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

2. ncRNA Structural 1.rRNA 2.tRNA 3.snRNA 4.snoRNA 5.cleavage: Rnases P & MRP, U3, snR30, etc Regulatory 1.Small siRNA miRNA 2.Long Activator Enhancer silencing

3. Regulatory ncRNA 1.SiRNA direct DNA-methylation via RNA-dependent DNA-methyltransferase 2. In other cases direct RNA degradation

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

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

6. 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’

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

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

9. 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’

10. 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

11. 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

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

13. 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

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

15. Other mRNA are targeted by small interfering RNA defense against RNA viruses DICERs cut dsRNA into 21-28 bp

16. Other mRNA are targeted by small interfering RNA defense against RNA viruses DICERs cut dsRNA into 21-28 bp helicase melts dsRNA

17. 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

18. 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

19. 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, then digested by XRN1 & exosome

20. Small RNA regulation siRNA: target RNA viruses (& transgenes) miRNA: arrest translation of targets created by digestion of foldback Pol II RNA with mismatch loop

21. Small RNA regulation siRNA: target RNA viruses (& transgenes) miRNA: arrest translation of targets created by digestion of foldback Pol II RNA with mismatch loop Mismatch is key difference: generated by different Dicer

22. Small RNA regulation siRNA: target RNA viruses (& transgenes) miRNA: arrest translation of targets created by digestion of foldback Pol II RNA with mismatch loop Mismatch is key difference: generated by different Dicer Arrest translation in animals, target degradation in plants

23. small interfering RNA mark specific targets once cut they are removed by endonuclease-mediated decay

24. Most RNA degradation occurs in P bodies recently identified cytoplasmic sites where exosomes & XRN1 accumulate when cells are stressed

25. Most RNA degradation occurs in P bodies recently identified cytoplasmic sites where exosomes & XRN1 accumulate when cells are stressed Also where AGO & miRNAs accumulate

26. Most RNA degradation occurs in P bodies recently identified cytoplasmic sites where exosomes & XRN1 accumulate when cells are stressed Also where AGO & miRNAs accumulate w/o miRNA P bodies dissolve!

28. Other ncRNA? Incredible diversity of functions! Epigenetic Directly regulating transcription Post-transcriptional regulation

29. Other ncRNA? Incredible diversity of functions! Epigenetic Directly regulating transcription Post-transcriptional regulation Some are made by Pol II, others by Pol III

30. Other ncRNA? Incredible diversity of functions!

31. Other ncRNA? Epigenetic regulation: guide &scaffold for chromatin mods XIST in mammals controls X-inactivation Transcribed by Pol II only from the inactive X 17 kb, -> Spliced, capped, poly-A Coats the inactive X Recruits PRC2, which silences the chromosome Ubiquitinates H2AK119 Tri-methylates H3K27 Hypermethylates H3K9

32. Other ncRNA? Epigenetic regulation: guide &scaffold for chromatin mods FLC blocks flowering in fall; after 20 days near 0˚C plants make COLDAIR ncRNA

33. FLC blocks flowering in fall; after 20 days near 0˚C plants make COLDAIR ncRNA: Targets PRC2 to FLC locus & silences it Flower next Spring Basic rule for epigentic ncRNA Bind target locus & recruit chromatin modifiers

34. ncRNA directly regulating transcription 1.Gene-specific Activating ncRNAs bind DNA & recruit Mediator

35. ncRNA directly regulating transcription 1.Gene-specific Activating ncRNAs bind DNA & recruit Mediator heat shock RNA1 (HSR1) mobilizes heat shock factor 1 (HSF1) upon heat shock

36. ncRNA directly regulating transcription 1.Gene-specific Activating ncRNAs bind DNA & recruit Mediator heat shock RNA1 (HSR1) mobilizes heat shock factor 1 (HSF1) upon heat shock The steroid receptor RNA activator 1 locus encodes both a protein and an ncRNA, depending on how it is spliced. The ncRNA acts as a coactivator for steroid hormone receptors

37. ncRNA directly regulating transcription 1.Gene-specific Activating ncRNAs bind DNA & recruit Mediator heat shock RNA1 (HSR1) mobilizes heat shock factor 1 (HSF1) upon heat shock The steroid receptor RNA activator 1 locus encodes both a protein and an ncRNA, depending on how it is spliced.The ncRNA acts as a coactivator for steroid hormone receptors. The protein acts as a repressor of the ncRNA!

38. ncRNA directly regulating transcription 1.Gene-specific Activating ncRNAs bind DNA & recruit Mediator heat shock RNA1 (HSR1) mobilizes heat shock factor 1 (HSF1) upon heat shock The steroid receptor RNA activator 1 locus encodes both a protein and an ncRNA, depending on how it is spliced.The ncRNA acts as a coactivator for steroid hormone receptors. The protein acts as a repressor of the ncRNA! ncRNA CCND1 are transcribed from the cyclin D locus during stress and remain tethered to DNA and recruit and allosterically modulate the protein TLS

39. ncRNA directly regulating transcription 1.Gene-specific Activating ncRNAs bind DNA & recruit Mediator heat shock RNA1 (HSR1) mobilizes heat shock factor 1 (HSF1) upon heat shock The steroid receptor RNA activator 1 locus encodes both a protein and an ncRNA, depending on how it is spliced.The ncRNA acts as a coactivator for steroid hormone receptors. The protein acts as a repressor of the ncRNA! ncRNA CCND1 are transcribed from the cyclin D locus during stress and remain tethered to DNA and recruit and allosterically modulate the protein TLS Other ncRNAs remain bound to the DNA coding sequence & prevent transcription

40. ncRNA directly regulating transcription 1.Gene-specific Other ncRNAs remain bound to the DNA coding sequence & prevent transcription Many sense and antisense ncRNA are found around promoters

41. ncRNA directly regulating transcription 1.Gene-specific Other ncRNAs remain bound to the DNA coding sequence & prevent transcription Many sense and antisense ncRNA are found around promoters Most are rapidly degraded: defective?

42. ncRNA directly regulating transcription 1.Gene-specific Other ncRNAs remain bound to the DNA coding sequence & prevent transcription Many sense and antisense ncRNA are found around promoters Most are rapidly degraded: defective? Loops may help identify correct direction

43. ncRNA directly regulating transcription 1.Gene-specific Many enhancer elements are transcribed by Pol II, this somehow enhances transcription from the core

44. ncRNA directly regulating transcription 1.Gene-specific Many enhancer elements are transcribed by Pol II, this somehow enhances transcription from the core

45. ncRNA directly regulating transcription 1.Gene-specific Many enhancer elements are transcribed by Pol II, this somehow enhances transcription from the core

46. ncRNA directly regulating transcription 1.Gene-specific Many enhancer elements are transcribed by Pol II, this somehow enhances transcription from the core Looping brings many related genes close, where [Pol II] is high

47. ncRNA directly regulating transcription 1.Gene-specific 2.General U1 snRNA associates with TFIIH to stimulate Pol II CTD phosphorylation hence transcription Couples splicing with transcription

48. ncRNA directly regulating transcription General U1 snRNA associates with TFIIH to stimulate Pol II CTD phosphorylation hence transcription Upon heat shock SINE (Alu) elements are transcribed by Pol III; Alu RNA binds Pol II and prevents initiation

49. ncRNA directly regulating transcription U1 snRNA associates with TFIIH to stimulate Pol II CTD phosphorylation hence transcription Upon heat shock SINE (Alu) elements are transcribed by Pol III; Alu RNA binds Pol II and prevents initiation P-TEFb (an elongation factor) reversibly associates with 7SK ncRNA to regulate Pol II elongation

50. ncRNA indirectly regulating transcription 1.Decoys For transcription factors: eg Gas5 RNA binds the DNA- binding domain of steroid hormone receptors NRON (noncoding repressor of NFAT) RNA prevents NFAT from shuttling into the nucleus

51. ncRNA regulating genes post-transcriptionally 1.Decoys for miRNAs (or siRNAs) Many mammalian pseudogenes, including PTENP1 and KRASP1 have miRNA binding sites

52. ncRNA regulating genes post-transcriptionally 1.Decoys for miRNAs (or siRNAs) Many mammalian pseudogenes, including PTENP1 and KRASP1 have miRNA binding sites 2.Thousands of genes are transcribed in both sense and antisense directions

53. Thousands of antisense transcripts in plants 1.Overlapping genes

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

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

56. Thousands of antisense transcripts in plants Hypotheses 1.Accident: transcription unveils “cryptic promoters” on opposite strand (Zilberman et al)

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

58. 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!

59. Detecting ncRNA 1. cDNA pairs

60. Detecting ncRNA 1.cDNA pairs 2.MPSS

61. Detecting ncRNA 1.cDNA pairs 2.MPSS 3.Tiling path microarrays

62. Detecting ncRNA 1.cDNA pairs 2.MPSS 3.Tiling path microarrays 4.RNA-seq (high-throughput RNA sequencing)

63. Our ncRNA 1.Extracted total RNA, separated out and sequenced 50- 300 nt

64. Our ncRNA 1.Extracted total RNA, separated out and sequenced 50- 300 nt 2.BLAST to find relatives (and discard tRNA, etc)

65. Our ncRNA 1.Extracted total RNA, separated out and sequenced 50- 300 nt 2.BLAST to find relatives (and discard tRNA, etc) 3.T-DNA express to find mutants

66. Our ncRNA 1.Extracted total RNA, separated out and sequenced 50- 300 nt 2.BLAST to find relatives (and discard tRNA, etc) 3.T-DNA express to find genes that weren’t near protein- coding genes

67. Our ncRNA 1.Extracted total RNA, separated out and sequenced 50- 300 nt 2.BLAST to find relatives (and discard tRNA, etc) 3.T-DNA express to find genes that weren’t near protein- coding genes 4.33 Hits: order seeds