W9

mRNA processing & regulation

- background
◼ DNA structure
◆ 5/3’ UTR: untranslated region
◆ Exon, intron
◼ Step: DNA → Pre-mRNA → mRNA → protein
◼ Addition: CAP, poly A tail
- Spliceosome
◼ Pre mRNA conserve
◆ Core splice sites: not enough to accurately identify by spliceosomes
⚫ AG/GU…branchpoint (A)… Y10-20(U/C polypyrimidine tract)
AG/G
⚫ Need other sequence within intons/exons
◼ Different types of splicing
◆ Self (no need energy) transesterification reactions
◆ Proteins
⚫ Small nuclear ribonucleoprotein (snRNP) * 5 = spliceosome
◆ Ribonucleoproteins
- Splicing
◼ Machinery:
◆ Intron definition: intron <250 nucleotides
⚫ no preform of catalytic center; it is formed by step by step
⚫ Recognition
◼ U2snRNP: Branch site without A
⚫ RNA helicase
◼ Structural rearrangement within the snRNP
⚫ Step
◼ 1.
◆ U1snRNP: 5’splice site
◆ BBP branchpoint binging protein: branchpoint
◆ U2AF(65+35): polyY and AG→ recruit and stabilize
U2snRNP
◼ 2. Destabilize, BBP leave and U2AF leave.
◆ U1, and U2
◼ 3. U4,5,6 complex bind
◆ U1,2,4,5,6
◼ 4. RNA helicase → destabilize U1, 4 leave
 ◆ U2,5,6
◼ 5. RNA helicase→ Confirmational change
◆ Catalytically activated
◼ 6. 2 steps Reaction
◆ Transesterification reaction 1
⚫ GU: phosphodiester bond + A: OH
◆ TE reaction 2 → intron in shape of a lariat
⚫ Exon1: 3’OH + 3’ splice site: phosphodiester
◼ 7. RNA helicase remove mRNA and release the snRNP
◆ Extron definition: intron>250
⚫ SR protein bind to extron ESE → U1 U2AF → U2
⚫ With unknown mechanism → intron definition
◆ Need ATP, GTP
◼ snRNPs = snRNA + protein
◆ snRNA U1, 2, 4, 5, 6
◆ bind to different types of protein
◆ structural arrangement
◆ different in 2nd structure
- Why intron?
◼ Alternative splicing
◆ Types: constitutively, alternative5’(E), alternative3’(D), cassette exon,
mutually exclusive

◆
◼ Not the only method to generate different mRNA
- How to regulate?
◼ 1. How to distinguish exon and intron? E.g. Dystrophin gene
◆ Why difficult?
 ⚫ Introns contain pseudo-exon (appear to be exons but are not
spliced) and cryptic (appear to be authentic splice sites but are
not used)
⚫ Core splice site contain only half of the information
◼ Nucleotide sequences: splicing code
◆ Made by splicing enhancer and splicing silencer sequences embed in
sequence.
◆ Cis-acting
⚫ 5’ss. Branchpoint A, polypyrimidine tract, 3’ss
⚫ splicing enhancer and silencer sequences
◼ ESE(SR), ESS(hnRNPs)
◼ ISE(SR), ISS(hnRNPs)
◼ pseudo-exon: no SE
◆ Transacting splicing regulator: RS, hnRNPs(heterogenous
ribonucleoprotein)
⚫ PTB polypyrimidine track binding protein
⚫ RS: U1, U2, U2AF
⚫ huRNPs: inhibit U1, U2, U2AF
◼ Regulation (tissue specific expression pattern)
◆ Change the expression of transacting splicing regulator
◆ SR: exon inclusion

How to detect?
- RT-PCR
◼ RNA→ DNA

◼
Pre-mRNA 3’end: Cleave and poly A
- Part at 3’ end
◼ Conserved hexamer AAUAAA, poly A signal
◼ GU/CU rich sequence
- Cleave
◼ CA in poly A side
- PolyA
◼ polyA added after CA by PAP(poly A polymerase +200A).
◆ need poly-A-binding protein
⚫ stabilize the poly-A-tail
⚫ bind initiation factors→ translate ()
◼ without poly A
◆ cannot translate
◆ degraded
◼ not in gene
1. Which three snRNPs together make the tri-snRNP particle?

Group of answer choices

U1, U2, U4
U2, U4, U5
U1, U5, U6
U4, U5, U6
2. Which of the following proteins is correctly paired to its function?
Group of answer choices
U2AF – binds to 5' splice site
DEAD-box – recognizes pyrimidine tract
BBP-binds to the branch point site
3. Select the three main functions of the small nuclear ribonuclear proteins (snRNPs)
Group of answer choices
Add poly-A tails to mRNA
Recognize the end of promoter sequences
Recognize the 5’ splice site and the branch site
Responsible for RNA methylation at splice sites
Bring the 5’ splice site and branch site together
Catalyze RNA cleavage and joining reactions
4. Which component of the spliceosome machinery is recruited by the SR protein?
Group of answer choices
U4snRNP
U3snRNP
U1snRNP
U6snRNP
5. What are the two important functions of Poly-A-binding proteins (PABPs)?
Group of answer choices
PABPs bind to the poly-A tail and stabilize the mRNA
PABPs facilitate production of more than one protein from a gene
PABPs promote mRNA degradtion
PABPs bnd to eukaryotic initation factors and promote initation of translation
6. 'snurps' are made of
Group of answer choices
RNA
Protein
DNA and protein
RNA and protein
7. Which of the following is not involved in creating the early spliceosome complex
or the E-
complex?
Group of answer choices
Base pairing of U1 and splice site
U2AF binding to 3’ splicing site
U2 binding to branch point site
BBP interaction with U2AF
8. In a mutually exclusive pre-mRNA splicing, if one exon is chosen, the alternative
exon is
Group of answer choices
also chosen
is chosen or excluded based on the gene
is exclude