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Regulating gene expression Goal is controlling Proteins How many? Where? How active? 8 levels (two not shown are mRNA localization & prot degradation)

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Presentation on theme: "Regulating gene expression Goal is controlling Proteins How many? Where? How active? 8 levels (two not shown are mRNA localization & prot degradation)"— Presentation transcript:

1 Regulating gene expression Goal is controlling Proteins How many? Where? How active? 8 levels (two not shown are mRNA localization & prot degradation)

2 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

3 mRNA Processing: Polyadenylation 1) CPSF (Cleavage and Polyadenylation Specificity Factor) binds AAUAAA in hnRNA

4 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

5 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

6 mRNA Processing: Polyadenylation 3) PAP (PolyA polymerase) binds & cleaves b 3’ to AAUAAA 4) PAP adds As slowly, CFI, CFII and CPSF fall off

7 mRNA Processing: Polyadenylation 4) PAP adds As slowly, CFI, CFII and CPSF fall off 5)PABII binds, add As rapidly until 250

8 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!

9 Export from Nucleus Occurs through nuclear pores anything > 40 kDa needs exportin protein bound to 5’ cap

10 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!

11 Cytoplasmic regulation lifetime localization initiation

12 Post-transcriptional regulation Nearly ½ of human genome is transcribed, only 1% is CDS 98% of RNA made is non-coding

13 Post-transcriptional regulation Nearly ½ of human genome is transcribed, only 1% is CDS 98% of RNA made is non-coding ~1/3 intron

14 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”

15 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

16 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)

17 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

18 Thousands of antisense transcripts in plants 1.Overlapping genes

19 Thousands of antisense transcripts in plants 1.Overlapping genes 2.Non-coding RNAs

20 Thousands of antisense transcripts in plants 1.Overlapping genes 2.Non-coding RNAs 3.cDNA pairs

21 Thousands of antisense transcripts in plants 1.Overlapping genes 2.Non-coding RNAs 3.cDNA pairs 4.MPSS

22 Thousands of antisense transcripts in plants 1.Overlapping genes 2.Non-coding RNAs 3.cDNA pairs 4.MPSS 5.TARs

23 Thousands of antisense transcripts in plants Hypotheses 1.Accident: transcription unveils “cryptic promoters” on opposite strand (Zilberman et al)

24 Hypotheses 1. Accident: transcription unveils “cryptic promoters” on opposite strand (Zilberman et al) 2. Functional a.siRNA b.miRNA c.Silencing

25 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!

26 Post-transcriptional regulation RNA degradation is crucial with so much “extra” RNA

27 Post-transcriptional regulation RNA degradation is crucial with so much “extra” RNA mRNA lifespan varies 100x Highly regulated! > 30 RNAses in Arabidopsis!

28 Post-transcriptional regulation mRNA degradation lifespan varies 100x Sometimes due to AU-rich 3' UTR sequences (DST)

29 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’

30 mRNA degradation Most are degraded by de-Adenylation pathway Deadenylase removes tail

31 mRNA degradation Most are degraded by de-Adenylation pathway Deadenylase removes tail Exosome digests 3’ -> 5’

32 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’

33 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

34 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

35 Post-transcriptional regulation mRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillance Non-stop decay: Ribosome goes to end & cleans off PABP

36 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

37 Post-transcriptional regulation mRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillance No-go decay: cut RNA 3’ of stalled ribosomes

38 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

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

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

41 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

42 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

43 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


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