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 b 3’ to AAUAAA

mRNA Processing: Polyadenylation 3) PAP (PolyA polymerase) binds & cleaves 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 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 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 bp

Post-transcriptional regulation Other mRNA are targeted by small interfering RNA defense against RNA viruses DICERs cut dsRNA into bp helicase melts dsRNA

Post-transcriptional regulation Other mRNA are targeted by small interfering RNA defense against RNA viruses DICERs cut dsRNA into 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 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 bp helicase melts dsRNA - RNA binds RISC complex binds target target is cut