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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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DNA-Binding Proteins / analysis
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DNA-Binding Proteins / genetics
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Electrophoresis, Polyacrylamide Gel
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Green Fluorescent Proteins / analysis
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Green Fluorescent Proteins / genetics
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Immunosorbent Techniques
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Mass Spectrometry
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Nuclear Proteins / analysis
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Nuclear Proteins / genetics
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Nucleocytoplasmic Transport Proteins / analysis
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Nucleocytoplasmic Transport Proteins / genetics
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Oligonucleotide Array Sequence Analysis
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Porins
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RNA Cap-Binding Proteins
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RNA, Fungal / analysis
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RNA, Messenger / analysis
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RNA, Ribosomal / analysis
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RNA-Binding Proteins / analysis
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RNA-Binding Proteins / genetics
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Ribonucleoproteins / analysis*
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Ribonucleoproteins / genetics
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Ribosomal Proteins / analysis
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Ribosomal Proteins / genetics
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins / analysis*
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Saccharomyces cerevisiae Proteins / genetics
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Staphylococcal Protein A / analysis
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Staphylococcal Protein A / genetics
Substances
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DNA-Binding Proteins
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MEX67 protein, S cerevisiae
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Nuclear Proteins
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Nucleocytoplasmic Transport Proteins
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Porins
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RNA Cap-Binding Proteins
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RNA, Fungal
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RNA, Messenger
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RNA, Ribosomal
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RNA-Binding Proteins
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Ribonucleoproteins
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Ribosomal Proteins
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SAC3 protein, S cerevisiae
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SHE2 protein, S cerevisiae
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STO1 protein, S cerevisiae
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Saccharomyces cerevisiae Proteins
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Staphylococcal Protein A
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Thp2 protein, S cerevisiae
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YRA1 protein, S cerevisiae
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messenger ribonucleoprotein
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Green Fluorescent Proteins