PIP-Seq

PIP-Seq

PIP-Seq sequences RBP interaction sites on both unprocessed and mature RNA species in crosslinked or noncrosslinked cells. In PIP-seq, both RNase-sensitive and RNase-insensitive fragments are isolated and processed separately to differentiate which sequences are actually bound to RBPs and which are just insensitive to RNases.

First, crosslinked (through UV or formaldehyde) or noncrosslinked cells are lysed. The lysates are split into 2 batches: experimental and RNase-insensitivity control. The experimental/footprint samples are digested with double-stranded RNase (dsRNase) or single-stranded RNase (ssRNase) and subsequently treated with proteinase K to remove the RBPs. However, to screen for regions insensitive to RNases, the controls are first treated with proteinase K and then with ssRNase or dsRNase. Both sets of fragments are reverse-crosslinked and used to prepare strand-specific libraries. Each library is normalized using rehybridization and a thermostable duplex-specific nuclease, and then sequenced.

Pros:

• Sequences RNA regions bound to RBPs in unprocessed or mature RNAs
• Identifies regions that are insensitive to RNases
• Can be used on tissues and whole organisms
• Strand-specific

Cons:

• Limited resolution of small nucleotide bulges and loops¹
• Formaldehyde crosslinking presents a risk of protein-protein linking, in addition to protein-RNA linking¹
• Nucleases have limited diffusability into plant cells; an advantage of chemical probes, such as dimethyl sulfate (DMS)¹

• PIP-Seq: Silverman I. M., Li F., Alexander A., Goff L., Trapnell C., et al. (2014) RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome. Genome Biology 15: R3
• PIP-Seq: Silverman I. M. and Gregory B. D. (2014) Transcriptome-wide ribonuclease-mediated protein footprinting to identify RNA-protein interaction sites. Methods
• 1. Foley S. W., Vandivier L. E., Kuksa P. P. and Gregory B. D. (2015) Transcriptome-wide measurement of plant RNA secondary structure. Curr Opin Plant Biol 27: 36-43

1. Anderson S. J., Willmann M. R. and Gregory B. D. Protein Interaction Profile Sequencing (PIP-seq) in Plants. Current Protocols in Plant Biology. 2016;
2. Nussbacher J. K., Batra R., Lagier-Tourenne C. and Yeo G. W. RNA-binding proteins in neurodegeneration: Seq and you shall receive. Trends Neurosci. 2015;38:226-236
3. Foley S. W., Vandivier L. E., Kuksa P. P. and Gregory B. D. Transcriptome-wide measurement of plant RNA secondary structure. Curr Opin Plant Biol. 2015;27:36-43
4. Popova V. V., Kurshakova M. M. and Kopytova D. V. [Methods to study the RNA-protein interactions]. Mol Biol (Mosk). 2015;49:472-481

1. Gosai S. J., Foley S. W., Wang D., et al. Global analysis of the RNA-protein interaction and RNA secondary structure landscapes of the Arabidopsis nucleus. Mol Cell. 2015;57:376-388