Abstract
Mammalian cells express hundreds of RNA binding proteins (RBPs) that are essential regulators of RNA metabolism. RBP activity plays a central role in the control of gene expression programs and identification of RNA-protein interactions is critical for comprehensive understanding of gene regulation in cells. In recent years, various tools and techniques to identify these RNA-protein interactions have been developed. Among those, RNA immunoprecipitation is a precise and powerful assay that can be used to establish the physical interaction of an individual RBP with its target RNAs in vivo. Here, we describe a quantitative method for determining RNA-protein interactions using RNA immunoprecipitation (RNA-IP) assay in mouse embryonic stem cells carrying ectopically expressed mutant constructs. This protocol is reliable and easily adaptable to identify the interactions of endogenous or ectopically expressed RNAs and proteins.
Keywords: RNA immunoprecipitation, RNA binding protein, SR protein, RNA-protein interaction, Antibody, Quantitative PCR
Background
Embryonic stem cells express more than 500 different RBPs highlighting the potential of RNA regulation in controlling pluripotency (Kwon et al., 2013). We have established serine-arginine-rich splicing factor 3 (SRSF3) as an essential RBP to maintain pluripotency in mouse embryonic stem cells (Ratnadiwakara et al., 2018). SRSF3 binds to RNAs encoding key components of mouse pluripotency circuitry and controls their expression via multiple RNA processing mechanisms. Among these, we have specifically demonstrated that SRSF3 directly binds to the core pluripotency transcription factor Nanog mRNA to facilitate its nucleo-cytoplasmic export. Our results have shown that SRSF3 binding is essential for the efficient export of the Nanog mRNA, targeted mutation of SRSF3 binding sites in Nanog mRNA abolishing SRSF3 binding and consequently the nucleo-cytoplasmic export of Nanog. The basic principle of RNA-IP is based on using an antibody recognizing the protein of interest to specifically pull down the RBP with the target RNA complexes. Therefore, any RNA that is physically associated with the protein can be isolated and further analyzed. We have used quantitative PCR for the downstream analysis. However, other methods such as sequencing or hybridization can also be used to identify the targeted RNAs. Here we use cells that express an SRSF3-GFP fusion protein and used an anti-GFP antibody for the immunoprecipitation. It is important to note that an antibody against an endogenous RBP may be successfully used for RNA-IP. However, we have observed that anti-RBP antibodies can compete with RNA and thus, for instance in the case of SRSF3, cannot be used to efficiently purify SRSF3 in complex with RNA. We have used RNA immunoprecipitation (RNA-IP) to detect the physical interaction of SRSF3 not only with the endogenous Nanog mRNA, but also ectopically expressed constructs carrying mutations within SRSF3 binding sites in the Nanog mRNA. This assay allows the assessment of direct RNA-protein interactions as well as the sequence specificity of RBPs in cells.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
Recipes
Acknowledgments
MLA was supported by National Health and Medical Research Council (NHMRC) GNT1043092 and GNT1138870, Aatos and Jane Erkko Foundation and Monash Biomedicine Discovery Fellowship. The protocol was adapted from a previously published study (Anko et al., 2010).
Competing interests
The authors have no conflicts of interest or competing interests.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.