1. Post-transcriptional regulation
Nearly ½ of human genome is transcribed, only 1% is coding
98% of RNA made is non-coding

2. Post-transcriptional regulation
Nearly ½ of human genome is transcribed, only 1% is coding
98% of RNA made is non-coding
Fraction increases with organism’s complexity

3. Transcription in Eukaryotes
3 RNA polymerases
all are multi-subunit
complexes
5 in common
3 very similar
variable # unique ones
Plants also have Pols IV & V
make siRNA

4. Processing rRNA
C/D box snoRNA direct methylation of ~100 bases
H/ACA box snoRNA direct change of ~ 100 Us to y
Some snoRNA direct modification of tRNA and snRNA

5. Processing rRNA
~ 200 bases are modified
2) Ribozymes cut 45S pre-rRNA into 28S, 18S and 5.8S
RNase MRP cuts between 18S & 5.8S
U3, U8, U14, U22, snR10 and snR30 also guide cleavage
U3 is precursor to C/D & snR30 is precursor to H/ACA snoRNA!

6. RNA Polymerase III
makes ribosomal 5S and tRNA
(+ some snRNA, scRNA, etc)
>100 different kinds of ncRNA
~10% of all RNA synthesis
Cofactor = Mn2+ cf Mg2+
sensitive to high [-aminitin]

7. Processing tRNA
tRNA is trimmed
5’ end by RNAse P
(1 RNA, 10 proteins)

8. Splicing
Many snRNA are involved in splicing
most are made by pol II
U1, U2, U4, U5, and U6 are involved in the major spliceosome cycle
U11, U12, U4atac, U6atac and U5 are involved in the minor spliceosome cycle (1% of major cycle)
U7 functions in histone pre-mRNA processing
HBII-52 snoRNA directs alternative splicing of serotonin receptor 5-HT2CR; absence causes Prader-Willi
All associate with proteins to do their job

9. The major spliceosome cycle
1) U1 snRNP (RNA/protein complex) binds 5’ splice site

10. Splicing:The spliceosome cycle
1) U1 snRNP binds 5’ splice site
2) U2 snRNP binds “branchpoint”
-> displaces A at branchpoint

11. 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

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

13. 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

14. Splicing:The spliceosome cycle

15. Splicing:
Some RNAs can self-splice!
role of snRNPs is to increase rate!
Why splice?

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

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

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

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

20. 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

21. 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

22. Why splice?
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

23. 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

24. 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

25. Regulatory ncRNA
SiRNA direct DNA-methylation via RNA-dependent DNA-methyltransferase
In other cases direct RNA degradation

26. Post-transcriptional regulation
RNA degradation is crucial with so much “extra” RNA
mRNA lifespan varies 100x
Highly regulated! > 30 RNAses in Arabidopsis!