Trends in Biochemical Sciences
Technology of the MonthIn-Cell Discovery of RNA–Protein Interactions
Section snippets
ADVANTAGES:
Does not rely on direct RNA pulldowns, thus circumventing a step that is often plagued by low efficiency.
Avoids crosslinking and the associated inefficiency and biases.
Not limited by low expression levels of the test RNA or proteins.
Can identify proteomes of specific RNA regions and provide RNA region-specific resolution of protein binding.
Can identify transient RNA–protein interactions.
One cell line is used for all applications.
The protein library is not limited in size and can be expanded or
CHALLENGES:
incPRINT is a primary binding assay that does not consider the developmental context or timing of RNA–protein interactions.
A subset of proteins may interact with MS2 stem-loops but not with the test RNA.
Protein tags may alter protein expression or binding.
Some interactions may be indirect: additional validation assays are necessary to establish direct RNA binding.
Some RNA–protein interactions may require additional factors that are not expressed in the cell line used for the assay.
Ectopic
Literature (10)
Systematic discovery of Xist RNA binding proteins
Cell
(2015)The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts
Mol. Cell.
(2012)Insights into RNA biology from an atlas of mammalian mRNA-binding proteins
Cell
(2012)Quantitative analysis of HSP90–client interactions reveals principles of substrate recognition
Cell
(2012)The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3
Nature
(2015)