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) Capping addition 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 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:
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: 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: found 248 genes that were consistently edited across more than five human cell types
Bazak et al (2014) Genome Res. 24: 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: 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 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 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 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 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