RNA-Binding Protein Antibodies Validated for RNA Immunoprecipitation

Validated RBP antibodies: Enliven your RIP protocols

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RNA-binding proteins (RBPs) recognize specific RNA sequences or structures and control many molecular functions, including transcription, splicing, 5′ RNA capping, polyadenylation, mRNA export, ribosome assembly, translation, and RNA decay. These diverse functions allow RBPs to wield exquisite control over gene expression that is either dependent on or independent of transcription. Because these proteins play significant roles in regulating gene expression, identifying which RNAs a specific RBP associates with can provide powerful information for dissecting downstream functions. Similar to chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP) often starts with the crosslinking of live cells, followed by immunoprecipitation of RNA–protein complexes using a specific RBP antibody, and recovery and identification of the RNA sequences [1]. Thermo Fisher Scientific has instituted validation* programs for Invitrogen RBP antibodies that use RIP and RIP-western (RIP followed by western blotting to determine the protein immunoprecipitated). Here we highlight a few targets in our rapidly growing RBP antibody portfolio.

RNA binding by the PRC2 complex

The polycomb repressive complex 2 (PRC2), which has histone methyltransferase activity, plays a critical role in gene regulation by controlling the trimethylation of histone H3 on lysine 27 (H3K27me3)—one of the hallmarks of transcriptionally silent chromatin. Dysregulation of PRC2 has been implicated in several types of cancer, and PRC2 subunits have long been targets for therapeutics. Although PRC2 subunits have been shown to bind to numerous types of RNA, the complex’s function is still rather elusive [2]. For example, PRC2 interacts with many nascent RNAs, leading researchers to question the function of this promiscuous binding.

The "junk-mail model" suggests that promiscuous binding of PRC2 serves as a checkpoint— PRC2 identifies target genes that have escaped repression by binding to the newly transcribed RNA and then scanning the local chromatin for H3K27me3, which signals that the gene should be silenced. Alternatively, the "antagonistic model" is based on competition of RNA and chromatin for the binding of PRC2. For highly expressed genes, PRC2 binding shifts to the nascent RNA; for transcriptionally silent genes, RNA levels are low so PRC2 binding shifts to chromatin. Furthermore, it has been proposed that PRC2 may prefer certain RNA secondary structures— such as RNA G-quadruplexes [3]. The ability to analyze which RNAs bind the PRC2 complex is essential for a better understanding of the biological consequences of these interactions. Figure 1 shows that SUZ12, a core component of PRC2, can be immunoprecipitated using a recombinant monoclonal SUZ12 antibody and then analyzed by RIP-western. RIP and other experiments that examine RNA–protein interactions will help to clarify the function of PRC2 binding to RNA.

mRNA binding by IMPs

The highly conserved IMP family of mRNA-binding proteins, also known as insulin-like growth factor 2 mRNA binding proteins (IGF2BPs), regulate RNA localization, translation, and stability, and associate with over 1,000 target mRNAs [4]. IMP1 (IGF2BP1) and IMP3 (IGF2BP3) are primarily expressed during embryogenesis, although re-expressed in various tumor tissues [5,6]. IMP2 (IGFBP2) has been observed during development and in adult mice and has been shown to be expressed at elevated levels in cancer cells [7]. Although their roles are still being investigated, the IMPs selectively bind to and regulate their RNA targets. Dysregulated binding due to aberrant expression is associated with different types of cancer.

Several target mRNAs were used with RIP to explore the specificity of recombinant polyclonal IMP antibodies in HepG2 cells (Figure 2), a human liver cancer cell line. The IGF2 transcript is a target for all three IMPs, although IMP1 inhibits translation, whereas IMP2 and IMP3 enhance translation. ACTB and MYC transcripts have been described as targets for IMP1, but enrichment of these mRNAs was seen for all three IMPs in HepG2 cells, which may be related to the role of IMP2 and IMP3 in liver cancer. IMPs have not been known to associate with 18S rRNA, and our RIP data are consistent with this observation.

Techniques for examining RNA–protein interactions

RIP is an excellent technique for identifying RNAs within an RNA–protein complex but may not be well suited for identifying direct RNA–protein interactions. A modification of RIP—the crosslinking and immunoprecipitation technique (CLIP)—uses UV crosslinking and stringent conditions to isolate only the specific RBP of interest and RNA species to which it is bound, and not other proteins and RNA contained within the complex. Several refinements have been developed, including the enhanced CLIP technique (eCLIP) shown in Figure 3 [8]. Immunoprecipitation with eIF4E and hnRNP K monoclonal antibodies demonstrate that they bind at different locations in TRA2A, as shown by shifts in peak density.

References

1. Selth LA, Gilbert C, Svejstrup JQ (2009) Cold Spring Harb Protoc 2009: pdb.prot5234. 
2. Yan J, Dutta B, Hee YT et al. (2019) RNA Biol 16:176–184. 
3. Wang X, Goodrich KJ, Gooding AR et al. (2017) Mol Cell 65:1056–1067. 
4. Degrauwe N, Suvà ML, Janiszewska M et al. (2016) Genes Dev 30:2459–2474. 
5. Bell JL, Wächter, K, Mühleck B et al. (2013) Cell Mol Life Sci 70:2657–2675. 
6. Huang X, Zhang H, Guo X et al. (2018) J Hematol Oncol 11:88. 
7. Cao J, Mu Q, Huang H (2018) Stem Cells Int 2018:4217259. 
8. Van Nostrand EL, Pratt GA, Shishkin AA et al. (2016) Nat Methods 13:508–514.