Regulating gene expression Goal is controlling Proteins How many? Where? How active? 8 levels (two not shown are mRNA localization & prot degradation)

Activated transcription by Pol II enhancers are sequences 5’ to TATAA transcriptional activators bind them have distinct DNA binding and activation domains activation domain interacts with mediator helps assemble initiation complex on TATAA Recently identified “activating RNA”: bind enhancers & mediator

Activated transcription by Pol II Other lncRNA “promote transcriptional poising” in yeast 71%2Fjournal.pbio lncRNA displaces glucose-responsive repressors & co- repressors from genes for galactose catabolism Speeds induction of GAL genes

Euk gene regulation Initiating transcription is 1 st & most important control Most genes are condensed only express needed genes not enough room in nucleus to access all genes at same time! must find & decompress gene

First “remodel” chromatin: some proteins reposition nucleosomes others acetylate histones Neutralizes +ve charge makes them release DNA

Epigenetics heritable chromatin modifications are associated with activated & repressed genes

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 AGO4- siRNA 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 meth- ylated region

mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol All three are coordinated with transcription & affect gene expression: enzymes piggy-back on POLII

mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol 1) Capping addition of 7-methyl G to 5’ end

mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol 1) Capping addition of 7-methyl G to 5’ end identifies it as mRNA: needed for export & translation

mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol 1) Capping addition of 7-methyl G to 5’ end identifies it as mRNA: needed for export & translation Catalyzed by CEC attached to POLII

mRNA PROCESSING 1) Capping 2) Splicing: removal of introns Evidence: electron microscopy sequence alignment

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 448 genes were expressed differently by gender (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 Trabzuni D, Ramasamy A, Imran S, Walker R, Smith C, Weale ME, Hardy J, Ryten M; North American Brain Expression Consortium. Widespread sex differences in gene expression and splicing in the adult human brain. Nat Commun Nov 22;4:2771.

Splicing: Why splice? 1) Generate diversity 2) Modulate gene expression introns affect amount of mRNA produced

mRNA Processing: RNA editing Two types: C->U and A->I

mRNA Processing: RNA editing Two types: C->U and A->I Plant mito and cp use C -> U >300 different editing events have been detected in plant mitochondria: some create start & stop codons

mRNA Processing: RNA editing Two types: C->U and A->I Plant mito and cp use C -> U >300 different editing events have been detected in plant mitochondria: some create start & stop codons: way to prevent nucleus from stealing genes!

mRNA Processing: RNA editing Human intestines edit APOB mRNA C -> U to create a stop aa 2153 (APOB48) cf full-length APOB100 APOB48 lacks the CTD LDL receptor binding site

mRNA Processing: RNA editing Human intestines edit APOB mRNA C -> U to create a stop aa 2153 (APOB48) cf full-length APOB100 APOB48 lacks the CTD LDL receptor binding site Liver makes APOB100 -> correlates with heart disease

mRNA Processing: RNA editing Two types: C->U and A->I Adenosine de-aminases (ADA) are ubiquitously expressed in mammals act on dsRNA & convert A to I (read as G)

mRNA Processing: RNA editing Two types: C->U and A->I Adenosine de-aminases (ADA) are ubiquitously expressed in mammals act on dsRNA & convert A to I (read as G) misregulation of A-to-I RNA editing has been implicated in epilepsy, amyotrophic lateral sclerosis & depression

mRNA Processing: Polyadenylation Addition of As to end of mRNA Why bother? helps identify as mRNA required for translation way to measure age of mRNA ->mRNA s with < 200 As have short half-life

mRNA Processing: Polyadenylation Addition of As to end of mRNA Why bother? helps identify as mRNA required for translation way to measure age of mRNA ->mRNA s with < 200 As have short half-life >50% of human mRNAs have alternative polyA sites!

mRNA Processing: Polyadenylation >50% of human mRNAs have alternative polyA sites!

mRNA Processing: Polyadenylation >50% of human mRNAs have alternative polyA sites! result : different mRNA, can result in altered export, stability or different proteins

mRNA Processing: Polyadenylation >50% of human mRNAs have alternative polyA sites! result : different mRNA, can result in altered export, stability or different proteins some thalassemias are due to mis-poly A

mRNA Processing: Polyadenylation some thalassemias are due to mis-poly A Influenza shuts down nuclear genes by preventing poly- Adenylation (viral protein binds CPSF)

mRNA Processing: Polyadenylation 1) CPSF (Cleavage and Polyadenylation Specificity Factor) binds AAUAAA in hnRNA

mRNA Processing: Polyadenylation 1) CPSF binds AAUAAA in hnRNA 2) CStF (Cleavage Stimulatory Factor) binds G/U rich sequence 50 bases downstream CFI, CFII bind in between

Polyadenylation 1) CPSF binds AAUAAA in hnRNA 2) CStF binds; CFI, CFII bind in between 3) PAP (PolyA polymerase) binds & cleaves b 3’ to AAUAAA

mRNA Processing: Polyadenylation 3) PAP (PolyA polymerase) binds & cleaves b 3’ to AAUAAA 4) PAP adds As slowly, CFI, CFII and CPSF fall off

mRNA Processing: Polyadenylation 4) PAP adds As slowly, CFI, CFII and CPSF fall off 5)PABII binds, add As rapidly until 250

Coordination of mRNA processing Splicing and polyadenylation factors bind CTD of RNA Pol II-> mechanism to coordinate the three processes Capping, Splicing and Polyadenylation all start before transcription is done!

Export from Nucleus Occurs through nuclear pores anything > 40 kDa needs exportin protein bound to 5’ cap

Export from Nucleus In cytoplasm nuclear proteins fall off, new proteins bind eIF4E/eIF-4F bind cap also new proteins bind polyA tail mRNA is ready to be translated!