Download Description Display Conventions and Configuration Methods

Description
This track is produced as part of the ENCODE Project. This track displays transcriptional
fragments associated with RNA binding proteins in different cell lines using RIP-Chip
(Ribonomic) profiling on Affymetrix GeneChip® Human Gene 1.0 ST Arrays. These sutracks
show the genomic location of transcripts associated with the array probes. Data for this track was
produced as part of the Encyclopedia of DNA Elements (ENCODE) Project.
In eukaryotic organisms, gene regulatory networks require an additional level of coordination
that links transcriptional and post-transcriptional processes. Messenger RNAs have traditionally
been viewed as passive molecules in the pathway from transcription to translation. However, it is
now clear that RNA-binding proteins play a major role in regulating multiple mRNAs in order to
facilitate gene expression patterns. These tracks show the associated mRNAs that co-precipitate
with the targeted RNA-binding proteins using RIP-Chip profiling.
Display Conventions and Configuration
This track has multiple subtracks that display individually in the browser. The subtracks within
this track correspond to different antibodies/target proteins tested in different cell lines. These
subtracks show the genomic location of the mRNA transcripts associated with RNA Binding
Proteins as determined by the Affymetrix GeneChip® Human Gene 1.0 ST Array probes. Items
are shaded by p-value using the formula (maxPossibleScore((maxPossibleScore/cutOffValue)*pValue)) so that items with more significant expression levels
are shaded darker. The p-values are displayed in the browser convention as -log10(pValue).
Methods
RBP-mRNA complexes were purified from cells grown according to the approved ENCODE cell
culture protocols. Antibodies specific to the RNA Binding Protein (RBP) in question were first
coated onto protein A/G containing magnetic beads and then used to immunoprecipitate the
targeted, endogenously-formed mRNP complexes. Antibody-coated beads were
incubated/tumbled with cell lysate overnight in the cold followed by extensive rinsing and
subsequent purification of associated RNA using Phenol/Chloroform extraction and ethanol
precipitation. The associated transcripts were identified using GeneChip® Human Gene 1.0 ST
Arrays. Arrays were analyzed using Agilent's GeneSpringGX software (version 11.0).
Arrays were analyzed a group at a time by applying the Iterative PLIER16 algorithm using
quantile normalization. Probesets whose normalized expression levels (signal value) fell within
the 18 to 98 percentile in at least two of the three replicates were retained for further analysis. A
TTest (T7-Tag and RIP-Input) or a one-way ANOVA (samples and controls) was applied to
these probesets and a p-value cutoff of .05 was applied. The Benjamini-Hochberg false discovery
rate algorithm was then applied to generate corrected p-values, also known as q-values.
The RIP-Input was summarized first and selected probesets were retained for further analysis.
Next, the arrays for T7Tag (background/negative control) RIPs were summarized with those
retained RIP-input probesets. Probesets that fit the above criteria for either group (RIP-Input or
T7Tag) were then filtered for those that showed a minimum 2 fold increase of expression in
T7Tag versus RIP-Input. Finally, arrays for treatment RIP samples were summarized together
with those for RIP-inputs and T7Tag RIPs. Probesets that fit the above criteria for any group
(RIP-Input or T7Tag or samples) were then filtered for probesets that showed a minimum 2 fold
increase of expression in treatment over total. A similar list was produced for probesets showing
the same enrichment in the T7Tag RIP set. Probesets which appeared in both treatment and
negative control at these cutoff stringencies were subtracted from the treatment results as
background noise, yielding the final data track.
Verification
All experiments (including controls) were performed in and analyzed as triplicates.
Release Notes
Release 2 (September 2011) of this track corrects the scores and the calculated P and Q values.
In this release, the calculated P and Q values are -log10(P) and -log10(Q), and the scores, and
therefore the shading of items, reflect the p-values as described in the Display Conventions and
Configuration section above.
Credits
These data were produced and analyzed by a collaboration between the Tenenbaum lab at the
University at Albany-SUNY, College of Nanoscale Science and Engineering, the Luiz Penalva
group at the Greehey Children's Cancer Research Institute, University of Texas Health Science
Center and the Microarray Core Facility at the Center for Functional Genomics, Rensselaer, NY .
Contact: Scott Tenenbaum
References
Baroni TE, Chittur SV, George AD, Tenenbaum SA. Advances in RIP-chip analysis : RNAbinding protein immunoprecipitation-microarray profiling. Methods Mol Biol. 2008;419:93-108.
George AD, Tenenbaum SA. MicroRNA modulation of RNA-binding protein regulatory
elements. RNA Biol. 2006;3(2):57-9. Epub 2006 Apr 1.
Jain R, Devine T, George AD, Chittur SV, Baroni TE, Penalva LO, Tenenbaum SA. RNABinding Protein Immunoprecipitation-Microarray (Chip) Profiling. Methods Mol Biol.
2011;703:247-63
Jayaseelan S, Doyle F, Currenti S, Tenenbaum SA. RIP: An mRNA Localization Technique.
Methods Mol Biol. 2011;714:407-422.
Keene JD, Tenenbaum SA. Eukaryotic mRNPs may represent posttranscriptional operons. Mol
Cell. 2002;9(6):1161-7.
Penalva LO, Tenenbaum SA, Keene JD. Gene expression analysis of messenger RNP
complexes. Methods Mol Biol. 2004;257:125-34.
Tenenbaum SA, Carson CC, Lager PJ, Keene JD. Identifying mRNA subsets in messenger
ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci U S A. 2000 Dec
19;97(26):14085-90.
Tenenbaum SA, Lager PJ, Carson CC, Keene JD. Ribonomics: identifying mRNA subsets in
mRNP complexes using antibodies to RNA-binding proteins and genomic arrays. Methods. 2002
Feb;26(2):191-8.
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