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 5)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 1.Overlapping genes

19. Thousands of antisense transcripts in plants 1.Overlapping genes 2.Non-coding RNAs

20. Thousands of antisense transcripts in plants 1.Overlapping genes 2.Non-coding RNAs 3.cDNA pairs

21. Thousands of antisense transcripts in plants 1.Overlapping genes 2.Non-coding RNAs 3.cDNA pairs 4.MPSS

22. Thousands of antisense transcripts in plants 1.Overlapping genes 2.Non-coding RNAs 3.cDNA pairs 4.MPSS 5.TARs

23. Thousands of antisense transcripts in plants Hypotheses 1.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 a.siRNA b.miRNA c.Silencing

25. Hypotheses 1. Accident: transcription unveils “cryptic promoters” on opposite strand (Zilberman et al) 2. Functional a.siRNA b.miRNA c.Silencing d.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 1.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 1.Nonsense-mediated decay:EJC @ each splice junction that is displaced by ribosome 2.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