Figure 3.

Confirmation of the subcellular location and molecular function of HPE1. A, Schematic diagram of the HPE1 protein, including the chloroplast transit peptide (CTP) and RNA recognition motif (RRM). B, Subcellular localization of HPE1 within the chloroplast using the GFP assay. GFP, Control with empty vector; Nuc-GFP, nuclear control; Chl-GFP, chloroplast control; HPE1-GFP, HPE1-GFP fusion. Bars = 10 μm. C, Analysis of splicing defects in the hpe1 mutants using RT-PCR. Products of RT-PCR were isolated and sequenced. Asterisks indicate unspliced mRNA precursors, and stars indicate spliced mature mRNA. D, Confirmation of the association of HPE1 with target RNA using RNA immunoprecipitation (RIP). The top gels show western blots of proteins present in crude leaf extracts derived from Col-0 and hpe1 mutant plants and proteins immunoprecipitated (IP) with the anti-HPE1 antibody. In the bottom gels, RT-PCR was used to detect the association of trnK, rpoC1, and atpF introns with HPE1. Five additional independent biological replicates were performed, and similar results were obtained.