Using the genomeStudying expression of all genes simultaneously1.Microarrays: “reverse Northerns”2.High-throughput sequencing3. Bisulfite sequencing to detect C methylation

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

Generating the histone codeHistone acetyltransferases add acetic acidDeacetylases “reset” by removing the acetate

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

Generating the histone codeRad6 proteins ubiquitinate histone H2B to repress transcriptionPolycomb proteins ubiquitinate histone H2A to silence genes

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

Generating the histone codeMany proteins methylate histones: highly regulated!

Generating the histone codeMany proteins methylate histones: highly regulated!Methylation status determines gene activity

Generating the histone codeMany proteins methylate histones: highly regulated!Methylation status determines gene activityMutants (eg Curly leaf) are unhappy!

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)

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

Generating methylated DNASi RNA are key: RNA Pol IV generates antisense or foldback RNA, often from TE

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

Generating methylated DNARDR2 makes it DS, 24 nt siRNA are generated by DCL3AGO4 binds siRNA, complex binds target & Pol V

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

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

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

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

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/

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

Using the genomeMany sites provide gene expression data onlineMany sites provide other kinds of genomic data online• http://encodeproject.org/ENCODE/

Post-transcriptional regulationNearly ½ of human genome is transcribed, only 1% is coding• 98% of RNA made is non-coding

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

Known NcRNAs classes and functions

Implication in diseases

Implication in diseases

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

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

Transcription in EukaryotesPol I: acts exclusively in nucleolus to make 45S-rRNA precursor•accounts for 50% of total RNA synthesis

Transcription in EukaryotesPol I: acts exclusively in nucleolus to make 45S-rRNA precursor• accounts for 50% of total RNA synthesis• insensitive to -aminitin

Transcription in EukaryotesPol I: only makes 45S-rRNA precursor• 50 % of total RNA synthesis• insensitive to -aminitin•Mg2+ cofactor•Regulated @ initiation frequency

Processing rRNA1) ~ 100 bases are methylated• C/D box snoRNA pick sites• One for each!

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!

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

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

Processing rRNA1) ~ 200 bases are methylated2) 45S pre-rRNA is cut into 28S, 18S and 5.8S products3) 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 synthesisCofactor = 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 + PPitRNA-C-C +ATP -> tRNA-C-C-A + PPi

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

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 cycle1) U1 snRNP binds 5’ splice site2) U2 snRNP binds “branchpoint”

-> displaces A at branchpoint

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

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

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

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 diversityexons often encode protein domainsIntrons = 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 ncRNA1. SiRNA direct DNA-methylation via RNA-dependent

DNA-methyltansferase2. In other cases direct RNA degradation

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

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 specifictargets•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 plants1. Overlapping genes

Thousands of antisense transcripts in plants1. Overlapping genes2. Non-coding RNAs

Thousands of antisense transcripts in plants1. Overlapping genes2. Non-coding RNAs3. cDNA pairs

Thousands of antisense transcripts in plants1. Overlapping genes2. Non-coding RNAs3. cDNA pairs4. MPSS

Thousands of antisense transcripts in plants1. Overlapping genes2. Non-coding RNAs3. cDNA pairs4. MPSS5. 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!