High-throughput methodology for identifying RNA-protein interactions transcriptome-wide

1. A method of transcriptome-wide identification of RNA-protein interaction sites, comprising:

(a) preparing an RNase footprinting library, wherein said preparation comprises:

i. exposing a eukaryotic cell to a crosslinking agent;

ii. obtaining a nucleic acid-containing sample from the cell;

iii. treating a first fraction of the sample with a single-stranded RNase;

iv. thereafter treating the first fraction with a protease or proteinase;

v. treating a second fraction of the sample with a double-stranded RNase;

vi. thereafter treating the second fraction with a protease or proteinase;

vii. isolating RNA from the first fraction and the second fraction; and

viii. preparing a strand specific sequence library from the isolated RNA;

(b) preparing an RNase control library, wherein said preparation comprises:

i. exposing a eukaryotic cell to a crosslinking agent;

ii. obtaining a nucleic acid-containing sample from the cell;

iii. treating a first fraction of the sample with protease or proteinase;

iv. thereafter treating the first fraction of the sample with a single-stranded RNase;

v. treating a second fraction of the sample with protease or proteinase;

vi. thereafter treating the second fraction of the sample with a double-stranded RNase;

vii. isolating RNA from the first fraction and the second fraction; and

viii. preparing a strand specific sequence library from the isolated RNA,

(c) identifying candidate protein binding sites within the RNase footprinting library; and

(d) identifying sequence level motifs within the candidate protein binding sites.

2. The method of claim 1 , wherein the sample is obtained by lysing the cell.

3. The method of claim 2 , further comprising treating the sample with a DNase following cell lysis.

4. The method of claim 1 , wherein identifying candidate protein binding sites comprises identifying protein protected sites; and comparing the protein protected sites with previously published protein data.

5. The method of claim 1 , wherein identifying sequence level motifs comprises selecting a site specific candidate protein binding site from the candidate protein binding sites; and using a motif finding algorithm to identify at least one motif from the site specific candidate protein binding site.

6. The method of claim 5 , wherein the site specific candidate binding site is found in the 5′ UTR, the protein coding region, or the 3′ UTR.

7. The method of claim 5 , further comprising validating the motif by performing a competitive pull-down assay.