Isolation and characterization of a plastid-targeted mRNA binding protein. A. rbcL 5′ UTR probe used for affinity purification of RNA binding proteins from plastid extracts. This probe contained a 12 carbon linker and biotin at the 3′ end for attachment to streptavadin magnetic beads. B. Biotinylated rbcL and control RNAs (7ZAS, is a yeast viral UTR of similar length and AU content) incubated with plastid extracts. RNA-protein complexes were “fished” from the extracts using streptavadin magnetic beads, and then analyzed using SDS-PAGE (10% gel). Bands were excised from this gel and used for identification by Maldi-Tof/amino acid sequence analysis. Red arrow shows the position of the p44 rbcL mRNA binding protein (now designated RLSB). C. Diagram of RLSB ortholog of Arabidopsis. Maldi-Tof mass spectrometry/amino acid sequence analysis (Custom Biologics, Toronto, CA), and comparisons of peptide sequences with the Arabidopsis and other plant databases, identified one of the purified proteins (p44, red arrow in 1B) as having properties of interest, with a plastid transit sequence and a conserved RNA binding domain. Green = plastid transit sequence (identified using (http://www.cbs.dtu.dk/services/TargetP/). Purple = conserved S1 RNA binding domain. Blue = 149 aa region expressed in E. coli used for affinity purification of p44 (RLSB) antibodies. The 447 nt region encoding this peptide sequence was also used for production of an RNA silencing vector in pHannibal. Underlined sequences within the blue region were used for production of peptide antibodies; the second underlined sequence (bold) also corresponds to a conserved 23 aa tryptic peptide identified in the purified amaranth protein that was identical in the Arabidopsis protein, and highly similar in orthologs from many other plant species (Additional file 1: Figure S1 and Additional file 2: Figure S2).