Transcript No Slide Title

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
Is capped, spliced and poly-adenylated before export to cytosol
• Many are also edited
All three are coordinated with transcription & affect gene
expression: enzymes piggy-back on POLII
mRNA Processing: Polyadenylation
1) CPSF (Cleavage and Polyadenylation Specificity
Factor) binds AAUAAA in hnRNA
mRNA Processing: Polyadenylation
1) CPSF binds AAUAAA in hnRNA
2) CStF (Cleavage Stimulatory Factor) binds G/U rich
sequence 50 bases downstream
CFI, CFII bind in between
Polyadenylation
1) CPSF binds AAUAAA in hnRNA
2) CStF binds; CFI, CFII bind in between
3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to
AAUAAA
mRNA Processing: Polyadenylation
3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to
AAUAAA
4) PAP adds As slowly, CFI, CFII and CPSF fall off
mRNA Processing: Polyadenylation
4) PAP adds As slowly, CFI, CFII and CPSF fall off
5) PABII binds, add
As rapidly until 250
Coordination of mRNA processing
Splicing and polyadenylation factors bind CTD of RNA
Pol II-> mechanism to coordinate the three processes
Capping, Splicing and Polyadenylation
all start before transcription is done!
Export from Nucleus
Occurs through nuclear
pores
anything >
40 kDa needs
exportin
protein
bound
to 5’ cap
Export from Nucleus
In cytoplasm nuclear proteins fall off, new proteins bind
• eIF4E/eIF-4F bind cap
• also new
proteins bind
polyA tail
• mRNA is
ready to be
translated!
Cytoplasmic regulation
• lifetime
• localization
• initiation
Post-transcriptional regulation
Nearly ½ of human genome is transcribed, only 1% is
CDS
• 98% of RNA made is non-coding
Post-transcriptional regulation
Nearly ½ of human genome is transcribed, only 1% is
CDS
• 98% of RNA made is non-coding
• ~1/3 intron
Post-transcriptional regulation
Nearly ½ of human genome is transcribed, only 1% is
CDS
• 98% of RNA made is non-coding
• ~1/3 intron
• ~2/3 “independently transcribed”
Post-transcriptional regulation
Nearly ½ of human genome is transcribed, only 1% is
CDS
• 98% of RNA made is non-coding
• ~1/3 intron
• ~2/3 “independently transcribed”
• Polymerases II & III (+ IV & V in plants) all help
Post-transcriptional regulation
Nearly ½ of human genome is transcribed, only 1% is
CDS
• 98% of RNA made is non-coding
• ~1/3 intron
• ~2/3 “independently transcribed”
• Polymerases II & III (+ IV & V in plants) all help
• many are from transposons or gene fragments
made by transposons (pack-MULES)
Post-transcriptional regulation
Nearly ½ of human genome is transcribed, only 1% is
CDS
• 98% of RNA made is non-coding
• ~1/3 intron
• ~2/3 “independently transcribed”
• Polymerases II & III (+ IV & V in plants) all help
• many are from transposons or gene fragments
made by transposons (pack-MULES)
• ~ 10-25% is anti-sense: same region is transcribed
off both strands
Thousands of antisense transcripts in plants
1. Overlapping genes
Thousands of antisense transcripts in plants
1. Overlapping genes
2. Non-coding RNAs
Thousands of antisense transcripts in plants
1. Overlapping genes
2. Non-coding RNAs
3. cDNA pairs
Thousands of antisense transcripts in plants
1. Overlapping genes
2. Non-coding RNAs
3. cDNA pairs
4. MPSS
Thousands of antisense transcripts in plants
1. Overlapping genes
2. Non-coding RNAs
3. cDNA pairs
4. MPSS
5. TARs
Thousands of antisense transcripts in plants
Hypotheses
1. Accident: transcription unveils “cryptic promoters” on
opposite strand (Zilberman et al)
Hypotheses
1. Accident: transcription unveils “cryptic promoters” on
opposite strand (Zilberman et al)
2. Functional
a. siRNA
b. miRNA
c. Silencing
Hypotheses
1. Accident: transcription unveils “cryptic promoters” on
opposite strand (Zilberman et al)
2. Functional
a. siRNA
b. miRNA
c. Silencing
d. Priming: chromatin remodeling requires
transcription!
Post-transcriptional regulation
RNA degradation is crucial with so much “extra” RNA
Post-transcriptional regulation
RNA degradation is crucial with so much “extra” RNA
• mRNA lifespan varies 100x
• Highly regulated! > 30 RNAses in Arabidopsis!
Post-transcriptional regulation
mRNA degradation
• lifespan varies 100x
• Sometimes due to AU-rich 3' UTR sequences (DST)
mRNA degradation
• lifespan varies 100x
• Sometimes due to AU-rich 3' UTR sequences (DST)
•Endonuclease cuts DST, then exosome digests 3’->5’ &
XRN1 digests 5’->3’
mRNA degradation
•Most are degraded by de-Adenylation pathway
•Deadenylase removes tail
mRNA degradation
•Most are degraded by de-Adenylation pathway
•Deadenylase removes tail
•Exosome digests 3’ -> 5’
mRNA degradation
•Most are degraded by de-Adenylation pathway
•Deadenylase removes tail
•Exosome digests 3’ -> 5’
•Or, decapping enz
removes cap & XRN1
digests 5’ ->3’
Post-transcriptional regulation
mRNA degradation: mRNA is checked &
defective transcripts are degraded
= mRNA surveillance
1.Nonsense-mediated decay:EJC @
each splice junction that is displaced by
ribosome
Post-transcriptional regulation
mRNA degradation: mRNA is checked &
defective transcripts are degraded
= mRNA surveillance
1.Nonsense-mediated decay:EJC @
each splice junction that is displaced by
ribosome
2.If not-displaced, is cut by
endonuclease & RNA is degraded
Post-transcriptional regulation
mRNA degradation: mRNA is checked &
defective transcripts are degraded
= mRNA surveillance
Non-stop decay:
Ribosome goes to end
& cleans off PABP
Post-transcriptional regulation
mRNA degradation: mRNA is checked &
defective transcripts are degraded
= mRNA surveillance
Non-stop decay:
Ribosome goes to end
& cleans off PABP
w/o PABP exosome
eats mRNA
Post-transcriptional regulation
mRNA degradation: mRNA is checked & defective
transcripts are degraded = mRNA surveillance
No-go decay: cut RNA 3’ of stalled ribosomes
Post-transcriptional regulation
mRNA degradation
• lifespan varies 100x
• Sometimes due to AU-rich 3'
UTR sequences
• Defective mRNA may be targeted
by NMD, NSD, NGD
Other RNA are targeted by
small interfering RNA
Post-transcriptional regulation
Other mRNA are targeted by
small interfering RNA
• defense against RNA viruses
• DICERs cut dsRNA into 21-28 bp
Post-transcriptional regulation
Other mRNA are targeted by
small interfering RNA
• defense against RNA viruses
• DICERs cut dsRNA into 21-28 bp
• helicase melts dsRNA
Post-transcriptional regulation
Other mRNA are targeted by
small interfering RNA
• defense against RNA viruses
• DICERs cut dsRNA into 21-28 bp
• helicase melts dsRNA
• - RNA binds RISC
Post-transcriptional regulation
Other mRNA are targeted by
small interfering RNA
• defense against RNA viruses
• DICERs cut dsRNA into 21-28 bp
• helicase melts dsRNA
• - RNA binds RISC
• complex binds target
Post-transcriptional regulation
Other mRNA are targeted by
small interfering RNA
• defense against RNA viruses
• DICERs cut dsRNA into 21-28 bp
• helicase melts dsRNA
• - RNA binds RISC
• complex binds target
• target is cut