Open Access Highly Accessed Methodology article

Identification of proteins binding coding and non-coding human RNAs using protein microarrays

Zurab Siprashvili1, Dan E Webster1, Markus Kretz1, Danielle Johnston1, John L Rinn2, Howard Y Chang13 and Paul A Khavari14*

Author Affiliations

1 The Program in Epithelial Biology, Stanford University School of Medicine, 269 Campus Drive, Room 2145, Stanford, CA 94305, USA

2 Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA

3 Howard Hughes Medical Institute, Stanford, CA 94305, USA

4 Veterans Affairs Palo Alto Healthcare System, Palo Alto, CA 94304

For all author emails, please log on.

BMC Genomics 2012, 13:633  doi:10.1186/1471-2164-13-633

Published: 16 November 2012

Additional files

Additional file 1:

Figure S1. Protein microarray incubation device and RNAs used for this work. (A) Modified Gentel SIMplex 16 device with microarray slide and assembly components. Schematic diagram of the custom-made silicone gasket and spacer with main dimensions indicated. (B) Expression vector pSPARTA. hPGK - human phosphoglycerate kinase promoter, SV40 polyA - simian virus 40 polyadenylation signal, bGlob polyA - beta-globin polyadenylation signal, Puromycin - resistance gene, EM7 - bacterial promoter, CMV Enh/Prom - cytomegalovirus enhancer promoter, Ori - origin of replication. Unique site depicted in black. Polylinker sites are in red. (C) Denaturing agarose gel electrophoresis of sense and antisense RNAs used in this work. M - RiboRuler RNA ladder (bp): 6000, 4000, 3000, 2000, 1500, 1000, 500, 200. (D) Denaturing agarose gel electrophoresis of biotin-16-UTP labeled RNAs TP53, HRAS, LacZ (1.2kb fragment of LacZ, experimental procedures). M - RiboRuler RNA ladder (bp): 6000, 4000, 3000, 2000, 1500, 1000, 500, 200.

Format: PDF Size: 380KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 2:

Table S1. Expression constructs and promoters used for the strand-specific RNA sequence production. The efficiency of RNA labeling with Cy5 dye was calculated as described in Experimental Procedures.

Format: XLS Size: 37KB Download file

This file can be viewed with: Microsoft Excel Viewer

Open Data

Additional file 3:

Table S2. Significant RNA binding proteins for all sense and antisense RNAs used in this work. Z-Score ≥ 3 and signal intensity above background ≥ 2.5 was used to filter RNA-protein binding events as described in the text.

Format: XLS Size: 144KB Download file

This file can be viewed with: Microsoft Excel Viewer

Open Data

Additional file 4:

Figure S2. Confirmation of RNA-protein binding on microarrays. The reciprocal pull-down assays for CNBP with HRAS mRNA. (A) Quantitation images of human microarray showing selective binding signal of HRAS mRNA sense strand to duplicate CNBP protein spots. The incubation signal shown with respect to adjacent protein controls in the same sub-array. (B) Pull-down of biotin labeled HRAS mRNA in vitro, but not TP53 or LacZ precipitates associated HA-CNBP protein; densitometry quantification of the immunoblots shown (right). (C) HA-CNBP protein pulls down HRAS mRNA in vitro after immunoprecipitation with HA Mab; immunoblots to HA-tagged CNBP verifying CNBP precipitation are shown on the left panel. (D) HA-CNBP protein pulls down HRAS mRNA in vivo, but not control TP53 and GAPDH mRNAs after immunoprecipitation with HA Mab; immunoblots to HA-tagged CNBP verifying CNBP precipitation from cell extracts are shown (left). (E) Incubation of TP53-ORF mRNA sense strand lacking 5′ and 3′ UTR regions to human protein microarrays. Panel at left shows the entire microarray spotted with ~9400 recombinant human proteins; the middle panels represent an enlargement of the sub-array containing Stau1 and WIT1 proteins. Note absence of the Stau1-TP53 mRNA association signal in comparison to Figure 2D when full-length TP53 mRNA was probed. [all proteins spotted in duplicate; Stau1 and WIT1 spots boxed in white; sub-array positive controls boxed in red]. The quantification of the incubation results shown on the right. (F) Contrary to STAU1 depletion, UPF1 KD has no affect on TP53 RNA decay in Primary Fibroblasts.

Format: PDF Size: 487KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data