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Comprehensive analysis of diverse ribonucleoprotein complexes

Abstract

The study of the dynamic interactome of cellular ribonucleoprotein (RNP) particles has been hampered by severe methodological limitations. In particular, the affinity purification of intact RNP complexes from cell lysates suffers from RNA degradation, loss of interacting macromolecules and poor overall yields. Here we describe a rapid affinity-purification method for efficient isolation of the subcomplexes that dynamically organize different RNP biogenesis pathways in Saccharomyces cerevisiae. Our method overcomes many of the previous limitations to produce large RNP interactomes with almost no contamination.

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Figure 1: Schematic outline of the single-step purification technique.
Figure 2: Isolation of PrA-tagged Nop15p complexes.
Figure 3: Quantitation of recovered RNA and protein-rearrangement analysis during complex isolation.
Figure 4: Analysis of specific complexes along the mRNA maturation pathway.

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Acknowledgements

We thank D. Zenklusen (Albert Einstein College of Medicine) for providing the She2p-PrA and Cbp80p-PrA strains, and F. Stutz (University of Geneva) for providing the antibody to Yra1. We thank all the members of the Rout, Chait and Aitchison laboratories for their support. This work was supported by a grant from the American Cancer Society (RSG0404251) to M.P.R.; by grants from the US National Institutes of Health to B.T.C. (RR00862), J.D.A. (GM067228 and GM076547), and M.P.R., J.D.A. and B.T.C. (RR022220); by a Tri-Institutional Training Fellowship in Chemical Biology to J.A.D.; and by a fellowship from the Charles Revson Foundation to M.O.

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Correspondence to Michael P Rout.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Table 4, Supplementary Methods (PDF 977 kb)

Supplementary Table 1

List of proteins identified with different PrA-tagged mRNP components. (XLS 56 kb)

Supplementary Table 2

List of proteins identified with Nop15-PrA. (XLS 44 kb)

Supplementary Table 3

Lists of transcripts enriched with Nop15p-PrA and She2p-PrA. (XLS 23 kb)

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Oeffinger, M., Wei, K., Rogers, R. et al. Comprehensive analysis of diverse ribonucleoprotein complexes. Nat Methods 4, 951–956 (2007). https://doi.org/10.1038/nmeth1101

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