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Regulating gene expression Goal is controlling Proteins How many? Where? How active?

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

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Regulating gene expression Goal is controlling Proteins How many? Where? How active?

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

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

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

  4. Splicing:The spliceosome cycle

  5. Splicing: • Why splice? • 1) Generate diversity • exons often encode protein domains

  6. Splicing: • Why splice? • 1) Generate diversity • exons often encode protein domains • Introns = larger target for insertions, recombination

  7. Why splice? 1) Generate diversity >94% of human genes show alternate splicing

  8. Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

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

  17. 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

  18. 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!

  19. 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

  20. 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

  21. 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

  22. 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

  23. 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

  24. 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

  25. 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

  26. 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

  27. 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!

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

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

  30. 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

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

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

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