title: Purification of an Arabidopsis chloroplast extract with in vitro RNA processing activity on psbA and petD 3'-untranslated regions.
abstract: The mature 3'-end of many chloroplast mRNAs is generated by the processing of the 3'-untranslated region (3'-UTR), which is a mechanism that involves the removal of a segment located downstream an inverted repeat sequence that forms a stem-loop structure. Nuclear-encoded chloroplast RNA binding proteins associate with the stem-loop to process the 3'-UTR or to influence mRNA stability. A spinach chloroplast processing extract (CPE) has been previously generated and used to in vitro dissect the biochemical mechanism underlying 3'-UTR processing. Being Arabidopsis thaliana an important genetic model, the development of a CPE allowing to correlate 3'-UTR processing activity with genes encoding proteins involved in this process, would be of great relevance. Here, we developed a purification protocol that generated an Arabidopsis CPE able to correctly process a psbA 3'-UTR precursor. By UV crosslinking, we characterized the protein patterns generated by the interaction of RNA binding proteins with Arabidopsis psbA and petD 3'-UTRs, finding that each 3'-UTR bound specific proteins. By testing whether Arabidopsis CPE proteins were able to bind spinach ortholog 3'-UTRs, we also found they were bound by specific proteins. When Arabidopsis CPE 3'-UTR processing activity on ortholog spinach 3'-UTRs was assessed, stable products appeared: for psbA, a smaller size product than the expected mature 3'-end, and for petD, low amounts of the expected product plus several others of smaller sizes. These results suggest that the 3'-UTR processing mechanism of these chloroplast mRNAs might be partially conserved in Arabidopsis and spinach.
authors.affiliation: Departamento de Bioquímica, Universidad Nacional Autónoma de México, Av. Universidad 3000, Ciudad Universitaria, Delegación Coyoacán, CP 04510, México, DF México.
authors.forename: Denia
authors.initials: D
authors.lastname: Coloapa-Soto
authors.forename: Martín
authors.initials: M
authors.lastname: Vargas-Suárez
authors.forename: Herminia
authors.initials: H
authors.lastname: Loza-Tavera
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D060365:Chloroplast Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism*; D010936:Plant Extracts / Q000302:isolation & purification / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018724:Spinacia oleracea / Q000235:genetics
chemical_list: D020413:3' Untranslated Regions; D029681:Arabidopsis Proteins; D060365:Chloroplast Proteins; D010936:Plant Extracts; D012333:RNA, Messenger; D018749:RNA, Plant; D016601:RNA-Binding Proteins