1, embryonic development
Primary Collaborators: Sean Ryder, U. Mass Medical School, firstname.lastname@example.org
In most organisms, the body axes and founding tissue types are formed prior to the onset of zygotic transcription. Thus, post-transcriptional regulation of maternal transcripts by maternally supplied RNA-binding proteins is crucial to early patterning events. Few tools exist to study specific regulatory networks guided by RNA-binding proteins during early development. Importantly, standard genetic analyses are complicated by the maternal effect, pleiotropy, and embryonic lethality. This screen aims to identify small molecule inhibitors of RNA-binding protein function for two proteins required for Caenorhabditis elegans early development. The rationale was two fold. First, inhibitory compounds would serve as a set of chemical tools to facilitate study of the RNA-binding proteins during subsequent aspects of development. Second, if the inhibitory compounds function across nematode species boundaries, then they may be useful as a new class of anti-parasitic agent that specifically targets Helminth embryos notoriously difficult to inactivate.
The two proteins chosen, MEX-5 and POS-1, belong to a family of conserved nematode CCCH class tandem zinc finger proteins. Both are critical to body axis formation and segregation of the germline from the soma. MEX-5 coordinates anterior cell fate specification, while POS-1 coordinates posterior patterning and germline specification. Both proteins can be expressed and purified from Escherichia coli as recombinant fusion proteins, and both proteins bind with high affinity to specific RNA sequences in vitro. Finally, fluorescence polarization can be used to effectively monitor the in vitro RNA-binding activity of both proteins using a standard fluorescence plate reader.
1) Probe attributes
Small molecule that inhibits MEX-5
Works as a transient inactivator
Does not bind to other zinc-finger proteins
The ability of purified MEX-5 protein to change the anisotropy of fluorescently labeled RNAs upon binding is used as the basis of the assay. This biochemical assay will be used to screen for inhibitors of this interaction.
7.5 nL of compound per well was pre-dispensed into black Aurora 1536 low-base plates (PN 00029844) in duplicate assay plates. Pre-dispensed plates were stored lidded at room temperature for up to two weeks.
MEX-5 protein, expressed in E. coli JM109 with a C-terminal fusion to maltose binding protein, was prepared by the assay provider at approximately 50 uM as previously described. A protein master mix was prepared at 200 nM in 1x buffer (20 mM tris pH 8.0 (Ambion), 40 mM NaCl (Ambion), 0.004% IPEGAL (Sigma), 100 uM zinc acetate dihydrate (Fluka)). The solution was loaded on the dispenser (Combi nL, Thermo) at room temperature.
The target binding sequence TCR2 is a Temporal Control Region in the 3' untranslated region of the C. elegans gene glp-1 (abnormal Germ Line Proliferation). Fluorescent TCR2 RNA (5'-UUUCUUUAUAACUUGUUACAAUUUUUGAAA-FITC-3', Dharmacon / Thermo) mastermix was prepared in 1x buffer and supplemented with 0.02 mg/mL tRNA type X (Sigma). The RNA mastermix was heated at 65 degrees for 2 minutes then cooled on ice prior to loading onto the dispenser (BioRaptr, Beckman Coulter) at room temperature. The dispenser bottle was wrapped in foil to protect from light.
Competing unlabeled TCR2 RNA (Dharmacon / Thermo) was prepared as a positive control solution at 25 uM in 1x buffer. The solution was heated to 65 degrees for 2 minutes and cooled on ice prior to loading onto the dispenser (BioRaptr) at room temperature.
1x buffer alone was also prepared and loaded onto the dispenser (BioRaptr).
The assay plates with pre-dispensed compound were filled with 4.5 uL / well of protein mastermix on the Combi nL and incubated for 5 minutes at room temperature.
The plates were then filled on the Bioratpr with 1.5 uL / well of labeled RNA mastermix and either 1.5 uL / well of 1x buffer only (compound wells and negative control DMSO wells) or 1.5 uL / well of competing RNA in 1x buffer (positive control wells).
The final assay concentrations in 7.5 uL total volume were:
20 mM tris pH 8.0
40 mM NaCl
100 uM zinc acetate
120 nM MEX5 protein
2 nM FITC labeled TCR2 RNA
4 ug/mL tRNA type X
10 uM test compound, 0.1% DMSO
For positive control wells: 5 uM unlabeled TCR2 RNA
Plates were spun at 1000 rpm (Beckman Coulter Allegra 6KR, GH3.8 rotor) for one minute.
One hour after the addition of the RNA to the plates, fluorescence polarization was read on a ViewLux reader (Perkin Elmer). Total fluorescence from the P and S polarization channels was recorded along with the Fluorescence Polarization calculation.
HTS Data Analysis:
128 negative control wells (DMSO) were included on every plate. 32 positive control wells (5 uM competing unlabaled RNA target TCR2) were included on every plate. Compound activity was scaled to these values, with DMSO activity measuring 0% and the competing RNA measuring 100% activity. Automatic masking of spot patterns was performed by Genedata software to remove bleed-over signal from very highly fluorescent wells. Pattern correction was performed by Genedata software using a runwise additive algorithm.
Total fluorescence intensity in the P and S was normalized to the negative control wells on each plate.
The PubChem_Activity_Score was derived using the follow procedure:
1. A background-subtracted value was calculated for each well by subtracting the median value of the negative control wells on each plate from the value of each well on that plate.
2. An activity score was derived for each well by dividing the background-subtracted value for each well by the median of the background-subtracted value of the positive control wells in the same run and multiplying the resulting fraction by 100.
3. Runwise patterns were smoothed using an additive algorithm in Genedata software
4. The final PubChem_Activity_Score represents the mean of all valid replicate activity scores obtained.
The PubChem_Activity_Outcome class was assigned as described below:
Activity_Outcome = 1 (inactive)
PubChem_Activity_Score for more than half the replicate activity scores <25.
Activity_Outcome = 2 (active)
PubChem_Activity_Score for more than half the replicate activity scores > 25 and average total fluorescence intensity values in the S channel less than twice the median S channel fluorescence intensity of the negative controls on each plate.
Activity_Outcome = 3 (inconclusive)
PubChem_Activity_Score > 25 for half the replicate activity scores and < 25 for half; or average total fluorescence intensity in the S channel > twice the median S channel fluorescence intensity of the negative controls on each plate.
5. Compounds with total fluorescence intensity in S channel > twice the median S channel fluorescence intensity of the negative controls on each plate were given an ASSAYDATA_COMMENT of "autoflorescent"
Data Table (Concise)