1. Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing to detect C methylation

2. Using the genome Bisulfite sequencing to detect C methylation ChIP-chip or ChIP-seq to detect chromatin modifications: 17 mods are associated with active genes in CD-4 T cells

3. Generating the histone code Histone acetyltransferases add acetic acid Deacetylases “reset” by removing the acetate

4. Generating the histone code CDK8 kinases histones to repress transcription Appears to interact with mediator to block transcription Phosphorylation of Histone H3 correlates with activation of heat shock genes! Phosphatases reset the genes

5. Generating the histone code Rad6 proteins ubiquitinate histone H2B to repress transcription Polycomb proteins ubiquitinate histone H2A to silence genes

6. Generating the histone code Rad6 proteins ubiquitinate histone H2B to repress transcription Polycomb proteins ubiquitinate histone H2A to silence genes A TFTC/STAGA module mediates histone H2A and H2B deubiquitination, coactivates nuclear receptors, and counteracts heterochromatin silencing

7. Generating the histone code Many proteins methylate histones: highly regulated!

8. Generating the histone code Many proteins methylate histones: highly regulated! Methylation status determines gene activity

9. Generating the histone code Many proteins methylate histones: highly regulated! Methylation status determines gene activity Mutants (eg Curly leaf) are unhappy!

10. Generating the histone code Many proteins methylate histones: highly regulated! Methylation status determines gene activity Mutants (eg Curly leaf) are unhappy! Chromodomain protein HP1 can tell the difference between H3K9me2 (yellow) & H3K9me3 (red)

11. Generating the histone code Chromodomain protein HP1 can tell the difference between H3K9me2 (yellow) & H3K9me3 (red) Histone demethylases have been recently discovered

12. Generating methylated DNA Si RNA are key: RNA Pol IV generates antisense or foldback RNA, often from TE

13. Generating methylated DNA Si RNA are key: RNA Pol IV generates antisense or foldback RNA, often from TE RDR2 makes it DS, 24 nt siRNA are generated by DCL3

14. Generating methylated DNA RDR2 makes it DS, 24 nt siRNA are generated by DCL3 AGO4 binds siRNA, complex binds target & Pol V

15. Generating methylated DNA RDR2 makes it DS, 24 nt siRNA are generated by DCL3 AGO4 binds siRNA, complex binds target & Pol V Pol V makes intergenic RNA, associates with AGO4- siRNA to recruit “silencing Complex” to target site

16. Generating methylated DNA RDR2 makes it DS, 24 nt siRNA are generated by DCL3 AGO4 binds siRNA, complex binds target & Pol V Pol V makes intergenic RNA, associates with AGO4-siRNA to recruit “silencing Complex” to target site Amplifies signal! extends meth- ylated region

17. Using the genome Many sites provide gene expression data online NIH Gene expression omnibus http://www.ncbi.nlm.nih.gov/geo/ provides access to many different types of gene expression data http://www.ncbi.nlm.nih.gov/geo/

18. Using the genome Many sites provide gene expression data online NIH Gene expression omnibus http://www.ncbi.nlm.nih.gov/geo/ provides access to many different types of gene expression data http://www.ncbi.nlm.nih.gov/geo/ Many different sites provide “digital Northerns” or other comparative analyses of gene expression http://cgap.nci.nih.gov/SAGE http://www.weigelworld.org/research/projects/geneexpr essionatlas http://www.weigelworld.org/research/projects/geneexpr essionatlas

19. Using the genome Many sites provide gene expression data online NIH Gene expression omnibus http://www.ncbi.nlm.nih.gov/geo/ provides access to many different types of gene expression data http://www.ncbi.nlm.nih.gov/geo/ Many different sites provide “digital Northerns” or other comparative analyses of gene expression http://cgap.nci.nih.gov/SAGE http://www.weigelworld.org/research/projects/geneexpr essionatlas http://www.weigelworld.org/research/projects/geneexpr essionatlas MPSS (massively-parallel signature sequencing) http://mpss.udel.edu/

20. Using the genome Many sites provide gene expression data online NIH Gene expression omnibus http://www.ncbi.nlm.nih.gov/geo/ provides access to many different types of gene expression data http://www.ncbi.nlm.nih.gov/geo/ Many different sites provide “digital Northerns” or other comparative analyses of gene expression http://cgap.nci.nih.gov/SAGE http://www.weigelworld.org/research/projects/geneexpr essionatlas http://www.weigelworld.org/research/projects/geneexpr essionatlas MPSS (massively-parallel signature sequencing) http://mpss.udel.edu/ http://mpss.udel.edu/ Use it to decide which tissues to extract our RNA from

21. Using the genome Many sites provide gene expression data online Many sites provide other kinds of genomic data online http://encodeproject.org/ENCODE/

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

23. Post-transcriptional regulation Nearly ½ of human genome is transcribed, only 1% is coding 98% of RNA made is non-coding Fraction increases with organism’s complexity

24. Known NcRNAs classes and functions

25. Implication in diseases

27. Transcription in Eukaryotes 3 RNA polymerases all are multi-subunit complexes 5 in common 3 very similar variable # unique ones Plants also have Pols IV & V make siRNA

28. Transcription in Eukaryotes RNA polymerase I: 13 subunits (5 + 3 + 5 unique) acts exclusively in nucleolus to make 45S-rRNA precursor

29. Transcription in Eukaryotes Pol I: acts exclusively in nucleolus to make 45S-rRNA precursor accounts for 50% of total RNA synthesis

30. Transcription in Eukaryotes Pol I: acts exclusively in nucleolus to make 45S-rRNA precursor accounts for 50% of total RNA synthesis insensitive to  -aminitin

31. Transcription in Eukaryotes Pol I: only makes 45S-rRNA precursor 50 % of total RNA synthesis insensitive to  -aminitin Mg 2+ cofactor Regulated @ initiation frequency

32. Processing rRNA 1)~ 100 bases are methylated C/D box snoRNA pick sites One for each!

33. Processing rRNA 1)~ 100 bases are methylated C/D box snoRNA pick sites One for each! 2)~ 100 Us are changed to PseudoU H/ACA box snoRNA pick sites One for each!

34. Processing rRNA 1)~ 100 bases are methylated C/D box snoRNA pick sites 2)~ 100 Us are changed to PseudoU H/ACA box snoRNA pick sites 3) Some snoRNA direct modification of tRNA and snRNA

35. Processing rRNA 1)~ 200 bases are modified 2) 45S pre-rRNA is cut into 28S, 18S and 5.8S products by ribozymes RNase MRP cuts between 18S & 5.8S U3, U8, U14, U22, snR10 and snR30 also guide cleavage

36. Processing rRNA 1)~ 200 bases are methylated 2) 45S pre-rRNA is cut into 28S, 18S and 5.8S products 3) Ribosomes are assembled w/in nucleolus

37. RNA Polymerase III makes ribosomal 5S and tRNA (+ some snRNA, scRNA, etc) >100 different kinds of ncRNA ~10% of all RNA synthesis Cofactor = Mn 2+ cf Mg 2+ sensitive to high [  -aminitin]

38. Processing tRNA 1)tRNA is trimmed 5’ end by RNAse P (1 RNA, 10 proteins)

39. Processing tRNA 1)tRNA is trimmed 2)Transcript is spliced Some tRNAs are assembled from 2 transcripts

40. Processing tRNA 1)tRNA is trimmed 2)Transcript is spliced 3)CCA is added to 3’ end By tRNA nucleotidyl transferase (no template) tRNA +CTP -> tRNA-C + PPi tRNA-C +CTP--> tRNA-C-C + PPi tRNA-C-C +ATP -> tRNA-C-C-A + PPi

41. Processing tRNA 1)tRNA is trimmed 2)Transcript is spliced 3)CCA is added to 3’ end 4)Many bases are modified Protects tRNA Tweaks protein synthesis

42. Processing tRNA 1)tRNA is trimmed 2)Transcript is spliced 3)CCA is added to 3’ end 4)Many bases are modified 5)No cap! -> 5’ P (due to 5’ RNAse P cut)

43. Splicing: the spliceosome cycle 1) U1 snRNP (RNA/protein complex) binds 5’ splice site

44. Splicing:The spliceosome cycle 1) U1 snRNP binds 5’ splice site 2) U2 snRNP binds “branchpoint” -> displaces A at branchpoint

45. Splicing:The spliceosome cycle 1) U1 snRNP binds 5’ splice site 2) U2 snRNP binds “branchpoint” -> displaces A at branchpoint 3) U4/U5/U6 complex binds intron displace U1 spliceosome has now assembled

46. Splicing: RNA is cut at 5’ splice site cut end is trans-esterified to branchpoint A

47. Splicing: 5) RNA is cut at 3’ splice site 6) 5’ end of exon 2 is ligated to 3’ end of exon 1 7) everything disassembles -> “lariat intron” is degraded

48. Splicing:The spliceosome cycle

49. Splicing: Some RNAs can self-splice! role of snRNPs is to increase rate! Why splice?

50. Splicing: Why splice? 1) Generate diversity exons often encode protein domains

51. Splicing: Why splice? 1) Generate diversity exons often encode protein domains Introns = larger target for insertions, recombination

52. Why splice? 1) Generate diversity >94% of human genes show alternate splicing

53. Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues

54. Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues Stressed plants use AS to make variant stress-response proteins

55. Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues Stressed plants use AS to make variant Stress-response proteins Splice-regulator proteins control AS: regulated by cell-specific expression and phosphorylation

56. Why splice? 1)Generate diversity Trabzuni D, et al (2013)Nat Commun. 22:2771. Found 448 genes that were expressed differently by gender in human brains (2.6% of all genes expressed in the CNS). All major brain regions showed some gender variation, and 85% of these variations were due to RNA splicing differences

57. Why splice? 1)Generate diversity Wilson LOW, Spriggs A, Taylor JM, Fahrer AM. (2014). A novel splicing outcome reveals more than 2000 new mammalian protein isoforms. Bioinformatics 30: 151-156 Splicing created a frameshift, so was annotated as “nonsense-mediated decay” an alternate start codon rescued the protein, which was expressed

58. Why splice? Splicing created a frameshift, so was annotated as “nonsense-mediated decay” an alternate start codon rescued the protein, which was expressed Found 1849 human & 733 mouse mRNA that could encode alternate protein isoforms the same way So far 64 have been validated by mass spec

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

60. 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

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

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

63. 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

64. 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

65. 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

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

67. 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

68. 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

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

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

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

73. 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!

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

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

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

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

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

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

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

81. 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!