1. Prepare about a 10 minute talk for Wednesday Feb 25 on something to do with ncRNA, RNA editing, alternative poly-adenylation, mRNA localization or mRNA transport.

2. Post-transcriptional regulation 1) mRNA processing 2) export from nucleus 3) mRNA degradation 4) mRNA localization 5) initiation of translation varies >10x 6) regulating enzyme activity activators Inhibitors Covalent mods

3. Post-transcriptional regulation Protein degradation rate varies 100x Some have motifs, eg Destruction box, marking them for polyubiquitination : taken to proteasome & destroyed

4. DWD Proteins Jae-Hoon Lee’s research putative substrate receptors for CUL4-based E3 ligases

5. DWD Proteins used bioinformatics to find all Arabidopsis & rice DWDs Placed in subgroups based on DWD sequence Tested members of each subgroup for DDB1 binding

6. DWD Proteins Tested members of each subgroup for DDB1 binding co-immunoprecipitation

7. DWD Proteins Tested members of each subgroup for DDB1 binding co-immunoprecipitation Two-hybrid: identifies interacting proteins

8. DWD Proteins Tested members of each subgroup for DDB1 binding co-immunoprecipitation Two-hybrid: identifies interacting proteins Only get transcription if one hybrid supplies Act D & other supplies DNA Binding Domain

9. DWD Proteins Two-hybrid libraries are used to screen for protein-protein interactions

10. DWD Proteins Tested members of each subgroup for DDB1 binding co-immunoprecipitation Two-hybrid

11. DWD Proteins Tested members of each subgroup for DDB1 binding co-immunoprecipitation Cul4cs &PRL1 (Pleiotropic Regulatory Locus 1) had Similar phenotypes

12. DWD Proteins Cul4cs &PRL1 (PleiotropicRegulatory Locus 1) had similar phenotypes PRL1 may be receptor for AKIN10 degradation (involved in sugar sensing)

13. DWD Proteins Found T-DNA insertions 3 were sensitive to ABA

14. DWD Proteins Found T-DNA insertions 3 were sensitive to ABA ABI5 was elevated in dwa mutants

15. DWD Proteins Found T-DNA insertions 3 were sensitive to ABA ABI5 was elevated in dwa mutants ABI5 was degraded more slowly in dwa extracts

16. DWD Proteins Found T-DNA insertions 3 were sensitive to ABA ABI5 was elevated in dwa mutants ABI5 was degraded more slowly in dwa extracts DWA1 & DWA2 target ABI5 for degradation

17. Regulating E3 ligases The COP9 signalosome (CSN), a complex of 8 proteins, regulates E3 ligases by removing Nedd8 from cullin

18. Regulating E3 ligases The COP9 signalosome (CSN), a complex of 8 proteins, regulates E3 ligases by removing Nedd8 from cullin CAND1 then blocks cullin

19. Regulating E3 ligases The COP9 signalosome (CSN), a complex of 8 proteins, regulates E3 ligases by removing Nedd8 from cullin CAND1 then blocks cullin Ubc12 replaces Nedd8

20. Regulating E3 ligases The COP9 signalosome (CSN), a complex of 8 proteins, regulates E3 ligases by removing Nedd8 from cullin CAND1 then blocks cullin Ubc12 replaces Nedd8 Regulates DNA-damage response, cell-cycle & gene expression

21. Regulating E3 ligases The COP9 signalosome (CSN), a complex of 8 proteins, regulates E3 ligases by removing Nedd8 from cullin CAND1 then blocks cullin Ubc12 replaces Nedd8 Regulates DNA-damage response, cell-cycle & gene expression Not all E3 ligases associate with Cullins!

22. COP1 is a non-cullin-associated E3 ligase Protein degradation is important for light regulation COP1/SPA1 tags transcription factors for degradation W/O COP1 they act in dark In light COP1 is exported to cytoplasm so TF can act

23. COP1 is a non-cullin-associated E3 ligase Recent data indicates that COP1 may also associate with CUL4

24. Protein degradation rate varies 100x Most have motifs marking them for polyubiquitination : taken to proteosome & destroyed Other signals for selective degradation include PEST & KFERQ PEST : found in many rapidly degraded proteins e.g. ABCA1 (which exports cholesterol in association with apoA-I) is degraded by calpain

25. Protein degradation rate varies 100x Other signals for selective degradation include PEST & KFERQ PEST : found in many rapidly degraded proteins e.g. ABCA1 (which exports cholesterol in association with apoA-I) is degraded by calpain Deletion increases t 1/2 10x, adding PEST drops t 1/2 10x

26. Protein degradation rate varies 100x Other signals for selective degradation include PEST & KFERQ PEST : found in many rapidly degraded proteins e.g. ABCA1 (which exports cholesterol in association with apoA-I) is degraded by calpain Deletion increases t 1/2 10x, adding PEST drops t 1/2 10x Sometimes targets poly-Ub

27. Protein degradation rate varies 100x Other signals for selective degradation include PEST & KFERQ PEST : found in many rapidly degraded proteins e.g. ABCA1 (which exports cholesterol in association with apoA-I) is degraded by calpain Deletion increases t 1/2 10x, adding PEST drops t 1/2 10x Sometimes targets poly-Ub Recent yeast study doesn’t support general role

28. Protein degradation rate varies 100x Other signals for selective degradation include PEST & KFERQ PEST : found in many rapidly degraded proteins e.g. ABCA1 (which exports cholesterol in association with apoA-I) is degraded by calpain Deletion increases t 1/2 10x, adding PEST drops t 1/2 10x Sometimes targets poly-Ub Recent yeast study doesn’t support general role KFERQ: cytosolic proteins with KFERQ are selectively taken up by lysosomes in chaperone-mediated autophagy under conditions of nutritional or oxidative stress.

29. Protein degradation in bacteria Also highly regulated, involves chaperone-like proteins 1.Lon

30. Protein degradation in bacteria Also highly regulated, involves chaperone like proteins 1.Lon 2.Clp

31. Protein degradation in bacteria Also highly regulated, involves chaperone like proteins 1.Lon 2.Clp 3.FtsH in IM

32. PROTEIN TARGETING All proteins are made with an “address” which determines their final cellular location Addresses are motifs within proteins

33. PROTEIN TARGETING All proteins are made with “addresses” which determine their location Addresses are motifs within proteins Remain in cytoplasm unless contain information sending it elsewhere

34. PROTEIN TARGETING Targeting sequences are both necessary & sufficient to send reporter proteins to new compartments.

35. PROTEIN TARGETING 2 Pathways in E.coli http://www.membranetransport.org/ 1.Tat: for periplasmic redox proteins & thylakoid lumen!

36. 2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen! Preprotein has signal seqS/TRRXFLK

37. 2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen! Preprotein has signal seqS/TRRXFLK Make preprotein, folds & binds cofactor in cytosol

38. 2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen! Preprotein has signal seqS/TRRXFLK Make preprotein, folds & binds cofactor in cytosol Binds Tat in IM & is sent to periplasm

39. 2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen! Preprotein has signal seqS/TRRXFLK Make preprotein, folds & binds cofactor in cytosol Binds Tat in IM & is sent to periplasm Signal seq is removed in periplasm

40. 2 Pathways in E.coli http://www.membranetransport.org/ 1.Tat: for periplasmic redox proteins & thylakoid lumen! 2.Sec pathway SecB binds preprotein as it emerges from rib

41. Sec pathway SecB binds preprotein as it emerges from rib & prevents folding

42. Sec pathway SecB binds preprotein as it emerges from rib & prevents folding Guides it to SecA, which drives it through SecYEG into periplasm using ATP

43. Sec pathway SecB binds preprotein as it emerges from rib & prevents folding Guides it to SecA, which drives it through SecYEG into periplasm using ATP In periplasm signal peptide is removed and protein folds

44. Sec pathway part deux SRP binds preprotein as it emerges from rib & stops translation Guides rib to FtsY FtsY & SecA guide it to SecYEG, where it resumes translation & inserts protein into membrane as it is made

45. Periplasmic proteins with the correct signals (exposed after cleaving signal peptide) are exported by XcpQ system