phusion pcr mix: for 20 µl on ice! 1.prepare 100 pmol/µl solutions of each of your primers with...

Phusion PCR mix: for 20 µlOn ICE!

1. Prepare 100 pMol/µl solutions of each of your primers with molecular grade water

2. prepare 10x dilution of F primer and add 2 µl to your PCR tube

3. prepare 10x dilution of R primer and add 2 µl to your PCR tube

4. Add 1 µl of suitable genomic DNA5. Add 15 µl Phusion master mix (for 140 µl total volume)

1. 28 µl 5x HF buffer2. 2.8 µl 10 mM dNTP3. 1.4 µl Phusion4. 72.8 µl molecular grade water

Cycle1x 60” @ 98˚ C35x: 30” @ 98˚ C : (55-cycle #)” @ Topt : 1 min/1000 bp@

72˚ C1x 5 ‘ @ 72˚ CTransfer 5 µl to fresh tube, add 1 µl dye & run on 2% gel

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• ~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

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


•Deadenylase removes tail•Exosome digests 3’ -> 5’


•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


• defense against RNA viruses• DICERs cut dsRNA into 21-28 bp• helicase melts dsRNA


• defense against RNA viruses• DICERs cut dsRNA into 21-28 bp• helicase melts dsRNA• - RNA binds RISC


• defense against RNA viruses• DICERs cut dsRNA into 21-28 bp• helicase melts dsRNA• - RNA binds RISC• complex binds target


• defense against RNA viruses• DICERs cut dsRNA into 21-28 bp• helicase melts dsRNA• - RNA binds RISC• complex binds target• target is cut


Small RNA regulation

• siRNA: target RNA viruses (& transgenes)

•miRNA: arrest translation of targets

• created by digestion of foldback

Pol II RNA with mismatch loop







•Mismatch is key difference:

generated by different Dicer







•Mismatch is key difference:

generated by different Dicer

•Arrest translation in animals,

target degradation in plants

small interfering RNA mark specifictargets•once cut they are removed by endonuclease-mediated decay

Most RNA degradation occurs in P bodies• recently identified cytoplasmic sites where exosomes & XRN1 accumulate when cells are stressed

Most RNA degradation occurs in P bodies• recently identified cytoplasmic sites where exosomes & XRN1 accumulate when cells are stressed •Also where AGO & miRNAs accumulate

Most RNA degradation occurs in P bodies• recently identified cytoplasmic sites where exosomes & XRN1 accumulate when cells are stressed •Also where AGO & miRNAs accumulate•w/o miRNA P bodies dissolve!

Post-transcriptional regulation1) mRNA processing2) export from nucleus3) mRNA degradation 4) mRNA localization• RNA-binding proteinslink it to cytoskeleton:bring it to correct siteor store it

4) mRNA localization• RNA-binding proteins link it to cytoskeleton:bring it to correct site or store it•Some RNA (eg Knotted) are transported into neighboring cells

4) mRNA localization• RNA-binding proteins link it to cytoskeleton:bring it to correct site or store it•Some RNA are transported into neighboring cells•Others are transported t/o theplant in the phloem (SUT1, KN1)

4) mRNA localization• RNA-binding proteins link it to cytoskeleton:bring it to correct site or store it•Some RNA are transported into neighboring cells•Others are transported t/o the plant in the phloem (SUT1, KN1)•Also some siRNA & miRNA!

4) mRNA localization• RNA-binding proteins link it to cytoskeleton:bring it to correct site or store it•Some RNA are transported into neighboring cells•Others are transported t/o the plant in the phloem (SUT1, KN1)•Also some siRNA & miRNA!•siRNA mediate silencing• Especially of viruses & TE

4) mRNA localization• RNA-binding proteins link it to cytoskeleton:bring it to correct site or store it•Some RNA are transported into neighboring cells•Others are transported t/o the plant in the phloem (SUT1, KN1)•Also some siRNA & miRNA!•siRNA mediate silencing•MiR399 moves to roots todestroy PHO2 mRNA upon Pi stress•PHO2 negatively regulates Pi uptake

Post-transcriptional regulationRNA in pollen controls first division after fertilization!

Post-transcriptional regulationRNA in pollen controls first division after fertilization!Delivery by pollen ensures correct development doesn’t happen unless egg is fertilized by pollen

Post-transcriptional regulation4) mRNA localization• RNA-binding proteins link it to cytoskeleton: bring it to correct site or store it• many are stored in P-bodies! More than just an RNA-destruction site

Post-transcriptional regulation4) mRNA localization• RNA-binding proteins link it to cytoskeleton: bring it to correct site or store it• many are stored in P-bodies! More than just an RNA-destruction site•Link with initiation of translation

Post-transcriptional regulationProtein degradation rate varies 100x• Some have motifs, eg Destruction box, marking them for

polyubiquitination: taken to proteasome & destroyed


polyubiquitination: taken to proteasome & destroyed• N-terminal rule: Proteins with N-terminal Phe, Leu,

Asp, Lys, or Arg have half lives of 3 min or less.


polyubiquitination: taken to proteasome & destroyed• N-terminal rule: Proteins with N-terminal Phe, Leu,

Asp, Lys, or Arg have half lives of 3 min or less.• Proteins with N-terminal Met, Ser, Ala, Thr, Val, or Gly

have half lives greater than 20 hours.

Protein degradationSome have motifs marking them for polyubiquitination:• E1 enzymes activate ubiquitin• E2 enzymes conjugate ubiquitin• E3 ub ligases determine specificity, eg for N-terminus

Protein degradationSome have motifs marking them for polyubiquitination:• E1 enzymes activate ubiquitin• E2 enzymes conjugate ubiquitin• E3 ub ligases determine specificity, eg for N-terminusDiscovered in plants: X-W Deng found COP1 mutant• Looks like light-grown plant in dark: tags proteins for

destruction

Protein degradation• E3 ub ligases determine specificity

• >1300 E3 ligases in Arabidopsis• 4 main classes according to cullin scaffolding protein

E3 ubiquitin ligases determine specificity>1300 E3 ligases in Arabidopsis4 main classes according to cullin scaffolding protein• RBX1 (or similar) positions E2

E3 ubiquitin ligases determine specificity>1300 E3 ligases in Arabidopsis4 main classes according to cullin scaffolding protein• RBX1 (or similar) positions E2• Linker (eg DDB1) positions substrate receptor

E3 ubiquitin ligases determine specificity>1300 E3 ligases in Arabidopsis4 main classes according to cullin scaffolding protein• RBX1 (or similar) positions E2• Linker (eg DDB1) positions substrate receptor• Substrate receptor (eg DCAF/DWD) picks substrate• >100 DWD in Arabidopsis

E3 ubiquitin ligases determine specificity>1300 E3 ligases in Arabidopsis4 main classes according to cullin scaffolding protein• RBX1 (or similar) positions E2• Linker (eg DDB1) positions substrate receptor• Substrate receptor (eg DCAF/DWD) picks substrate• NOT4 is an E3 ligase & a component of the CCR4–NOT

de-A complex

E3 ubiquitin ligases determine specificity>1300 E3 ligases in Arabidopsis4 main classes according to cullin scaffolding protein• RBX positions E2• DDB1 positions DCAF/DWD• DCAF/DWD picks substrate: >85 DWD in rice• NOT4 is an E3 ligase & a component of the CCR4–NOT

de-A complex• CCR4–NOT de-A Complex regulates pol II

E3 ubiquitin ligases determine specificity>1300 E3 ligases in Arabidopsis4 main classes according to cullin scaffolding protein• RBX positions E2• DDB1 positions DCAF/DWD• DCAF/DWD picks substrate• NOT4 is an E3 ligase & a component of the CCR4–NOT

de-A complex• CCR4–NOT de-A Complex regulates pol II• Transcription, mRNAdeg & prot deg are linked!

E3 ubiquitin ligases determine specificityCell cycle: Anaphase Promoting Complex is an E3 ligase.MPF induces APCAPC inactive until all kinetochores are boundAPC then tags securin to free separase: cuts proteins linking chromatids

E3 ubiquitin ligases determine specificityMPF induces APCAPC inactive until all kinetochores are boundAPC then tags securin to free separase: cuts proteins

linking chromatidsAPC next swaps Cdc20 for Cdh1 & tags cyclin B to enter

G1

E3 ubiquitin ligases determine specificityAPC next tags cyclin B (destruction box) to enter G1APC also targets Sno proteins in TGF- signaling• Sno proteins prevent Smad from activating genes

E3 ubiquitin ligases determine specificityAPC also targets Sno proteins in TGF- signaling• Sno proteins prevent Smad from activating genes• APC/Smad2/Smad3 tags Sno for destruction

E3 ubiquitin ligases determine specificityAPC also targets Sno proteins in TGF- signaling• Sno proteins prevent Smad from activating genes• APC/Smad2/Smad3 tags Sno for destruction• Excess Sno = cancer

E3 ubiquitin ligases determine specificityAPC also targets Sno proteins in TGF- signaling• Sno proteins prevent Smad from activating genes• APC/Smad2/Smad3 tags Sno for destruction• Excess Sno = cancerAngelman syndrome = bad UBE3A • Only express maternal allele because paternal allele is

methylated

Auxin signalingAuxin receptors eg TIR1 are E3 ubiquitin ligasesUpon binding auxin they activate complexes targeting AUX/IAA proteins for degradation

Auxin signalingAuxin receptors eg TIR1 are E3 ubiquitin ligases!Upon binding auxin they activate complexes targeting AUX/IAA proteins for degradationAUX/IAA inhibit ARF transcription factors,so this turns on "early genes"

Auxin signalingAuxin receptors eg TIR1 are E3 ubiquitin ligases!Upon binding auxin they activate complexes targeting AUX/IAA proteins for degradation!AUX/IAA inhibit ARF transcription factors,so this turns on "early genes"Some early genes turn on 'late genes" needed for development

DWD ProteinsJae-Hoon Lee’s research• putative substrate receptors for CUL4-based E3 ligases

DWD ProteinsJae-Hoon Lee’s research• putative substrate receptors for CUL4-based E3 ligases• used bioinformatics to find all Arabidopsis & rice DWDs

DWD Proteins• used bioinformatics to find all Arabidopsis & rice DWDs•Placed in subgroups based on DWD sequence

DWD Proteins• used bioinformatics to find all Arabidopsis & rice DWDs•Placed in subgroups based on DWD sequence•Tested members of eachsubgroup for DDB1 binding

DWD Proteins•Tested members of each subgroup for DDB1 binding

• co-immunoprecipitation


• co-immunoprecipitation•Two-hybrid: identifiesinteracting proteins


• co-immunoprecipitation•Two-hybrid: identifiesinteracting proteins•Only get transcription ifone hybrid supplies Act D& other supplies DNABinding Domain

DWD ProteinsTwo-hybrid libraries are used to screen for protein-protein interactions


• co-immunoprecipitation•Two-hybrid


• co-immunoprecipitation•Cul4cs &PRL1 (PleiotropicRegulatory Locus 1) hadSimilar phenotypes

DWD ProteinsCul4cs &PRL1 (PleiotropicRegulatory Locus 1) hadsimilar phenotypesPRL1 may be receptor for AKIN10 degradation (involved in sugar sensing)

DWD Proteins•Found T-DNA insertions

•3 were sensitive to ABA


•3 were sensitive to ABA• ABI5 was elevated in dwa mutants


•3 were sensitive to ABA• ABI5 was elevated in dwa mutants•ABI5 was degraded more slowly in dwa extracts


•3 were sensitive to ABA• ABI5 was elevated in dwa mutants•ABI5 was degraded more slowly in dwa extracts•DWA1 & DWA2 target ABI5 for degradation

Regulating E3 ligasesThe COP9 signalosome (CSN), a complex of 8 proteins,

regulates E3 ligases by removing Nedd8 from cullin


regulates E3 ligases by removing Nedd8 from cullinCAND1 then blocks cullin


regulates E3 ligases by removing Nedd8 from cullinCAND1 then blocks cullinUbc12 replaces Nedd8


regulates E3 ligases by removing Nedd8 from cullinCAND1 then blocks cullinUbc12 replaces Nedd8Regulates DNA-damage response, cell-cycle & gene expression


regulates E3 ligases by removing Nedd8 from cullinCAND1 then blocks cullinUbc12 replaces Nedd8Regulates DNA-damage response, cell-cycle & gene expressionNot all E3 ligases associate withCullins!

COP1 is a non-cullin-associated E3 ligase• Protein degradation is important for light regulation• COP1/SPA1 tags transcription factors for degradation• W/O COP1 they act in dark• In light COP1 is exported to cytoplasm so TF can act

COP1 is a non-cullin-associated E3 ligase• Recent data indicates that COP1 may also associate

with CUL4

Protein degradation rate varies 100xMost have motifs marking them for polyubiquitination:

taken to proteosome & destroyedOther signals for selective degradation include PEST &

KFERQ• PEST : found in many rapidly degraded proteins

• e.g. ABCA1 (which exports cholesterol in association with apoA-I) is degraded by calpain

Protein degradation rate varies 100xOther signals for selective degradation include PEST &



• Deletion increases t1/2 10x, adding PEST drops t1/2 10x




• Deletion increases t1/2 10x, adding PEST drops t1/2 10x• Sometimes targets poly-Ub




• Deletion increases t1/2 10x, adding PEST drops t1/2 10x• Sometimes targets poly-Ub• Recent yeast study doesn’t support general role




• Deletion increases t1/2 10x, adding PEST drops t1/2 10x• Sometimes targets poly-Ub• Recent yeast study doesn’t support general role

• KFERQ: cytosolic proteins with KFERQ are selectively taken up by lysosomes in chaperone-mediated autophagy under conditions of nutritional or oxidative stress.

phusion pcr mix: for 20 µl on ice! 1.prepare 100 pmol/µl solutions of each of your primers with...

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