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

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

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? Generate diversity 448 genes 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 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. 2013 Nov 22;4:2771.

Why splice? 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

Splicing: Why splice? 1) Generate diversity 2) Modulate gene expression introns increase amount of mRNA produced Especially introns near the 5’ end of coding sequence

Splicing: Why splice? 1) Generate diversity 2) Modulate gene expression introns increase amount of mRNA produced Especially introns near the 5’ end of coding sequence Also increase export from nucleus, translation efficiency & half-life

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 Two types: C->U and A->I Adenosine de-aminases acting on RNA (ADAR) 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: RNA editing Peng et al (2012) Nature Biotechnology 30, 253–260 found 22,688 RNA editing events in a human male 93 % were A-> I Most were in introns and non-coding regions

mRNA Processing: RNA editing Peng et al (2012) Nature Biotechnology 30: 253–260 found 22,688 RNA editing events in a human male 93 % were A-> I Most were in introns and non-coding regions Park et al (2012) Genome Res. 22: 1626-1633 found 248 genes that were consistently edited across more than five human cell types

mRNA Processing: RNA editing Peng et al (2012) Nature Biotechnology 30: 253–260 found 22,688 RNA editing events in a human male 93 % were A-> I (ADAR : A deaminase acting on RNA) Most were in introns and non-coding regions Park et al (2012) Genome Res. 22: 1626-1633 found 248 genes that were consistently edited across more than five human cell types Bazak et al (2014) Genome Res. 24: 365-376 found A-to-I editing occurs at > 108 genomic sites, located in a majority of human genes

mRNA Processing: RNA editing Bazak et al (2014) Genome Res. 24: 365-376 found A-to-I editing occurs at > 108 genomic sites, located in a majority of human genes Daniel et al (2014) Genome Biology 15:R28 found Alu elements cause cis-regulation of RNA editing Form hairpins that attract ADAR which then edits As in nearby ds-loops as well

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

mRNA Processing: Polyadenylation Addition of 200- 250 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 200- 250 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