オリゴヌクレオチド指向溶出を用いて細胞から同族RNA-タンパク質複合体の単離

Post-transcriptional gene expression is precisely regulated, beginning with DNA transcription in the nucleus. Controlled by RNA binding proteins (RBPs), mRNA biogenesis and metabolism occur in highly dynamic ribonucleoprotein particles (RNPs), which associate and dissociate with a substrate precursor mRNA during the progression of RNA metabolism^1-3. Dynamic changes in RNP components affect the post-transcriptional fate of an mRNA and provide quality assurance during the processing of primary transcripts, their nuclear trafficking and localization, their activity as mRNA templates for translation, and the eventual turnover of mature mRNAs.

Numerous proteins are designated as RBPs by virtue of their conserved amino acid domains, including the RNA recognition motif (RRM), the double-stranded RNA binding domain (RBD), and stretches of basic residues (e.g., arginine, lysine, and glycine)⁴. RBPs are routinely isolated by immunoprecipitation strategies and are screened to identify their cognate RNAs. Some RBPs co-regulate pre-mRNAs that are functionally-related, designated as RNA regulons^5-8. These RBPs, their cognate mRNAs, and sometimes non-coding RNA, form catalytic RNPs that vary in composition; their uniqueness is due to various combinations of associated factors, as well as to the temporal sequence, location, and duration of their interactions⁹.

RNA immunoprecipitation (RIP) is a powerful technique to isolate RNPs from cells and to identify associated transcripts using sequence analysis^10-13. Moving from candidate to genome-wide screening is feasible through RIP combined with a microarray analysis¹⁴ or high-throughput sequencing (RNAseq)¹⁵. Likewise, co-precipitating proteins may be identified by mass spectrometry, if they are sufficiently abundant and separable from the co-precipitating antibody^16,17. Here, we address the methodology for isolating RNP components of a specific cognate RNA from cultured human cells, although the approach is alterable for soluble lysates of plant cells, fungi, viruses, and bacteria. Downstream analyses of the material include candidate identification and validation by immunoblot, mass spectrometry, biochemical enzymatic assay, RT-qPCR, microarray, and RNAseq, as summarized in Figure 1.

Given the fundamental role of RNPs in controlling gene expression at the post-transcriptional level, alterations in the expression of component RBPs or their accessibility to cognate RNAs can be detrimental for the cell and are associated with several types of disorders, including neurological disease¹⁸. DHX9/RNA helicase A (RHA) is necessary for the translation of selected mRNAs of cellular and retroviral origins⁶. These cognate RNAs exhibit structurally-related cis-acting elements within their 5' UTR, which is designated as the post-transcriptional control element (PCE)¹⁹. RHA-PCE activity is necessary for the efficient cap-dependent translation of many retroviruses, including HIV-1, and of growth regulatory genes, including junD^6,20,21. Encoded by an essential gene (dhx9), RHA is essential to cell proliferation and its down-regulation eliminates cell viability²². The molecular analysis of RHA-PCE RNPs is an essential step to understanding why RHA-PCE activity is necessary to control cell proliferation.

The precise characterization of the RHA-PCE RNP components at steady state or upon physiological perturbation of the cell requires the selective enrichment and capture of the RHA-PCE RNPs in sufficient abundance for downstream analysis. Here, retroviral PCEgag RNA was tagged with 6 copies of the cis-acting RNA binding site for the MS2 coat protein (CP) within the open reading frame. The MS2 coat protein was exogenously co-expressed with PCEgag RNA by plasmid transfection to facilitate the RNP assembly in growing cells. RNPs containing the MS2 coat protein with cognate MS2-tagged PCEgag RNA were immunoprecipitated from the cell extract and captured on magnetic beads (Figure 2a). To selectively capture the RNP components bound to the PCE, the immobilized RNP was incubated with an oligonucleotide complementary to sequences distal to the PCE, forming an RNA-DNA hybrid that is the substrate for RNase H activity. Since PCE is positioned in the 5' terminal of the 5' untranslated region, the oligonucleotide was complementary to the RNA sequences adjacent to the retroviral translation start site (gag start codon). RNase H cleavage near the gag start codon released the 5' UTR complex from the immobilized RNP, which was collected as the eluent. Thereafter, the sample was evaluated by RT-PCR to confirm the capture of PCEgag and by SDS PAGE and immunoblot to confirm the capture of the target MS2 coat protein. A validation of the PCE-associated RNA binding protein, DHX9/RNA helicase A, was then performed.