Regulating gene expression Goal is controlling Proteins How many? Where? How active?

1. Regulating gene expression • Goal is • controlling • Proteins • How many? • Where? • How active? • 8 levels (two not • shown are mRNA • localization & prot • degradation)

2. mRNA PROCESSING • Primary transcript is hnRNA • Is capped, spliced and poly-adenylated before export to cytosol • Many are also edited • All three are coordinated with transcription & affect gene expression: enzymes piggy-back on POLII

3. mRNA Processing: Polyadenylation 1) CPSF (Cleavage and Polyadenylation Specificity Factor) binds AAUAAA in hnRNA

4. mRNA Processing: Polyadenylation 1) CPSF binds AAUAAA in hnRNA 2) CStF (Cleavage Stimulatory Factor) binds G/U rich sequence 50 bases downstream CFI, CFII bind in between

5. Polyadenylation 1) CPSF binds AAUAAA in hnRNA 2) CStF binds; CFI, CFII bind in between 3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to AAUAAA

6. mRNA Processing: Polyadenylation 3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to AAUAAA 4) PAP adds As slowly, CFI, CFII and CPSF fall off

7. mRNA Processing: Polyadenylation 4) PAP adds As slowly, CFI, CFII and CPSF fall off PABII binds, add As rapidly until 250

8. Coordination of mRNA processing • Splicing and polyadenylation factors bind CTD of RNA Pol II-> mechanism to coordinate the three processes • Capping, Splicing and Polyadenylation all start before transcription is done!

9. Export from Nucleus Occurs through nuclear pores anything > 40 kDa needs exportin protein bound to 5’ cap

10. Export from Nucleus • In cytoplasm nuclear proteins fall off, new proteins bind • eIF4E/eIF-4F bind cap • also new • proteins bind • polyA tail • mRNA is • ready to be • translated!

11. Cytoplasmic regulation • lifetime • localization • initiation

12. Post-transcriptional regulation • Nearly ½ of human genome is transcribed, only 1% is CDS • 98% of RNA made is non-coding

13. Post-transcriptional regulation • Nearly ½ of human genome is transcribed, only 1% is CDS • 98% of RNA made is non-coding • ~1/3 intron

14. Post-transcriptional regulation • Nearly ½ of human genome is transcribed, only 1% is CDS • 98% of RNA made is non-coding • ~1/3 intron • ~2/3 “independently transcribed”

15. Post-transcriptional regulation • Nearly ½ of human genome is transcribed, only 1% is CDS • 98% of RNA made is non-coding • ~1/3 intron • ~2/3 “independently transcribed” • Polymerases II & III (+ IV & V in plants) all help

16. Post-transcriptional regulation • Nearly ½ of human genome is transcribed, only 1% is CDS • 98% of RNA made is non-coding • ~1/3 intron • ~2/3 “independently transcribed” • Polymerases II & III (+ IV & V in plants) all help • many are from transposons or gene fragments made by transposons (pack-MULES)

17. Post-transcriptional regulation • Nearly ½ of human genome is transcribed, only 1% is CDS • 98% of RNA made is non-coding • ~1/3 intron • ~2/3 “independently transcribed” • Polymerases II & III (+ IV & V in plants) all help • many are from transposons or gene fragments made by transposons (pack-MULES) • ~ 10-25% is anti-sense: same region is transcribed off both strands

18. Thousands of antisense transcripts in plants • Overlapping genes

19. Thousands of antisense transcripts in plants • Overlapping genes • Non-coding RNAs

20. Thousands of antisense transcripts in plants • Overlapping genes • Non-coding RNAs • cDNA pairs

21. Thousands of antisense transcripts in plants • Overlapping genes • Non-coding RNAs • cDNA pairs • MPSS

22. Thousands of antisense transcripts in plants • Overlapping genes • Non-coding RNAs • cDNA pairs • MPSS • TARs

23. Thousands of antisense transcripts in plants • Hypotheses • Accident: transcription unveils “cryptic promoters” on opposite strand (Zilberman et al)

24. Hypotheses • 1. Accident: transcription unveils “cryptic promoters” on opposite strand (Zilberman et al) • 2. Functional • siRNA • miRNA • Silencing

25. Hypotheses • 1. Accident: transcription unveils “cryptic promoters” on opposite strand (Zilberman et al) • 2. Functional • siRNA • miRNA • Silencing • Priming: chromatin remodeling requires transcription!

26. Post-transcriptional regulation RNA degradation is crucial with so much “extra” RNA

27. Post-transcriptional regulation • RNA degradation is crucial with so much “extra” RNA • mRNA lifespan varies 100x • Highly regulated! > 30 RNAses in Arabidopsis!

28. Post-transcriptional regulation • mRNA degradation • lifespan varies 100x • Sometimes due to AU-rich 3' UTR sequences (DST)

29. mRNA degradation • lifespan varies 100x • Sometimes due to AU-rich 3' UTR sequences (DST) • Endonuclease cuts DST, then exosome digests 3’->5’ & XRN1 digests 5’->3’

30. mRNA degradation • Most are degraded by de-Adenylation pathway • Deadenylase removes tail

31. mRNA degradation • Most are degraded by de-Adenylation pathway • Deadenylase removes tail • Exosome digests 3’ -> 5’

32. mRNA degradation • Most are degraded by de-Adenylation pathway • Deadenylase removes tail • Exosome digests 3’ -> 5’ • Or, decapping enz • removes cap & XRN1 • digests 5’ ->3’

33. Post-transcriptional regulation mRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillance Nonsense-mediated decay:EJC @ each splice junction that is displaced by ribosome

34. Post-transcriptional regulation • mRNA degradation: mRNA is checked & • defective transcripts are degraded • = mRNA surveillance • Nonsense-mediated decay:EJC @ • each splice junction that is displaced by • ribosome • If not-displaced, is cut by • endonuclease & RNA is degraded

35. Post-transcriptional regulation mRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillance Non-stop decay: Ribosome goes to end & cleans off PABP

36. Post-transcriptional regulation mRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillance Non-stop decay: Ribosome goes to end & cleans off PABP w/o PABP exosome eats mRNA

37. Post-transcriptional regulation mRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillance No-go decay: cut RNA 3’ of stalled ribosomes

38. Post-transcriptional regulation • mRNA degradation • lifespan varies 100x • Sometimes due to AU-rich 3' • UTR sequences • Defective mRNA may be targeted • by NMD, NSD, NGD • Other RNA are targeted by • small interfering RNA

39. Post-transcriptional regulation • Other mRNA are targeted by • small interfering RNA • defense against RNA viruses • DICERs cut dsRNA into 21-28 bp

40. Post-transcriptional regulation • Other mRNA are targeted by • small interfering RNA • defense against RNA viruses • DICERs cut dsRNA into 21-28 bp • helicase melts dsRNA

41. Post-transcriptional regulation • Other mRNA are targeted by • small interfering RNA • defense against RNA viruses • DICERs cut dsRNA into 21-28 bp • helicase melts dsRNA • - RNA binds RISC

42. Post-transcriptional regulation • Other mRNA are targeted by • small interfering RNA • defense against RNA viruses • DICERs cut dsRNA into 21-28 bp • helicase melts dsRNA • - RNA binds RISC • complex binds target

43. Post-transcriptional regulation • Other mRNA are targeted by • small interfering RNA • defense against RNA viruses • DICERs cut dsRNA into 21-28 bp • helicase melts dsRNA • - RNA binds RISC • complex binds target • target is cut