uHTS identification of microRNA-mediated mRNA deadenylation inhibitors by fluoresence polarization assay
The regulation of gene expression is a mechanism that allows cells to respond to growth and proliferation stimuli, stress, and nutrient availability. It is managed at multiple levels: mRNA expression, mRNA translation initiation and mRNA decay (1). MicroRNAs (miRNAs) are endogenous small RNAs that post-transcriptionally regulate gene expression to control a wide range of biological processes including cell growth, division and differentiation, as well as metabolism and development. ..more
BioActive Compounds: 1307
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network(MLPCN)
Grant Number: R03 MH094198-01
Assay Provider: Kalle Gehring, Ph.D., McGill University
The regulation of gene expression is a mechanism that allows cells to respond to growth and proliferation stimuli, stress, and nutrient availability. It is managed at multiple levels: mRNA expression, mRNA translation initiation and mRNA decay (1). MicroRNAs (miRNAs) are endogenous small RNAs that post-transcriptionally regulate gene expression to control a wide range of biological processes including cell growth, division and differentiation, as well as metabolism and development.
The poly(A) tail of mRNA is bound by several molecules of the poly(A)-binding protein (PABP), an abundant cytoplasmic protein in eukaryotes that promotes translation (2). PABPC1 is a multi-domain protein that contains four phylogenetically conserved RNA recognition motifs (RRM1-4) (3,4) a proline-rich unstructured region and a 70-residue C-terminal domain termed Mlle or PABC (5). Mlle (Mademoiselle) refers to the conserved KITGMLLE signature motif of this domain. The Mlle domain binds proteins with a 12-15 amino acid motif termed PAM2 to regulate PABPC1 involvement in mRNA decay. The goal of this grant is to screen for small molecule modulators of Mlle interactions as chemical probes for the study of PABP regulation in translation and mRNA silencing. This will open new therapeutic opportunities for altering gene expression in cancer, neurodegeneration, and other diseases.
1. Sonenberg N, Hinnebusch AG. (2007) Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets. Cell 136: 731-745.
2. Kahvejian A, Roy G, Sonenberg N. (2001) The mRNA closed-loop model: the function of PABP and PABP-interacting proteins in mRNA translation. Cold Spring Harbor Symp. Quant. Biol. 66: 293-300.
3. Adam SA, Nakagawa T, Swanson MS, Woodruff TK, Dreyfuss G. (1986) mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence. Mol. Cell. Biol. 6: 2932-2943.
4. Sachs AB, Davis RW, Kornberg RD. (1987) A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability. Mol. Cell. Biol. 7: 3268-3276.
5. Kozlov G, Trempe JF, Khaleghpour K, Kahvejian A, I. Ekiel I, Gehring K. (2001) Structure and function of the C-terminal PABC domain of human poly(A)-binding protein. Proc. Natl Acad. Sci. USA 98: 4409-4413.
EDTA (Gibco 15575)
10% Tween 20 (MP biomedicals ICN10316890)
GST-Mlle protein (Assy Provider)
FITC Pam2 peptide (Assay Provider)
Molecular grade water (CellGro 46-000-CM)
Assay plate: Aurora 1536 Black Solid Bottom (00019120BX)
I. Prepare Solutions:
1- Prepare 1Xassay buffer in molecular grade water (20mM Hepes pH 7.4, 150 mM NaCl, 0.5mM EDTA, 0.05% Tween-20)
2- Prepare 2x protein solution (1.35uM) in assay buffer
3- Prepare 2x substrate solution (0.01uM) in assay buffer
II. Compound Addition:
4- Using LabCyte Echo 555, transfer 60 nL from 2 mM compound source plate into assay plate Columns 5-48 (final concentration of test compounds is 20 uM, 1.0% DMSO), and 60 nL of DMSO to control wells in Columns 1-4.
III. Reagent Addition
5- Dispense 3uL/well of assay buffer in columns 1 and 2 and 3ul/well protein solution in columns 3-48 using BiorapTR.
6- Dispense 3ul/well of FITC-PAM2 substrate into all wells using BiorapTR.
9- Spin down plates without lids on Vspin at 1000 rpm for 1 min
10- incubate plates at room temp for 2 hours.
IV. Reading plates:
11- Read the plate on PerkinElmer-EnVision plate reader FP protocol with dual mirror
The experimental values were normalized by difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.
Compounds that demonstrated % activity of >= 40 and F-ratio between 0.5 and 1.5 at 20 uM in either normalized or corrected results are defined as active in this assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
** Test Concentration.
Data Table (Concise)