Post-transcriptional Control of Chloroplast Gene Expression via RNA-Protein Complexes

1. Post-transcriptional Control of Chloroplast Gene Expression via RNA-Protein Complexes Current Past Josh Baecker Dawn Both Patricia Merhige Tracy Hotchkiss Brandon Stilb Mark Robida Department of Energy

2. Chloroplasts are like big green prokaryotes Spinach Chloroplast genome: 150,725 bp However, the majority of chloroplast polypeptides are encoded by the nucleus.

3. Post-transcriptional Mechanisms Have Important Effects on Chloroplast Gene Expression RNA processing RNA stability Translation Protein modification/complex assembly Protein turnover Mediated by RNA-protein Complexes

4. RNA-protein Complexes Can Form in 5’Untranslated Regions (5’UTRs) 5’UTRs are sequences upstream of open reading frames In chloroplasts, they often encode ribosome binding sites. Proteins that bind to chloroplast 5’UTRs have been shown to affect RNA stability and/or translation initiation.

5. RNA-protein Complexes Can Be Analyzed by Gel Mobility Shift Assays + RNAsPotential binding proteins (specific, radiolabeled) (specific) (non-specific) (non-specific) (non-binding) Incubation Self competition: - - + Chloroplast extract: - + + { RNA-protein complexes Native gel Unbound RNA:

6. Questions to Be Answered About Chloroplast RNA-protein Complexes What are the cis-acting elements that affect complex formation? What are the proteins involved in the complexes? What is the function of these complexes?

7. What are the cis-acting elements that affect complex formation? Is there conserved RNA structure? Where do the proteins bind?

8. The atpI 5’UTR Is Our Baseline Binding Substrate

9. THERE ARE TWO BINDING SITES IN THE atpI 5’UTR C: + - - E: + + - ORF * -155 -52 -1 +15 * ORF -155 -58 -52 -1 +15 C: + - - E: + + - C: + - - E: + + - * * C: self-competitor E: chloroplast extract *: RNA-protein complex

10. What are the proteins involved in the complexes? Are they ATP synthase-specific?

11. Competition Binding Assays Revealed that the Same Chloroplast Proteins Bind Diverse 5’UTRs Good Competitors; similar binding proteins atpI ATP synthase subunit CFo-IV atpA ATP synthase subunit CF1-α atpE ATP synthase subunit CF1-ε atpF ATP synthase subunit CFo- I atpH ATP synthase subunit CFo-III clpP protease subunit infA translation initiation factor ndhD NADH dehydrogenase subunit petL cytochrome b/f 3.5 kDa subunit psaC photosystem I 9 kDa protein rbcL large subunit, carboxylase rpl22 large ribosome subunit protein 22 rpoA RNA polymerase α-subunit rpoB RNA polymerase β-subunit rps2 small ribosome subunit protein 2 rps11 small ribosomal subunit 11 Partial competitor; some shared proteins rps16 small ribosome subunit 16 Poor competitor; few shared proteins psbA Photosystem II D1 protein Supershift atpB ATP synthase subunit CF1-β

12. S mag bead Affinity purification of binding proteins atpI 5’UTR Streptavidin- binding motif Chloroplast extract Incubate Isolate

13. SDS-PAGE of affinity-isolated 5’UTR-binding proteins MW Unbound W1 W2 W3 Isolated 66 55 42 37 27 14 20

14. What is the function of these complexes? Do they affect translation? Do they affect RNA stability?

15. Chloroplast Transformation Antibiotic-resistant transformants (Week 6) Target Plants (Week 2) Second Selection (Week 10) Rooting (Week 12) Roots

16. Wild-type D Deletion Chimeric Complete deletion (except for RBS) D 5’ UTR deletion Variations on a single 5’UTR rps16 3’ UTR misc. sequence atpI 5’ UTR uidA reporter

17. The atpI 5’UTR Affects Translation and RNA Abundance

18. Conclusions & Projects Cis-acting elements There are two binding sites and extensive phylogentically conserved sequence in the atpI 5’UTR Structure probing studies will be used to identify specific structure(s) necessary for binding. Trans-acting factors The same polypeptides bind many chloroplast 5’UTRs. Affinity-based methods will be used to isolate the binding protein(s) Once the genes encoding them are cloned, they will be mutated and expressed in vivo Function Preliminary data demonstrate an effect of the atpI 5’UTR on both RNA stability and translation. In vivo experiments will be used to dissect 5’UTR function.