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![Page 1: Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing.](https://reader036.fdocuments.us/reader036/viewer/2022062321/56649ee15503460f94bf266f/html5/thumbnails/1.jpg)
Using the genomeStudying expression of all genes simultaneously1.Microarrays: “reverse Northerns”2.High-throughput sequencing3. Bisulfite sequencing to detect C methylation
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Using the genomeBisulfite sequencing to detect C methylationChIP-chip or ChIP-seq to detect chromatin modifications: 17 mods are associated with active genes in CD-4 T cells
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Generating the histone codeHistone acetyltransferases add acetic acidDeacetylases “reset” by removing the acetate
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Generating the histone codeCDK8 kinases histones to repress transcriptionAppears to interact with mediator to block transcriptionPhosphorylation of Histone H3 correlates with activation of heat shock genes!Phosphatases reset the genes
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Generating the histone codeRad6 proteins ubiquitinate histone H2B to repress transcriptionPolycomb proteins ubiquitinate histone H2A to silence genes
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Generating the histone codeRad6 proteins ubiquitinate histone H2B to repress transcriptionPolycomb proteins ubiquitinate histone H2A to silence genesA TFTC/STAGA module mediates histone H2A and H2B deubiquitination, coactivates nuclear receptors, and counteracts heterochromatin silencing
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Generating the histone codeMany proteins methylate histones: highly regulated!
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Generating the histone codeMany proteins methylate histones: highly regulated!Methylation status determines gene activity
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Generating the histone codeMany proteins methylate histones: highly regulated!Methylation status determines gene activityMutants (eg Curly leaf) are unhappy!
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Generating the histone codeMany proteins methylate histones: highly regulated!Methylation status determines gene activityMutants (eg Curly leaf) are unhappy!Chromodomain protein HP1 can tell the difference between H3K9me2 (yellow)& H3K9me3 (red)
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Generating the histone codeChromodomain protein HP1 can tell the difference between H3K9me2 (yellow) & H3K9me3 (red)Histone demethylases have been recently discovered
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Generating methylated DNASi RNA are key: RNA Pol IV generates antisense or foldback RNA, often from TE
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Generating methylated DNASi RNA are key: RNA Pol IV generates antisense or foldback RNA, often from TERDR2 makes it DS, 24 nt siRNA are generated by DCL3
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Generating methylated DNARDR2 makes it DS, 24 nt siRNA are generated by DCL3AGO4 binds siRNA, complex binds target & Pol V
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Generating methylated DNARDR2 makes it DS, 24 nt siRNA are generated by DCL3AGO4 binds siRNA, complex binds target & Pol VPol V makes intergenic RNA, associates with AGO4-siRNA to recruit “silencing Complex” to target site
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Generating methylated DNARDR2 makes it DS, 24 nt siRNA are generated by DCL3AGO4 binds siRNA, complex binds target & Pol VPol V makes intergenic RNA, associates with AGO4-siRNA to recruit “silencing Complex” to target siteAmplifies signal!extends meth-ylated region
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Using the genomeMany 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
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Using the genomeMany 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/
geneexpressionatlas
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Using the genomeMany 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/
geneexpressionatlas• MPSS (massively-parallel signature sequencing)
http://mpss.udel.edu/
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Using the genomeMany 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/
geneexpressionatlas• MPSS (massively-parallel signature sequencing)
http://mpss.udel.edu/• Use it to decide which tissues to extract our RNA from
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Using the genomeMany sites provide gene expression data onlineMany sites provide other kinds of genomic data online• http://encodeproject.org/ENCODE/
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Post-transcriptional regulationNearly ½ of human genome is transcribed, only 1% is coding• 98% of RNA made is non-coding
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Post-transcriptional regulationNearly ½ of human genome is transcribed, only 1% is coding• 98% of RNA made is non-coding•Fraction increases with organism’s complexity
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Known NcRNAs classes and functions
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Implication in diseases
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Implication in diseases
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Transcription in Eukaryotes3 RNA polymerasesall are multi-subunit complexes 5 in common 3 very similar variable # unique onesPlants also have Pols IV & V •make siRNA
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Transcription in EukaryotesRNA polymerase I: 13 subunits (5 + 3 + 5 unique) acts exclusively in nucleolus to make 45S-rRNA precursor
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Transcription in EukaryotesPol I: acts exclusively in nucleolus to make 45S-rRNA precursor•accounts for 50% of total RNA synthesis
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Transcription in EukaryotesPol I: acts exclusively in nucleolus to make 45S-rRNA precursor• accounts for 50% of total RNA synthesis• insensitive to -aminitin
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Transcription in EukaryotesPol I: only makes 45S-rRNA precursor• 50 % of total RNA synthesis• insensitive to -aminitin•Mg2+ cofactor•Regulated @ initiation frequency
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Processing rRNA1) ~ 100 bases are methylated• C/D box snoRNA pick sites• One for each!
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Processing rRNA1) ~ 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!
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Processing rRNA1) ~ 100 bases are methylated• C/D box snoRNA pick sites2) ~ 100 Us are changed to PseudoU• H/ACA box snoRNA pick sites3) Some snoRNA direct modification
of tRNA and snRNA
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Processing rRNA1) ~ 200 bases are modified2) 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
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Processing rRNA1) ~ 200 bases are methylated2) 45S pre-rRNA is cut into 28S, 18S and 5.8S products3) Ribosomes are assembled w/in
nucleolus
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RNA Polymerase III makes ribosomal 5S and tRNA (+ some snRNA, scRNA, etc)>100 different kinds of ncRNA ~10% of all RNA synthesisCofactor = Mn2+ cf Mg2+
sensitive to high [-aminitin]
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Processing tRNA
1) tRNA is trimmed
• 5’ end by RNAse P
(1 RNA, 10 proteins)
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Processing tRNA
1) tRNA is trimmed
2) Transcript is spliced
• Some tRNAs are
assembled from 2 transcripts
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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 + PPitRNA-C-C +ATP -> tRNA-C-C-A + PPi
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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
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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)
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Splicing: the spliceosome cycle
1) U1 snRNP (RNA/protein complex) binds 5’ splice site
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Splicing:The spliceosome cycle1) U1 snRNP binds 5’ splice site2) U2 snRNP binds “branchpoint”
-> displaces A at branchpoint
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Splicing:The spliceosome cycle1) U1 snRNP binds 5’ splice site2) U2 snRNP binds “branchpoint”
-> displaces A at branchpoint3) U4/U5/U6 complex binds intron
displace U1spliceosome has now assembled
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Splicing:RNA is cut at 5’ splice sitecut end is trans-esterified to branchpoint A
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Splicing:5) RNA is cut at 3’ splice site6) 5’ end of exon 2 is ligated to 3’ end of exon 17) everything disassembles -> “lariat intron” is degraded
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Splicing:The spliceosome cycle
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Splicing:
Some RNAs can self-splice!
role of snRNPs is to increase rate!
Why splice?
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Splicing:
Why splice?
1) Generate diversity
exons often encode protein domains
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Splicing:Why splice?
1) Generate diversityexons often encode protein domainsIntrons = larger target for insertions, recombination
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Why splice?
1) Generate diversity
>94% of human genes show alternate splicing
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Why splice?
1) Generate diversity
>94% of human genes show alternate splicing
same gene encodes
different protein
in different tissues
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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
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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
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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
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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
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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
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Regulatory ncRNA1. SiRNA direct DNA-methylation via RNA-dependent
DNA-methyltansferase2. In other cases direct RNA degradation
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mRNA degradation• lifespan varies 100x• Sometimes due to AU-rich 3' UTR sequences • Defective mRNA may be targetedby NMD, NSD, NGD
Other RNA are targeted by small interfering RNA
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Other mRNA are targeted by small interfering RNA• defense against RNA viruses• DICERs cut dsRNA into 21-28 bp
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Other mRNA are targeted by small interfering RNA• defense against RNA viruses• DICERs cut dsRNA into 21-28 bp• helicase melts dsRNA
![Page 63: Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing.](https://reader036.fdocuments.us/reader036/viewer/2022062321/56649ee15503460f94bf266f/html5/thumbnails/63.jpg)
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
![Page 64: Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing.](https://reader036.fdocuments.us/reader036/viewer/2022062321/56649ee15503460f94bf266f/html5/thumbnails/64.jpg)
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
![Page 65: Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing.](https://reader036.fdocuments.us/reader036/viewer/2022062321/56649ee15503460f94bf266f/html5/thumbnails/65.jpg)
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
![Page 66: Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing.](https://reader036.fdocuments.us/reader036/viewer/2022062321/56649ee15503460f94bf266f/html5/thumbnails/66.jpg)
Small RNA regulation
• siRNA: target RNA viruses (& transgenes)
•miRNA: arrest translation of targets
• created by digestion of foldback
Pol II RNA with mismatch loop
![Page 67: Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing.](https://reader036.fdocuments.us/reader036/viewer/2022062321/56649ee15503460f94bf266f/html5/thumbnails/67.jpg)
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
![Page 68: Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing.](https://reader036.fdocuments.us/reader036/viewer/2022062321/56649ee15503460f94bf266f/html5/thumbnails/68.jpg)
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
![Page 69: Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing.](https://reader036.fdocuments.us/reader036/viewer/2022062321/56649ee15503460f94bf266f/html5/thumbnails/69.jpg)
small interfering RNA mark specifictargets•once cut they are removed by endonuclease-mediated decay
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Most RNA degradation occurs in P bodies• recently identified cytoplasmic sites where exosomes & XRN1 accumulate when cells are stressed
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Most RNA degradation occurs in P bodies• recently identified cytoplasmic sites where exosomes & XRN1 accumulate when cells are stressed •Also where AGO & miRNAs accumulate
![Page 73: Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing.](https://reader036.fdocuments.us/reader036/viewer/2022062321/56649ee15503460f94bf266f/html5/thumbnails/73.jpg)
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!
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Thousands of antisense transcripts in plants1. Overlapping genes
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Thousands of antisense transcripts in plants1. Overlapping genes2. Non-coding RNAs
![Page 76: Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing.](https://reader036.fdocuments.us/reader036/viewer/2022062321/56649ee15503460f94bf266f/html5/thumbnails/76.jpg)
Thousands of antisense transcripts in plants1. Overlapping genes2. Non-coding RNAs3. cDNA pairs
![Page 77: Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing.](https://reader036.fdocuments.us/reader036/viewer/2022062321/56649ee15503460f94bf266f/html5/thumbnails/77.jpg)
Thousands of antisense transcripts in plants1. Overlapping genes2. Non-coding RNAs3. cDNA pairs4. MPSS
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Thousands of antisense transcripts in plants1. Overlapping genes2. Non-coding RNAs3. cDNA pairs4. MPSS5. TARs
![Page 79: Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing.](https://reader036.fdocuments.us/reader036/viewer/2022062321/56649ee15503460f94bf266f/html5/thumbnails/79.jpg)
Thousands of antisense transcripts in plants
Hypotheses
1. Accident: transcription unveils “cryptic promoters” on opposite strand (Zilberman et al)
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Hypotheses
1. Accident: transcription unveils “cryptic promoters” on opposite strand (Zilberman et al)
2. Functional
a. siRNA
b. miRNA
c. Silencing
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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!