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What was the biological question addressed? What major claims to the authors make? What are the major results that the authors obtained? Did the experiments presented justify the claims made? What major unanswered questions were raised by this work?

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What was the biological question addressed what major claims to the authors make

  • What was the biological question addressed?

  • What major claims to the authors make?

  • What are the major results that the authors obtained?

  • Did the experiments presented justify the claims made?

  • What major unanswered questions were raised by this work?

  • What experiments need to be done to answer such questions?


Background prokaryotic translation
Background: Prokaryotic translation

IF1=eIF1A IF2=eIF5B IF3=eIF1

Larson, B. et. al (2005) Microbiol Mol Biol Rev


Background hcv
Background: HCV

  • HCV is a flavivirus that contains an IRES in the 5’ UTR

  • HCV and HCV-like IRES only need a small sub set of initiation factors

Lukavsky, PJ (2008) Virus Research


Background hcv1
Background: HCV

  • The HCV IRES can bind to the 40S subunit

  • eIF3 and eIF2-GTP-Met-tRNAiMet are needed to form the 48S pre-initiation complex

Lukavsky, PJ (2008) Virus Research


Hcv after 48s complex formation
HCV after 48S complex formation

  • Would expect that:

    • eIF5 would hydrolyze GTP on eIF2-GTP-Met-tRNAiMet

    • eIF2-GDP would disassociate

    • 60S and 48S complex would release eIFs except eIF5B

    • GTP hydrolysis of eIF5B and 80S formation


80s complex can be formed on hcv ires without eif2
80S complex can be formed on HCV IRES without eIF2

  • In vitro assembly reactions

    • Add in 40S, 60S, eIF2, Met-tRNAiMet, and GTP or GMPPNP

  • Eliminating eIF5 still get 80S formation

  • Eliminating eIF5B no 80S complexes formed


80s complex can be formed on hcv ires without eif21
80S complex can be formed on HCV IRES without eIF2

  • In then presence of GMPPNP and all the factors still get 80S formation

  • Need eIF3, eIF5B and GTP or analog to get 80S formation

  • Hydrolysis of GTP is not required for 80S formation


Eif5b promotes trna binding to 40s eif3 hcv ires complex
eIF5B promotes tRNA binding to 40S-eIF3-HCV IRES complex

  • Two things could be going on

    • eIF5B is just involved in 80S complex formation

    • Like IF2, eIF5B could be involved in 80S complex formation and stabilizing Met-tRNAiMet

  • [S35]-Met-tRNAiMet,40S subunits, HCV IRES and eIF3

  • eIF5B is required to place the Met-tRNAiMet


Elongation with 80s complexes assembled without eif2
Elongation with 80S complexes assembled without eIF2

  • 80S subunits form, but are they functional?

  • In vitro translation elongation reconstituted using HCV ORF with a stop codon added in

  • Used toeprinting assay to identify pretermination complexes


Elongation with 80s complexes assembled without eif21
Elongation with 80S complexes assembled without eIF2

  • Pretermination complexes formed with eIF3 and eIF5B

  • eIF2 is not needed for elongation


Eif2 independent mechanism operates in cell free system
eIF2-independent mechanism operates in cell free system

  • eIF2-indpendent initiation is physiologically relevant

  • Using rabbit reticulocyte lysate with labeled HCV-IRES RNA

  • GMPPNP blocks eIF2 dependent pathway at 48S formation

  • dsRNA will activate PKR, which then phosphorylate eIF2

    • Depleting eIF2 from the system

U

T


Hcv ires driven translation in stressed transfected cells
HCV IRES-driven translation in stressed transfected cells

  • Cells were transfected with a cap-dependent mRNA and a monocistronic HCV IRES mRNA

  • eIF2-a phosphorylation was induced

  • HCV IRES-mediated translation is relatively resistant to eIF2 inactivation


Hcv ires driven translation in stressed transfected cells1
HCV IRES-driven translation in stressed transfected cells

  • HCV IRES-mediated translation is relatively resistant to eIF2 inactivation

  • Including IFNa, which is used as a treatment



Conclusion1
Conclusion

  • The HCV IRES needs eIF3, eIF5B, and GTP form 80S complex

  • The HCV IRES does not need GTP hydrolysis or eIF2

  • What does eIF3 do?


Background ash1 mrna
Background:ASH1 mRNA

  • ASH1 mRNA is localized to the bud tip during late anaphase

  • Translation of the mRNA is repressed until the mRNA is anchored at the bud tip

  • Puf6 and Khd1p are required for localization and translation repression

Paquin, N. & Chartand (2008) Trends in Cell Biology


Background khd1p repression
Background: Khd1p repression

  • Khd1p binds ASH1 mRNA and represses translation

  • ASH1 mRNA localizes to the bud tip

  • Khd1p gets phosphorylated by casein kinase I at the bud tip, and releases the mRNA

  • ASH1 mRNA then able to be translated

Paquin, N., et. al (2007) Cell


Background puf6
Background: Puf6

  • Puf6 binds a conserved UUGU sequence in the E3 domain of the ASH mRNA 3’UTR.

  • When bound Puf6 represses translation of the ASH1 mRNA

  • What is the mechanism of translational repression by Puf6?


Puf6 interfere with 80s assembly
Puf6 interfere with 80S assembly

  • The reporter: mRNA with Renilla luciferase and E3 localization element of ASH1 mRNA

  • The reporter R-luc-E3 mRNA incubated with Puf6 does not translate in vitro as well as R-luc alone.


Puf6 interfere with 80s assembly1
Puf6 interfere with 80S assembly

  • Sucrose gradients for in vitro translation assays done with E3 containing mRNA

    • With H2O get an 80S peak

    • GMPPNP blocks 60S joining, get increased 48S peak

    • E3 RNA with Puf6 increases 48S complex and decrease 80S


Puf6 interfere with 80s assembly2
Puf6 interfere with 80S assembly

  • Competition with cold E3 RNA and Puf6 recovers the 80S peak

  • In the presence of EDTA 48S complex was not detected with or without Puf6

  • Puf6 blocks 80S complex assembly, but not 48S formation.

  • 60S joining?


Fun12p associates with puf6 and is required for ash1 translation and localization
Fun12p associates with puf6 and is required for ASH1 translation and localization

  • Puf6 and Fun12 have been found to interact

  • Puf6 and Fun12 Co-IP together in an RNA dependent manner


Fun12p associates with puf6 and is required for ash1 translation and localization1
Fun12p associates with puf6 and is required for ASH1 translation and localization

  • In fun12 knockout strains:

    • Ash1p expression in down >80% compared to Pgk1p, and Nop1p

    • ASH1 mRNA decreased 50% compared to ACT1


Fun12p associates with puf6 and is required for ash1 translation and localization2
Fun12p associates with puf6 and is required for ASH1 translation and localization

  • ASH1 mRNA delocalizes in fun12 mutants

    • 62% in bud tip

    • 16% in neck

    • 12% in mother and bud


Both the n terminal region and puf domain of puf6p are required for translational repression
Both the N-terminal region and PUF domain of puf6p are required for translational repression

  • C536 (contain PUF domain)

    • Binds E3 RNA

    • Binds eIF5B

    • Represses both R-luc constructs

  • N120

    • Does not contain PUF domain

  • Need more than the PUF domain


Puf6p is phosphorylated by protein kinase ck2
Puf6p is phosphorylated by protein kinase CK2 required for translational repression

  • Puf6p is phosphorylated in yeast cells

  • lPPAse will remove the phosphates

  • N120 is phosphorylated when incubated with yeast extracts


Puf6p is phosphorylated by protein kinase ck21
Puf6p is phosphorylated by protein kinase CK2 required for translational repression

  • N120 is phosphorylated by casein kinase 2 (CK2) from sea stars

  • Phosphorylation occurs by ATP or GTP

  • Phosphorylation sites on N120 match previous predictions for puf6p phosphorylation


Puf6p is phosphorylated by protein kinase ck22
Puf6p is phosphorylated by protein kinase CK2 required for translational repression

  • Recombinant Puf6 was phosphorylated in yeast extracts and run on Mass spec

  • Two site identified Ser34 and Ser35

  • These sites

    • Are in the N120 region

    • match previous predictions for puf6p phosphorylation

  • Ser31 could also be a possible phosphorylation site based on previous research


Ck2 phosphorylation of puf6p relives translational repression
CK2 phosphorylation of Puf6p relives translational repression

  • Puf6 point mutations

    • Ser to Ala mutation in all three possible phosphorylation sites

  • Mutant puf6 phosphorylation is down ~90%

  • Mutant puf6 repressed translation better

  • Puf6 phosphorylation was decreased by DMAT


Ck2 phosphorylation of puf6p relives translational repression1
CK2 phosphorylation of Puf6p relives translational repression

  • Wild type Puf6 phosphorylation was decreased by DMAT

  • Translation of R-luc-E3 was reduced in the presence of DMAT


Ck2 phosphorylation of puf6p relives translational repression2
CK2 phosphorylation of Puf6p relives translational repression

  • RNA-binding:

    • When treated with lppase, Puf6p-TAP purified from yeast extracts retained E3-RNA

    • Puf6-TAP was not able to maintain mutation E3 RNA

    • His-Puf6 phosphorylated by CK2 bound E3 less efficiently

  • Puf6 phosphorylation by CK2 reduces RNA binding, which affects the repressing role.


Ck2 phosphorylation of puf6p is required for ash1 mrna localization and translation
CK2 phosphorylation of Puf6p is required for ASH1 mRNA localization and translation

  • Localization of Ash1 mRNA:

    • WT Puf6  bud tip

    • SApuf6  diffuse

    • Cka1  diffuse

    • Cka2 diffuse

  • CK2 phosphorylation of Pus6 required for localization


Ck2 phosphorylation of puf6p is required for ash1 mrna localization and translation1
CK2 phosphorylation of Puf6p is required for ASH1 mRNA localization and translation

  • CK2 colocalizes in the bud cortex with ASH1 mRNA

  • CK2 is detectable in the bud cortex before ASH1 mRNA expression began


Ck2 phosphorylation of puf6p is required for ash1 mrna localization and translation2
CK2 phosphorylation of Puf6p is required for ASH1 mRNA localization and translation

  • CK2 moves to the cortex prior to ASH1 mRNA localization

  • Puf6 is phosphorylated at the bud tip, which allows for localized translation


Conclusions
Conclusions localization and translation

  • Puf6 binds to the 3’ UTR of ASH1, and blocks translation, by blocking 80S complex formation

  • Deleting Fun12 decreases ASH1 mRNA expression

  • The binding of fun12 and mRNA to puf6 is not sufficient for translation repression

  • CK2 phosphorylation of puf6 is critical for puf6 repression

  • CK2 catalytic subunits localize to the bud tip prior to ASH1 mRNA localization