Phusion PCR mix: for 20

1

• Phusion PCR mix for 20 µl
• On ICE!
• Prepare 100 pMol/µl solutions of each of your
  primers with molecular grade water
• prepare 10x dilution of F primer and add 2 µl to
  your PCR tube
• prepare 10x dilution of R primer and add 2 µl to
  your PCR tube
• Add 1 µl of suitable genomic DNA
• Add 15 µl Phusion master mix (for 140 µl total
  volume)
• 28 µl 5x HF buffer
• 2.8 µl 10 mM dNTP
• 1.4 µl Phusion
• 72.8 µl molecular grade water

2

• Cycle
• 1x 60 _at_ 98 C
• 35x 30 _at_ 98 C (55-cycle ) _at_ Topt 1
  min/1000 bp_at_ 72 C
• 1x 5 _at_ 72 C
• Transfer 5 µl to fresh tube, add 1 µl dye run
  on 2 gel

3

• Cytoplasmic regulation
• lifetime
• localization
• initiation

4

• 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

5

• Hypotheses
• 1. Accident transcription unveils cryptic
  promoters on opposite strand (Zilberman et al)
• 2. Functional
• siRNA
• miRNA
• Silencing
• Priming chromatin remodeling requires
  transcription!

6
Post-transcriptional regulation RNA degradation
is crucial with so much extra RNA
7

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

8

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

9

• mRNA degradation
• lifespan varies 100x
• Sometimes due to AU-rich 3' UTR sequences (DST)
• Endonuclease cuts DST, then exosome digests
  3-gt5 XRN1 digests 5-gt3

10

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

11

• mRNA degradation
• Most are degraded by de-Adenylation pathway
• Deadenylase removes tail
• Exosome digests 3 -gt 5

12

• mRNA degradation
• Most are degraded by de-Adenylation pathway
• Deadenylase removes tail
• Exosome digests 3 -gt 5
• Or, decapping enz
• removes cap XRN1
• digests 5 -gt3

13

• Post-transcriptional regulation
• mRNA degradation mRNA is checked
• defective transcripts are degraded
• mRNA surveillance
• Nonsense-mediated decayEJC _at_
• each splice junction that is displaced by
• ribosome

14

• Post-transcriptional regulation
• mRNA degradation mRNA is checked
• defective transcripts are degraded
• mRNA surveillance
• Nonsense-mediated decayEJC _at_
• each splice junction that is displaced by
• ribosome
• If not-displaced, is cut by
• endonuclease RNA is degraded

15

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

16

• 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

17

• Post-transcriptional regulation
• mRNA degradation mRNA is checked defective
  transcripts are degraded mRNA surveillance
• No-go decay cut RNA 3 of stalled ribosomes

18

• 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

19

• Post-transcriptional regulation
• Other mRNA are targeted by
• small interfering RNA
• defense against RNA viruses
• DICERs cut dsRNA into 21-28 bp

20

• 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

21

• 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

22

• 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

23

• 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

24

• Cytoplasmic regulation
• Small RNA regulation
• siRNA target RNA viruses ( transgenes)
• miRNA arrest translation of targets
• created by digestion of foldback
• Pol II RNA with mismatch loop

25

• Cytoplasmic regulation
• 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

26

• Cytoplasmic regulation
• 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
• Arrest translation in animals,
• target degradation in plants

27

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

28
(No Transcript)
29

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

30

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

31

• 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!

32

• Post-transcriptional regulation
• 1) mRNA processing
• 2) export from nucleus
• 3) mRNA degradation
• 4) mRNA localization
• RNA-binding proteins
• link it to cytoskeleton
• bring it to correct site
• or store it

33

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

34

• 4) mRNA localization
• RNA-binding proteins link it to
  cytoskeletonbring 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)

35

• 4) mRNA localization
• RNA-binding proteins link it to
  cytoskeletonbring 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!

36

• 4) mRNA localization
• RNA-binding proteins link it to
  cytoskeletonbring 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

37

• 4) mRNA localization
• RNA-binding proteins link it to
  cytoskeletonbring 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 to
• destroy PHO2 mRNA upon Pi stress
• PHO2 negatively regulates
• Pi uptake

38
Post-transcriptional regulation RNA in pollen
controls first division after fertilization!
39
Post-transcriptional regulation RNA in pollen
controls first division after fertilization! Deliv
ery by pollen ensures correct development doesnt
happen unless egg is fertilized by pollen
40

• Post-transcriptional regulation
• 4) 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

41

• Post-transcriptional regulation
• 4) 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

42

• Post-transcriptional regulation
• Protein degradation rate varies 100x
• Some have motifs, eg Destruction box, marking
  them for polyubiquitination taken to proteasome
  destroyed

43

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

44

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

45

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

46

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

47

• Protein degradation
• E3 ub ligases determine specificity
• gt1300 E3 ligases in Arabidopsis
• 4 main classes according to cullin scaffolding
  protein

48

• E3 ubiquitin ligases determine specificity
• gt1300 E3 ligases in Arabidopsis
• 4 main classes according to cullin scaffolding
  protein
• RBX1 (or similar) positions E2

49

• E3 ubiquitin ligases determine specificity
• gt1300 E3 ligases in Arabidopsis
• 4 main classes according to cullin scaffolding
  protein
• RBX1 (or similar) positions E2
• Linker (eg DDB1) positions substrate receptor

50

• E3 ubiquitin ligases determine specificity
• gt1300 E3 ligases in Arabidopsis
• 4 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
• gt100 DWD in Arabidopsis

51

• E3 ubiquitin ligases determine specificity
• gt1300 E3 ligases in Arabidopsis
• 4 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
  CCR4NOT de-A complex

52

• E3 ubiquitin ligases determine specificity
• gt1300 E3 ligases in Arabidopsis
• 4 main classes according to cullin scaffolding
  protein
• RBX positions E2
• DDB1 positions DCAF/DWD
• DCAF/DWD picks substrate gt85 DWD in rice
• NOT4 is an E3 ligase a component of the
  CCR4NOT de-A complex
• CCR4NOT de-A
• Complex regulates pol II

53

• E3 ubiquitin ligases determine specificity
• gt1300 E3 ligases in Arabidopsis
• 4 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
  CCR4NOT de-A complex
• CCR4NOT de-A
• Complex regulates pol II
• Transcription, mRNA
• deg prot deg are
• linked!

54

• E3 ubiquitin ligases determine specificity
• Cell cycle Anaphase Promoting Complex is an E3
  ligase.
• MPF induces APC
• APC inactive until all kinetochores are bound
• APC then tags securin to free
• separase cuts proteins linking
• chromatids

55

• E3 ubiquitin ligases determine specificity
• MPF induces APC
• APC inactive until all kinetochores are bound
• APC then tags securin to free separase cuts
  proteins linking chromatids
• APC next swaps Cdc20 for Cdh1 tags cyclin B to
  enter G1

56

• E3 ubiquitin ligases determine specificity
• APC next tags cyclin B (destruction box) to enter
  G1
• APC also targets Sno proteins in TGF-b signaling
• Sno proteins prevent Smad from activating genes

57

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

58

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

59

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

60
Auxin signaling Auxin receptors eg TIR1 are E3
ubiquitin ligases Upon binding auxin they
activate complexes targeting AUX/IAA proteins for
degradation
61
Auxin signaling Auxin 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"
62
Auxin signaling Auxin 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
63

• DWD Proteins
• Jae-Hoon Lees research
• putative substrate receptors for CUL4-based E3
  ligases

64

• DWD Proteins
• Jae-Hoon Lees research
• putative substrate receptors for CUL4-based E3
  ligases
• used bioinformatics to find all Arabidopsis
  rice DWDs

65

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

66

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

67

• DWD Proteins
• Tested members of each subgroup for DDB1 binding
• co-immunoprecipitation

68

• DWD Proteins
• Tested members of each subgroup for DDB1 binding
• co-immunoprecipitation
• Two-hybrid identifies
• interacting proteins

69

• DWD Proteins
• Tested members of each subgroup for DDB1 binding
• co-immunoprecipitation
• Two-hybrid identifies
• interacting proteins
• Only get transcription if
• one hybrid supplies Act D
• other supplies DNA
• Binding Domain

70
DWD Proteins Two-hybrid libraries are used to
screen for protein-protein interactions
71

• DWD Proteins
• Tested members of each subgroup for DDB1 binding
• co-immunoprecipitation
• Two-hybrid

72

• DWD Proteins
• Tested members of each subgroup for DDB1 binding
• co-immunoprecipitation
• Cul4cs PRL1 (Pleiotropic
• Regulatory Locus 1) had
• Similar phenotypes

73

• DWD Proteins
• Cul4cs PRL1 (PleiotropicRegulatory Locus 1) had
• similar phenotypes
• PRL1 may be receptor for
• AKIN10 degradation
• (involved in sugar sensing)

74

• DWD Proteins
• Found T-DNA insertions
• 3 were sensitive to ABA

75

• DWD Proteins
• Found T-DNA insertions
• 3 were sensitive to ABA
• ABI5 was elevated in dwa mutants

76

• DWD Proteins
• Found T-DNA insertions
• 3 were sensitive to ABA
• ABI5 was elevated in dwa mutants
• ABI5 was degraded more slowly in dwa extracts

77

• DWD Proteins
• Found T-DNA insertions
• 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

78
Regulating E3 ligases The COP9 signalosome (CSN),
a complex of 8 proteins, regulates E3 ligases by
removing Nedd8 from cullin
79
Regulating E3 ligases The COP9 signalosome (CSN),
a complex of 8 proteins, regulates E3 ligases by
removing Nedd8 from cullin CAND1 then blocks
cullin
80
Regulating E3 ligases The COP9 signalosome (CSN),
a complex of 8 proteins, regulates E3 ligases by
removing Nedd8 from cullin CAND1 then blocks
cullin Ubc12 replaces Nedd8
81
Regulating E3 ligases The COP9 signalosome (CSN),
a complex of 8 proteins, regulates E3 ligases by
removing Nedd8 from cullin CAND1 then blocks
cullin Ubc12 replaces Nedd8 Regulates DNA-damage
response, cell-cycle gene expression
82
Regulating E3 ligases The COP9 signalosome (CSN),
a complex of 8 proteins, regulates E3 ligases by
removing Nedd8 from cullin CAND1 then blocks
cullin Ubc12 replaces Nedd8 Regulates DNA-damage
response, cell-cycle gene expression Not all
E3 ligases associate with Cullins!
83

• 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

84

• COP1 is a non-cullin-associated E3 ligase
• Recent data indicates that COP1 may also
  associate with CUL4

85

• Protein degradation rate varies 100x
• Most have motifs marking them for
  polyubiquitination taken to proteosome
  destroyed
• Other 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

86

• Protein degradation rate varies 100x
• Other 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
• Deletion increases t1/2 10x, adding PEST drops
  t1/2 10x

87

• Protein degradation rate varies 100x
• Other 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
• Deletion increases t1/2 10x, adding PEST drops
  t1/2 10x
• Sometimes targets poly-Ub

88

• Protein degradation rate varies 100x
• Other 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
• Deletion increases t1/2 10x, adding PEST drops
  t1/2 10x
• Sometimes targets poly-Ub
• Recent yeast study doesnt support general role

89

• Protein degradation rate varies 100x
• Other 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
• Deletion increases t1/2 10x, adding PEST drops
  t1/2 10x
• Sometimes targets poly-Ub
• Recent yeast study doesnt 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.