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Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing to detect C methylation 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 Generating the histone code Histone acetyltransferases add acetic acid Deacetylases “reset” by removing the acetate 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 Generating the histone code Rad6 proteins ubiquitinate histone H2B to repress transcription Polycomb proteins ubiquitinate histone H2A to silence genes 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 Generating the histone code Many proteins methylate histones: highly regulated! Generating the histone code Many proteins methylate histones: highly regulated! Methylation status determines gene activity Generating the histone code Many proteins methylate histones: highly regulated! Methylation status determines gene activity Mutants (eg Curly leaf) are unhappy! 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) Generating the histone code Chromodomain protein HP1 can tell the difference between H3K9me2 (yellow) & H3K9me3 (red) Histone demethylases have been recently discovered Generating methylated DNA Si RNA are key: RNA Pol IV generates antisense or foldback RNA, often from TE 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 Generating methylated DNA RDR2 makes it DS, 24 nt siRNA are generated by DCL3 AGO4 binds siRNA, complex binds target & Pol V 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 AGO4siRNA to recruit “silencing Complex” to target site 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 methylated region 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 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 •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 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 •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 • MPSS (massively-parallel signature sequencing) http://mpss.udel.edu/ 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 •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 • MPSS (massively-parallel signature sequencing) http://mpss.udel.edu/ • Use it to decide which tissues to extract our RNA from Using the genome Many sites provide gene expression data online Many sites provide other kinds of genomic data online • http://encodeproject.org/ENCODE/ Post-transcriptional regulation Nearly ½ of human genome is transcribed, only 1% is coding • 98% of RNA made is non-coding 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 Known NcRNAs classes and functions Implication in diseases Implication in diseases 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 Transcription in Eukaryotes RNA polymerase I: 13 subunits (5 + 3 + 5 unique) acts exclusively in nucleolus to make 45S-rRNA precursor Transcription in Eukaryotes Pol I: acts exclusively in nucleolus to make 45S-rRNA precursor •accounts for 50% of total RNA synthesis Transcription in Eukaryotes Pol I: acts exclusively in nucleolus to make 45S-rRNA precursor • accounts for 50% of total RNA synthesis • insensitive to -aminitin Transcription in Eukaryotes Pol I: only makes 45S-rRNA precursor • 50 % of total RNA synthesis • insensitive to -aminitin •Mg2+ cofactor •Regulated @ initiation frequency Processing rRNA 1) ~ 100 bases are methylated • C/D box snoRNA pick sites • One for each! 1) • • 2) • • Processing rRNA ~ 100 bases are methylated C/D box snoRNA pick sites One for each! ~ 100 Us are changed to PseudoU H/ACA box snoRNA pick sites One for each! 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 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 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 RNA Polymerase III makes ribosomal 5S and tRNA (+ some snRNA, scRNA, etc) >100 different kinds of ncRNA ~10% of all RNA synthesis Cofactor = Mn2+ cf Mg2+ sensitive to high [-aminitin] Processing tRNA 1) tRNA is trimmed • 5’ end by RNAse P (1 RNA, 10 proteins) Processing tRNA 1) tRNA is trimmed 2) Transcript is spliced • Some tRNAs are assembled from 2 transcripts 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 Processing tRNA 1) 2) 3) 4) • • tRNA is trimmed Transcript is spliced CCA is added to 3’ end Many bases are modified Protects tRNA Tweaks protein synthesis 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) Splicing: the spliceosome cycle 1) U1 snRNP (RNA/protein complex) binds 5’ splice site Splicing:The spliceosome cycle 1) U1 snRNP binds 5’ splice site 2) U2 snRNP binds “branchpoint” -> displaces A at branchpoint 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 Splicing: RNA is cut at 5’ splice site cut end is trans-esterified to branchpoint A 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 Splicing:The spliceosome cycle Splicing: Some RNAs can self-splice! role of snRNPs is to increase rate! Why splice? Splicing: Why splice? 1) Generate diversity exons often encode protein domains Splicing: Why splice? 1) Generate diversity exons often encode protein domains Introns = larger target for insertions, recombination Why splice? 1) Generate diversity >94% of human genes show alternate splicing Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues 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 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 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 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 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 Regulatory ncRNA 1. SiRNA direct DNA-methylation via RNA-dependent DNA-methyltansferase 2. In other cases direct RNA degradation 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 Other mRNA are targeted by small interfering RNA • defense against RNA viruses • DICERs cut dsRNA into 21-28 bp Other mRNA are targeted by small interfering RNA • defense against RNA viruses • DICERs cut dsRNA into 21-28 bp • helicase melts dsRNA 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 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 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 Small RNA regulation • siRNA: target RNA viruses (& transgenes) •miRNA: arrest translation of targets • created by digestion of foldback Pol II RNA with mismatch loop 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 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 small interfering RNA mark specific targets •once cut they are removed by endonuclease-mediated decay Most RNA degradation occurs in P bodies • recently identified cytoplasmic sites where exosomes & XRN1 accumulate when cells are stressed Most RNA degradation occurs in P bodies • recently identified cytoplasmic sites where exosomes & XRN1 accumulate when cells are stressed •Also where AGO & miRNAs accumulate 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! Thousands of antisense transcripts in plants 1. Overlapping genes Thousands of antisense transcripts in plants 1. Overlapping genes 2. Non-coding RNAs Thousands of antisense transcripts in plants 1. Overlapping genes 2. Non-coding RNAs 3. cDNA pairs Thousands of antisense transcripts in plants 1. Overlapping genes 2. Non-coding RNAs 3. cDNA pairs 4. MPSS Thousands of antisense transcripts in plants 1. Overlapping genes 2. Non-coding RNAs 3. cDNA pairs 4. MPSS 5. TARs Thousands of antisense transcripts in plants Hypotheses 1. Accident: transcription unveils “cryptic promoters” on opposite strand (Zilberman et al) Hypotheses 1. Accident: transcription unveils “cryptic promoters” on opposite strand (Zilberman et al) 2. Functional a. siRNA b. miRNA c. Silencing 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!
