How did the authors elute the protein complex from the beads?

The authors eluted the isolated protein complex from the beads twice for 20 min at room temperature in fresh aqueous 500 mM NH4OH, 0.5 mM EDTA solution.

How do the authors isolate the tagged complexes?

The authors isolate the tagged complexes using antibody-conjugated magnetic beads, rather than more commonly used antibody-conjugated resins such as Sepharose.

What is the rRNA that is associated with the nop15p?

To monitor recoveries of Nop15p-associated RNAs, the authors followed the ribosomal pre-RNA 27SB, which is the precursor to mature 5.8S and 25S rRNAs and a component of pre-60S particles.

What are the main issues that the authors found during their RNP isolation procedure?

The authors showed that neither protein nor RNA rearrangements (leading to false positives) are issues during their RNP isolation procedure.

What is the purpose of the primer extension analysis?

The authors also used primer extension analyses to check for crosscontamination of RNAs between complexes during the isolation procedure, asking whether rRNAs are found in an mRNP complex isolation and vice versa.

What is the importance of ensuring that RNPs do not rearrange on RNAs?

For the isolated RNP pool to represent the in vivo situation, the authors must ensure that RNP proteins do not rearrange on RNAs during the isolation procedure.

How did the authors identify the proteins She3p and Myo4p?

Using PrA-tagged She2p, their proteomic analysis identified the expected proteins She3p and Myo4p; in addition the authors found two other proteins, Myo2p and Act1p (Fig. 4).

How many of these proteins are NPC components?

Consistent with previous studies and in accordance with the function of Mex67p in transporting mRNAs across the NPC, 23 of these proteins are NPC components (Supplementary Table 2).

Open AccessJournal Article10.1038/NMETH1101

Comprehensive analysis of diverse ribonucleoprotein complexes

Marlene Oeffinger, +6 more

- 01 Nov 2007

- Nature Methods

- Vol. 4, Iss: 11, pp 951-956

300

TL;DR: This work describes a rapid affinity-purification method for efficient isolation of the subcomplexes that dynamically organize different RNP biogenesis pathways in Saccharomyces cerevisiae that overcomes many of the previous limitations to produce large RNP interactomes with almost no contamination.

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.

1. What are the contributions in "Npgrj_nmeth_1101 951..956" ?

The study of the dynamic interactome of cellular ribonucleoprotein ( RNP ) particles has been hampered by severe methodological limitations.. Here the authors describe a rapid affinity-purification method for efficient isolation of the subcomplexes that dynamically organize different RNP biogenesis pathways in Saccharomyces cerevisiae.

2. What is the mRNA content of the yeast complexes?

A recent study has shown that B70% of mRNA within a yeast cell is polysome-associated, with B10% suggested to be nuclear mRNAs and the rest being free cytoplasmic mRNAs waiting to be either translated or degraded18.

3. Why do the authors avoid damage to RNP complexes?

Because cell breakage occurs in the frozen state, the authors avoid damage to RNPcomplexes by released nucleases and proteases, as well as redistribution of proteins within the extract.

4. What is the role of She2p in the localization of ASH1 mRNA?

She2p is involved in the localization of ASH1 mRNA to the distal tip of daughter cells during anaphase28, which is accomplished by She2p binding directly to the RNA and engaging it via the linker protein She3p to the myosin Myo4p that then transports the complex along actin cables to the bud tip29.

Comprehensive analysis of diverse ribonucleoprotein complexes

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Most frequently asked questions

1. What are the contributions in "Npgrj_nmeth_1101 951..956" ?

2. What is the mRNA content of the yeast complexes?

3. Why do the authors avoid damage to RNP complexes?

4. What is the role of She2p in the localization of ASH1 mRNA?

5. How did the authors elute the protein complex from the beads?

6. How do the authors isolate the tagged complexes?

7. What is the rRNA that is associated with the nop15p?

8. What are the main issues that the authors found during their RNP isolation procedure?

9. What is the purpose of the primer extension analysis?

10. What is the importance of ensuring that RNPs do not rearrange on RNAs?

11. How did the authors identify the proteins She3p and Myo4p?

12. How many of these proteins are NPC components?

Figures

Citations

A census of human RNA-binding proteins.

Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding

Integrative structure and functional anatomy of a nuclear pore complex

CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1

Upf1 senses 3'UTR length to potentiate mRNA decay.

References

Functional organization of the yeast proteome by systematic analysis of protein complexes

Global analysis of protein expression in yeast

A comprehensive two-hybrid analysis to explore the yeast protein interactome

Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry

Global landscape of protein complexes in the yeast Saccharomyces cerevisiae

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