Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.
Pool design and oligo recovery
Co-immunoprecipitation of the oligo pool with an RNP of interest
Reverse transcription and amplification of co-IP samples in preparation for aminoallyl labeling
Amino-allyl labeling of RNA with Cy Dyes
Hybridize samples to the microarray using the Agilent Array Hybridization Kit
Interpreting the Microarray
Figure 1. Experimental schematic
A. Tiling scheme. After selecting and downloading pre-mRNA sequences of interest, the selecting area is tiled through in 30 nucleotide windows that shift in 10 nucleotide increments. Universal primer binding sites flank each side of the window.
B. Synthesis Array. Tiled oligo orders should be submitted to Agilent, where the sequences will be printed on a custom oligonucleotide microarray.
C. Co-immunoprecipitation. After boiling the oligos off the array, incubate in HeLa nuclear extract, then add the extract to magnetic beads that are prepared with an antibody against the RNP of interest. Label the starting oligo pool with Cy3 and the bound IP pool with Cy5 and apply to the detection array and scan.
Figure 2. Annotating the genome with and analysis of enrichment data.
A. Convert the average score at each genomic coordinate from the array data to base-ten log and map that score to the given coordinates. An illustration of this averaging step is given for 3 overlapping 30-nt oligos with scores of 2, 4, and 0.5, where the average enrichment score for each 10-nt window is graphed above. The selected genomic regions can be visualized using a Custom Track in the USCS Genome Browser. The example browser window given is in the clta gene, where gene features (exons/introns/alternative splicing etc.) are given along the bottom and log average enrichment scores from the PTB-bound oligos are represented by dark red vertical bars. PTB strongly binds just upstream of exon 2 in the polypyrimidine tract.
When doing this procedure it’s important to remember that creation of the pool is flexible and open to modification at either the RNA or protein level. At the RNA level orthologous regions, disease mutations, polymorphisms, or random mutations can be introduced into the oligonucleotide sequence. At the protein level the RNA binding factor can be modified by phosphorylation or other post translational modifications. Additionally, the binding environment can be manipulated to either increase or decrease the levels of additional factors that interact or compete with the RNA binding protein of interest.