Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression.
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 His(6)-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 RPs 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.
Zanetti
María Eugenia
ME
Department of Botany and Plant Science, Center for Plant Cell Biology, University of California, Riverside, California 92521-0124, USA.
Chang
Ing-Feng
IF
Gong
Fangcheng
F
Galbraith
David W
DW
Bailey-Serres
Julia
J
D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000032:analysis; D002458:Cell Fractionation / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D007120:Immunochemistry; D020411:Oligonucleotide Array Sequence Analysis; D011132:Polyribosomes / Q000737:chemistry*; D011485:Protein Binding; D012333:RNA, Messenger / Q000302:isolation & purification; D018749:RNA, Plant / Q000302:isolation & purification
D029681:Arabidopsis Proteins; D012333:RNA, Messenger; D018749:RNA, Plant