title: A novel plant in vitro assay system for pre-mRNA cleavage during 3'-end formation.
abstract: Messenger RNA (mRNA) maturation in eukaryotic cells requires the formation of the 3' end, which includes two tightly coupled steps: the committing cleavage reaction that requires both correct cis-element signals and cleavage complex formation, and the polyadenylation step that adds a polyadenosine [poly(A)] tract to the newly generated 3' end. An in vitro biochemical assay plays a critical role in studying this process. The lack of such an assay system in plants hampered the study of plant mRNA 3'-end formation for the last two decades. To address this, we have now established and characterized a plant in vitro cleavage assay system, in which nuclear protein extracts from Arabidopsis (Arabidopsis thaliana) suspension cell cultures can accurately cleave different pre-mRNAs at expected in vivo authenticated poly(A) sites. The specific activity is dependent on appropriate cis-elements on the substrate RNA. When complemented by yeast (Saccharomyces cerevisiae) poly(A) polymerase, about 150-nucleotide poly(A) tracts were added specifically to the newly cleaved 3' ends in a cooperative manner. The reconstituted polyadenylation reaction is indicative that authentic cleavage products were generated. Our results not only provide a novel plant pre-mRNA cleavage assay system, but also suggest a cross-kingdom functional complementation of yeast poly(A) polymerase in a plant system.
authors: Department of Botany, Miami University, Oxford, Ohio 45056, USA.
authors: Hongwei
authors: H
authors: Zhao
authors: Jun
authors: J
authors: Zheng
authors: Qingshun Quinn
authors: QQ
authors: Li
mesh_terms: D017360:Arabidopsis / Q000166:cytology / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D002478:Cells, Cultured; D004720:Endonucleases / Q000378:metabolism; D007700:Kinetics; D009687:Nuclear Proteins / Q000378:metabolism; D026723:Polyadenylation / Q000235:genetics; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012995:Solubility; D013379:Substrate Specificity
chemical_list: D009687:Nuclear Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D011062:Polynucleotide Adenylyltransferase; D004720:Endonucleases