Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression

María Eugenia Zanetti, Ing Feng Chang, Fangcheng Gong, David W Galbraith, Julia Bailey-Serres

Research output: Contribution to journalArticle

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Abstract

Immunoaffinity purification of polyribosomes (polysomes) from crude leaf extracts of Arabidopsis (Arabidopsis thaliana) was achieved with transgenic genotypes that overexpress a translational fusion of a ribosomal protein (RP) with a His6-FLAG dual epitope tag. In plants with a cauliflower mosaic virus 35S:HF-RPL18 transgene immunopurification with anti-FLAG agarose beads yielded 60-Svedberg ribosomal subunits, intact 80-Svedberg monosomes and polysomes. Sucrose density gradient fractionation of the purified complexes demonstrated that the distribution of polysome size was similar in crude cell extracts and the purified complexes. The immunopurified complexes included putative cytosolic KPs of Arabidopsis and ribosome-associated proteins, as well as full-length transcripts of high and low abundance. Whole-genome profiling using long DNA oligonucleotide-based microarrays provided a high level of reproducibility between polysomal mRNA samples immunopurified from two independent biological replicates (r approximately 0.90). Comparison of immunopurified and total cellular RNA samples revealed that for most of the genes, the mRNAs were associated with the epitope-tagged polysomal complexes, with an average relative level of association of 62.06% + 4.39%. The results demonstrate that the immunopurification of polysomes can be a valuable tool for the quantification of mRNAs present in translation complexes in plant cells. This technology can be extended to evaluation of mRNA populations at the cell- or tissue-specific level by regulation of the tagged RP with distinct promoters.

Original languageEnglish (US)
Pages (from-to)624-635
Number of pages12
JournalPlant Physiology
Volume138
Issue number2
DOIs
StatePublished - 2005

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polyribosomes
Polyribosomes
Arabidopsis
Gene Expression
gene expression
Messenger RNA
Ribosomal Proteins
ribosomal proteins
Complex Mixtures
Epitopes
Caulimovirus
Ribosome Subunits
Cauliflower mosaic virus
Plant Cells
cells
oligonucleotides
Cell Extracts
Oligonucleotide Array Sequence Analysis
ribosomes
Transgenes

ASJC Scopus subject areas

  • Plant Science

Cite this

Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression. / Zanetti, María Eugenia; Chang, Ing Feng; Gong, Fangcheng; Galbraith, David W; Bailey-Serres, Julia.

In: Plant Physiology, Vol. 138, No. 2, 2005, p. 624-635.

Research output: Contribution to journalArticle

Zanetti, María Eugenia ; Chang, Ing Feng ; Gong, Fangcheng ; Galbraith, David W ; Bailey-Serres, Julia. / Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression. In: Plant Physiology. 2005 ; Vol. 138, No. 2. pp. 624-635.
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