1. Bio 402/502 Section II, Lecture 4
mRNA processing events and export
Dr. Michael C. Yu
Lectures powerpoint files & readings (in PDF files) are located in
Office hours: Weds & Thurs, 9am-12pm, or by appt ( for appointment)

2. Recap from the last lecture - alternative splicing
Alternative splicing: a single gene can encode many messages depending on how the message is spliced
What are the molecular mechanisms governing alternative splicing?

3. Recognition of splice site occurs in pairs
1. Sequence composition of exons and proximity of splice site signals promote exon rather than intron recognition U1 U1
U2AF
cryptic 5’ ss GU AG
On rate favored because interaction is stabilizing
Off rate favored because no stabilizing interaction or it is blocked
2. SR proteins enhances this interaction
ESE: exonic splicing enhancer U1
Cryptic splice sites exist adjacent to strong hnRNP binding sites (splicing inhibitors) U1 SR
U2AF
hnRNP GU AG
ESE
ISS
(Cote, Univ. of Ottawa)

4. Why are exons preferentially recognized?
Differential size distributions of exons (~50 to 300 nt) vs. introns (< ,000 nt)
SR protein - preferentially binds to exon sequences
- mark the 5’ & 3’ splicing sites in conjunction w/ U1 & U2 during transcription
hnRNP - heterogenous nuclear ribonucleoproteins (twice the diameter of nucleosome)
- consists at least eight different proteins
- compacts introns, thereby masking cryptic splicing sites
- preferentially binds to introns, but also bind to exons, although less frequently
(Cote, Univ. of Ottawa)

5. Intron Definition Exon Definition
Models for splice site recognition: intron & exon definition
Boundaries between introns & exons are recognized through its interaction with multiple proteins either across exon or intron
Intron definition:
Uses intron as the unit of recognition mechanism. Complex forms through stabilized protein interactions across the intron SR SR SR RS
70K
U2AF35 RS
U2AF RS
SF2
Exon 1 A
Exon 2
SF1 U1
snRNP
Intron Definition SR
Exon Definition:
Complex can easily form stabilized protein interactions across the exon. Excises out the flanking introns SR RS
70K SR
U2AF35 RS
U2AF RS
SF2 A
Exon
SF1 U1
snRNP
Exon Definition
(Cote, Univ. of Ottawa)
Stable interaction confirms accuracy of splice site choice

6. Important point to remember about alternative splicing
Regulation of alternative splicing involves the specific stabilization or destabilization of splice site recognition
Stabilization: exon inclusion
Destabilization: exon skipping
(Cote, Univ. of Ottawa)

7. Approaches used to study alternative splicing
How would you identify cis-regulatory sequences responsible for cell-specific splicing ?
Mutational analysis finds intronic splicing silencer (ISS)
Transfection  
  
Reporter
Plasmid  
Alternatively spliced
Not alternatively spliced
Examine RNA Splicing of Transfected Splicing Reporters to identify cis-regulatory regions
(Cote, Univ of Ottawa)

8. Use of deletion analysis to identify intronic splicing silencer
First create deletional mutations across a DNA sequence
:alternatively spliced exon   
RSV 
hMT-1
hMT-2
hMT-3 WT D1   WT
101 bp
225 bp deletion  D1
Splicing Reporter Constructs were transfected into the cells and RNA splicing examined by RT-PCR
20% 80%
Inclusion
D1 contains a sequence element that regulates splicing blue exon
(Cote, Univ. of Ottawa)

9. How can we figure out where it is? ISS allows exon to be skipped  
Determining the sequence elements involved in alternative splicing
How can we figure out where it is? ISS allows exon to be skipped  
RSV
ISS 
hMT-1
hMT-2
hMT-3
Deletions often guided by conservation of sequence
ISS 
Exon Skipped (ES) - wild-type
101 bp
225 bp deletion 
Exon Incuded (EI) - deletion  ES
1. Deletion arrow the Region to Identify the ISS  ES
ISS  EI  ES
2. Point mutagenesis to Identify Key Nucleotides  EI
(Cote, Univ. of Ottawa)

10. UV Irradiate - crosslinks protein to RNA
Identifying trans-factors involved in alternative splicing
Identify trans-regulatory factors that demonstrate sequence-specific binding. Several Methods: UV Cross-linking, Isolation Assembled RNA/Protein Complexes (Affinity Selection or Gel Shift).
Separate & visualize by
PAGE
UV Cross-linking Assay
kDa WT
MUT
UV Irradiate - crosslinks protein to RNA
220 -
97.4 -
Prepare Nuclear
Splicing Extract
Digest RNA
66 -
Normal ISS
46 -
Incubate with
Normal and Mutant
Labeled RNA
30 -
Mutant ISS
Cell Line
(Cote, Univ. of Ottawa)
The identity of this protein can be determined candidate approach using antibodies, perform standard protein purification approaches, or affinity approaches followed by microsequencing

11. Studying alternative splicing using microarrays
Comparison: which spliced variants are produced in tissue X vs. tissue Y?
Splice variants produced from both tissues
Competitive hybridization to special high-definition tiling arrays
Verify signals - determine whether exon is skipped/included in a specific tissue type.
Can also test splicing changes under specific conditions: nutrients, temperature, response to pharmacological agents, etc
(Cote, Univ. of Ottawa)

12. Overview of mRNA processing steps
(McKee & Silver, 2007)

13. What if the message is not termianted properly?
Pre-mRNAs are cleaved and processed at its 3’-end
What if the message is not termianted properly?
Non-precise termination
Precise end after
cleavage and polyadenylation
AAAAAAAAAAAAA

14. Pre-mRNA cleavage process involves a set of trans-factors
Recognition of target sequences on the RNA by cleavage factors
3’-end cleavage and polyadenylation occur as a single complex
Cleavage event provides an indirect signal for transcription termination

15. Transcription termination model: the allosteric model
During transcriptonal elongation, RNA pol II is in a processive/stable conformation
As RNA pol II transcribes through the poly A site (AATAAA), it becomes less processive
Result: RNA pol II falls off, transcription is terminated

16. Transcription termination model: the torpedo model
After transcribing through the poly A site (AATAAA), downstream RNA sequence is digested by a 5’-3’ exonuclease
Once the cleavage occurs, the exonuclease continues degradation of nascent RNA to guide itself to its target--RNA pol II
Once it reaches RNA pol II, it dissociates the RNA pol II (torpedoed it!). Transcription is terminated

17. ? Transcription termination model: combined model
Cooperation between exonuclease and an unknown helicase or other factors affecting RNA pol II
Together, they convert RNA pol II from processive to non-processive form
This conversion results in the disruption of DNA:RNA hybrid and releasing of RNA pol II from the DNA template ?

18. Polyadenylation of pre-mRNAs stabilizes the newly synthesized messages
Occurs at the 3’-end of virtually all eukaryotes
Only occurs in mRNAs - not in other RNAs produced
Polyadenylation is correlated with half-life of mRNA
Short poly A = short half-life = little proteins made
Long poly A = long half-life = more proteins made

19. Steps involved in polyadenylation of pre-mRNAs
CPSF – cleavage & polyadenylation specificity factor
CstF – cleavage stimulation factor
CF – cleavage factors
PAP – poly(A) polymerase
PAB – Poly(A) binding protein
G/U – GU or U-rich sequence element
CPSF binds to upstream AAUAAA polyadenylation signal in RNA transcript
CstF interacts w/ a downstream GU- or U-rich seq., & w/ bound CPSF
Physical links between polyadenylation & transcription machinery - CTD of pol II required for efficient capping, splicing, 3’ end processing
CPSF co-purifies with TFIID  association of pol II w/ cleavage factors
CBC also physically contacts polyadenylation machinery (via assoc. w/CTD) and participates in cleavage
All these interactions may stabilize polyadenylation complex
Transcription termination dependent on a functional poly(A) signal

20. AAAAA TTTTTTTT Polyadenylation is useful for isolating mRNA population
Cell culture
Lyse cells & extract total RNA population
mRNAs
Chromatograhy column/solid support
AAAAA
TTTTTTTT
Cellulose beads
containing poly-dT
High temprature: elute mRNAs from the column

21. RT-PCR of total mRNA population can determine transcription on/off
Determining changes with a specific transcript - is it produced under a given condition/treatment? Answers a YES/NO question.
RT-PCR: reverse transcribe mRNA (using Oligo-dTs), PCR amplified reverse-transcribed cDNA population, electrophoresis to compare expression
No amplification =
no mRNA produced
Good: detect low levels of expression, only small amounts of mRNA needed
Bad: Non-linear comparison, multiple primers needed per gene

22. A better comparison in terms of relative transcript levels
To determine more quantitatively the changes with a specific transcript produced under a given condition: Northern blot/RNA hybridization
Steps: isolate total RNA, gel electrophoresis, probe for a specific message (radiolabelled or chemi-illuminescence)
Presence/absence of message picked up by hybridizing probe
Always normalize the RNA loading by probing for a “control” transcript
(Zhang et al, 2000)
Not great in comparing small changes in the level of expression

23. A highly sensitive assay to detect the presence of a specific transcript
RNAse Protection Assay - higher sensitivity than Northern blot, but not useful in picking up sizes of target messages
Make radioactive labeled RNA from cDNA (your gene of interest)
Total mRNA population extracted from cells
RNAse treatment removes unpaired probe
If a specific mRNA is present, it will “protect” the radiolabelled probe and does not allow it to be digested by RNAse treatment
Very sensitive and specific!

24. Real-time PCR offers quantitative result on # of copies of message
Real-time PCR: traditional PCR looks at “end-pt” analysis of products, but real-time PCR allows for detection and quantitation of PCR amplicon throughout a reaction (thus, “real-time”)
Theoretically there is a quantitative relationship between the amount of starting sample and the
amount of PCR product at any given sample.
Real-time PCR detects the accumulation of amplicon during the reaction. The data is then measured at the exponential phase of the PCR reaction rather than end-point plateau. The exponential phase is the optimal point for analyzing data.
Area of detection for
real-time PCR
Exponential: exact doubling of product is accumulating ea cycle w/ 100% reaction efficiency assumption
Linear: reaction components are being consumed, reaction is slowing, products are starting to degrade
Plateau: end point of PCR
(Kapa biosystems)

25. Real-time PCR: SYBR green method
SYBR Green I binds to double-
stranded DNA. The resulting DNA-
dye-complex absorbs blue light
(max = 498 nm) and emits green light
(max = 522 nm)
As DNA is amplified SYBR
fluorescence increases proportionally
(Kapa biosystems)

26. Real-time PCR: Sequence-specific probe (e.g. Taqman)
Dual fluorophore-labeled oligonucleotide probe: e.g. TaqMan
5’3’ exonuclease activity of
DNA polymerase cleaves reporter
dye from quencher and allowing
fluorescence.
Specific sequences are able to be
detected in the real-time reaction.
As reporter shifts away from quencher, fluorescence signal is detected
(Kapa biosystems)

27. Information you can learn from total mRNA population
Total gene expression using DNA microarrays
Able to determine complete gene expression program of a cell at once