Biochemical approach for isolation of polyadenylated RNAs with bound proteins from yeast

Abstract

In vivo characterization of RNA-protein interactions is the key for understanding RNA regulatory mechanisms. Herein, we describe a protocol for detection of proteins interacting with polyadenylated RNAs in the yeast Saccharomyces cerevisiae. Proteins are crosslinked to nucleic acids in vivo by ultraviolet (UV) irradiation of cells, and poly(A)-containing RNAs with bound proteins are isolated from cell lysates using oligo[dT]₂₅ beads. RBPs can be detected by immunoblot analysis or with mass spectrometry to define the mRNA-binding proteome (mRBPome) and its changes under stress. For complete details on the use and execution of this protocol, please refer to Matia-González et al. (2021, 2015).

Keywords: Gene Expression; Model Organisms; Molecular Biology; Protein Biochemistry.

Graphical abstract

Figure 1

RNA analysis after UV-irradiation of cells (A) Testing UV-irradiation and RNase control digest. One μg of total RNA was electrophoresed on a 1% agarose gel and stained with peqGREEN DNA/RNA dye. Lane 1: RNA from non-irradiated cells; Lane 2: RNA from UV-irradiated cells. Lane 3: RNA from UV-irradiated cells and RNase ONE treated extracts. M: molecular weight marker. (B) RNA degradation is prevented by UV-irradiation on ice. Lane 1: RNA after exposure of cells to 1,200 mJ without any breaks. Lane 2: one break on ice for 2 min after 600 mJ. Lane 3: 2 breaks on ice (after 400 mJ). M: molecular weight marker.

Figure 2

RNA and protein analysis of poly(A) RIC samples (A) RT-PCR on total RNA isolated from the extract (input) and RIC-eluates with ACT1 specific primers. Products were resolved on a 2% agarose gel and stained with peqGREEN DNA/RNA dye. Poly(A) designates the addition of excess competitor poly(A). A control reaction without RT was included to monitor potential DNA contamination. M: molecular weight marker. (B) Silver stained PAA gel. Lanes 1–2 correspond to 0.05% of input extract and poly(A) treated control samples; lanes 3–4, 10% of eluates from poly(A) mRNA isolation. A Marker (M) with molecular weights (MW) in kilodaltons (KDa) is indicated to the left. (C) Immunoblot analysis to monitor the indicated proteins in 0.1% of the inputs (lanes 1–2) and 40% of the RIC eluates (lanes 3–4). Pab1, poly(A) binding protein 1; Puf3:TAP, tandem affinity purification-tagged Pumilio family protein; Pgk1, 3-phosphoglycerate kinase (a non-conventional RBP); Act1, actin (non-RNA binding control protein). Poly(A) designates the addition of excess competitor poly(A). MW is indicated to the left.