Regulating gene expression Goal is controlling Proteins •How many? •Where? •How active? 8 levels (two not shown are mRNA localization & prot degradation)
Regulating gene expression Goal is controlling Proteins How many? Where? How active?
Jan 19, 2016
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 - PowerPoint PPT Presentation
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Regulating gene expressionGoal is controlling Proteins•How many?•Where?•How active?8 levels (two notshown are mRNAlocalization & protdegradation)
mRNA PROCESSINGPrimary transcript is hnRNAIs 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: Polyadenylation1) CPSF binds AAUAAA in hnRNA2) CStF (Cleavage Stimulatory Factor) binds G/U rich sequence 50 bases downstream
CFI, CFII bind in between
Polyadenylation1) CPSF binds AAUAAA in hnRNA2) CStF binds; CFI, CFII bind in between3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to AAUAAA
mRNA Processing: Polyadenylation3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to AAUAAA4) PAP adds As slowly, CFI, CFII and CPSF fall off
mRNA Processing: Polyadenylation4) PAP adds As slowly, CFI, CFII and CPSF fall off5) PABII binds, add As rapidly until 250
Coordination of mRNA processingSplicing 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 NucleusOccurs through nuclear poresanything > 40 kDa needs exportinproteinbound 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 regulationNearly ½ of human genome is transcribed, only 1% is CDS• 98% of RNA made is non-coding
Post-transcriptional regulationNearly ½ of human genome is transcribed, only 1% is CDS• 98% of RNA made is non-coding• ~1/3 intron
Post-transcriptional regulationNearly 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 regulationNearly 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 regulationNearly 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 regulationNearly 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 plants1. Overlapping genes
Thousands of antisense transcripts in plants1. Overlapping genes2. Non-coding RNAs
Thousands of antisense transcripts in plants1. Overlapping genes2. Non-coding RNAs3. cDNA pairs
Thousands of antisense transcripts in plants1. Overlapping genes2. Non-coding RNAs3. cDNA pairs4. MPSS
Thousands of antisense transcripts in plants1. Overlapping genes2. Non-coding RNAs3. cDNA pairs4. MPSS5. 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 regulationRNA degradation is crucial with so much “extra” RNA
Post-transcriptional regulationRNA degradation is crucial with so much “extra” RNA• mRNA lifespan varies 100x• Highly regulated! > 30 RNAses in Arabidopsis!
Post-transcriptional regulationmRNA 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 enzremoves cap & XRN1digests 5’ ->3’
Post-transcriptional regulationmRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillance1.Nonsense-mediated decay:EJC @ each splice junction that is displaced by ribosome
Post-transcriptional regulationmRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillance1.Nonsense-mediated decay:EJC @ each splice junction that is displaced by ribosome2.If not-displaced, is cut by endonuclease & RNA is degraded
Post-transcriptional regulationmRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillanceNon-stop decay:Ribosome goes to end & cleans off PABP
Post-transcriptional regulationmRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillanceNon-stop decay:Ribosome goes to end & cleans off PABPw/o PABP exosomeeats mRNA
Post-transcriptional regulationmRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillanceNo-go decay: cut RNA 3’ of stalled ribosomes
Post-transcriptional regulationmRNA degradation• lifespan varies 100x• Sometimes due to AU-rich 3' UTR sequences • Defective mRNA may be targetedby NMD, NSD, NGD
Other RNA are targeted by small interfering RNA
Post-transcriptional regulationOther mRNA are targeted by small interfering RNA• defense against RNA viruses• DICERs cut dsRNA into 21-28 bp
Post-transcriptional regulationOther mRNA are targeted by small interfering RNA• defense against RNA viruses• DICERs cut dsRNA into 21-28 bp• helicase melts dsRNA
Post-transcriptional regulationOther 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 regulationOther 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 regulationOther 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
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