A quantitative view on mRNA translation: the relative role of initiation and elongation - Luca Ciandrini

A quantitative view on mRNA translation: the relative role of initiation and elongation

Luca Ciandrini ([email protected])University of Montpellier, France

13th June 2016Quantitative Laws II, Villa del Grumello, Como

Translation from nucleotide sequences to functional proteins

Study the ribosome traffic to quantify the protein production rate (at the level of translation)

ribosomes

growing polypeptide

mRNA

5’

3’

coding sequence

ribosomeSTART

elongatingprotein

initiation(recruitment)

tRNAamino-acid

elongation (usage)

termination

STOP


elongation (usage)

termination

coding sequence

ribosomeSTART

elongatingprotein


tRNAamino-acid

elongation (usage)

termination

STOP


elongation (usage)

termination

How can we predict/vary the strength of initiation?

Ribosome recruitment Ribosome usage

How depletion of ribosomes affects gene expression and growth rates?

Codon usage and bias? Evolution? Does noise depend

on codon sequences, etc etc?

Can we tune and predict, just by varying the sequence, “optimal” protein production rates?P. Geulich, L. Ciandrini, R.J. Allen and M. C. Romano, Phys Rev E (2012)

Shah et al, Cell (2013)

Flow of ribosomes ~ driven lattice gas

Macdonald et al., Biopolymers (1968)Totally Asymmetric Simple Exclusion Process

coding sequence

ribosomeSTART

elongatingprotein


tRNAamino-acid

elongation (usage)

termination

STOP


elongation (usage)

termination

The model can predict translation efficiency depending on initiation and elongation rates

ribosomal current (mRNA efficiency)

initiation rate codon dependent elongation rates

termination rate

L. Ciandrini, I. Stansfield and M. C. Romano, PLoS Comp Bio (2013)

initia

tion

lim

ited elongation

limited

The model can predict ribosome density depending on initiation and elongation rates

ribosomal density

initiation rate codon dependent elongation rates

termination rate


initia

tion

lim

ited ribosomes

queueing

1. How initiation affects mRNA efficiency

L. Dias Fernandes, A. de Moura, L. Ciandrini, to be submitted J.C. Walter, L. Ciandrini, in preparation

Observation: the density of ribosomes depends on the length of the mRNA

data (yeast) from MacKay et al. (2004)

The local concentration of ribosomes determines translation initiation

↵ = ↵o

c = ↵o

(c1 + cR

)

initiation rate

overall ribosome concentration

contribution due to terminating ribosomes

(feedback)

Initiation depends on local concentrations of ribosomes

R

r

Contribution due to terminating ribosomes depends on the end-to-end distance and the production rate

↵ = ↵o

c = ↵o

(c1 + cR

)

initiation rate

R

r

The probability to end up in the reaction volume is ∝ r/R

J

current of the driven lattice gas

end-to-end distanceis a constant depending on r, the affinity mRNA-ribosome,…

The mRNA persistence length depends on the ribosomal density

R ⇠p

`pL R ⇠q`polyp L

`p= persistence length `polyp = f`+ (1� f)`p

f = fraction of transcript occupied by ribosomes

The initiation rate α is therefore length-dependent (via “recycling” and finite-size effects)

• Fix a tentative initiation rate

• Simulate the system and extract J and R

• Compute the initiation rate

• Compare to the tentative oneJ

We develop a self-consistent method to compute the initiation rate

initiation rate:

L. Dias Fernandes, A. De Moura, L. Ciandrini, to be submitted

finite size + local concentration

The theory is consistent with the ribosome density data

initiation rate:

The theory is consistent with the ribosome density data

finite size + local concentration

initiation rate:

L. Dias Fernandes, A. De Moura, L. Ciandrini, to be submitted

Short transcripts are less sensitive to changes in the ribosomal pool

decreasing concentration of available ribosomes

Short transcripts are less sensitive to changes in the ribosomal pool

mRNA efficiency short transcript/ mRNA efficiency long transcript

2. How elongation affects mRNA efficiency

AJ Kemp, R Betney, LC, ACM Schwenger, MC Romano,I Stansfield, Molecular Microbiology (2013) B Gorgoni, L. Ciandrini, M Mc Farland, M C Romano, I Stansfield, submitted Y. Meriguet, S. Guiziou, J. Bonnet, L. Ciandrini, in preparation

sup70-65 mutants exhibits slow translation of the cognate CAG codon

AJ Kemp, R Betney, L Ciandrini, ACM Schwenger, MC Romano,I Stansfield, Molecular Microbiology (2013)

How do we affect translation if we insert CAA and CAG repeats at the 5’end?

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

SUP70wild-type

multicopytRNACUG

sup70-65

Fold

-cha

nge

inlu

cife

rase

expr

essi

on

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

Mod

elpr

edic

tion:

fold

-cha

nge

inlu

cife

rase

expr

essi

on

B

C

A

luc

luc

luc

luc

5 x CAA

10 x CAA

5 x CAG

10 x CAG

*** *********

*** *********

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

0.0 0.5 1.0 1.5 2.0 6 9 12 150.00

0.02

0.04

0.06

0.08

0.10

Fold concentration of tRNACUGGln

relative to wild type

luci

fera

seex

pres

sion

rate

10 20 30 40 500.00

0.05

0.10

0.15

codon number

rib

oso

ma

lde

nsi

ty

0 2 4 6 8 100.020

0.040

0.060

0.080

Number of CAG codons in tandem repeats

Luci

fera

seex

pres

sion

rate

D

F

E

Slow CAG translation in yeast pseudohyphal growth mutants 293

© 2012 Blackwell Publishing Ltd, Molecular Microbiology, 87, 284–300

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

SUP70wild-type

multicopytRNACUG

sup70-65

Fold

-cha

nge

inlu

cife

rase

expr

essi

on

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

Mod

elpr

edic

tion:

fold

-cha

nge

inlu

cife

rase

expr

essi

on

B

C

A

luc

luc

luc

luc

5 x CAA

10 x CAA

5 x CAG

10 x CAG

*** *********

*** *********

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

0.0 0.5 1.0 1.5 2.0 6 9 12 150.00

0.02

0.04

0.06

0.08

0.10



luci

fera

seex

pres

sion

rate

10 20 30 40 500.00

0.05

0.10

0.15

codon number

ribo

som

ald

en

sity

0 2 4 6 8 100.020

0.040

0.060

0.080


Luci

fera

seex

pres

sion

rate

D

F

E



5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

SUP70wild-type

multicopytRNACUG

sup70-65

Fold

-cha

nge

inlu

cife

rase

expr

essi

on

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

Mod

elpr

edic

tion:

fold

-cha

nge

inlu

cife

rase

expr

essi

on

B

C

A

luc

luc

luc

luc

5 x CAA

10 x CAA

5 x CAG

10 x CAG

*** *********

*** *********

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

0.0 0.5 1.0 1.5 2.0 6 9 12 150.00

0.02

0.04

0.06

0.08

0.10



luci

fera

seex

pres

sion

rate

10 20 30 40 500.00

0.05

0.10

0.15

codon number

ribo

som

ald

en

sity

0 2 4 6 8 100.020

0.040

0.060

0.080


Luci

fera

seex

pres

sion

rate

D

F

E



5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

SUP70wild-type

multicopytRNACUG

sup70-65

Fold

-cha

nge

inlu

cife

rase

expr

essi

on5 x

CAA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

Mod

elpr

edic

tion:

fold

-cha

nge

inlu

cife

rase

expr

essi

on

B

C

A

luc

luc

luc

luc

5 x CAA

10 x CAA

5 x CAG

10 x CAG

*** *********

*** *********

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

0.0 0.5 1.0 1.5 2.0 6 9 12 150.00

0.02

0.04

0.06

0.08

0.10



luci

fera

seex

pres

sion

rate

10 20 30 40 500.00

0.05

0.10

0.15

codon number

rib

oso

ma

lde

nsi

ty

0 2 4 6 8 100.020

0.040

0.060

0.080


Luci

fera

seex

pres

sion

rate

D

F

E



5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

SUP70wild-type

multicopytRNACUG

sup70-65

Fold

-cha

nge

inlu

cife

rase

expr

essi

on

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

Mod

elpr

edic

tion:

fold

-cha

nge

inlu

cife

rase

expr

essi

on

B

C

A

luc

luc

luc

luc

5 x CAA

10 x CAA

5 x CAG

10 x CAG

*** *********

*** *********

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

0.0 0.5 1.0 1.5 2.0 6 9 12 150.00

0.02

0.04

0.06

0.08

0.10



luci

fera

seex

pres

sion

rate

10 20 30 40 500.00

0.05

0.10

0.15

codon number

rib

oso

ma

lde

nsi

ty

0 2 4 6 8 100.020

0.040

0.060

0.080


Luci

fera

seex

pres

sion

rate

D

F

E



modelexperiments

sup70-65 mutants exhibits slow translation of the cognate CAG codon

The method: we simulated the translation in WT and sup70-65 mutant to perform an in silico genetic screen

sensitiv

e genesins

ensitiv

e gene

s

The predicted targets of tRNAGlnCUG regulation are confirmed by experiments

54% relative to WT control 103% relative to WT control

predicted to be sensitive predicted to be insensitive

We used constitutively active mutants of the Gcn2 protein kinase to decrease the initiation

Gcn2 reduces (globally) the translation initiation rate via the phosphorylation of the essential translation initiation factor eIF2-αWe tested our hypothesis on a gene (FAR7)

Also, we observed a significant (almost 2-fold) shift in the population away from chains and towards single cells

initiation rate

mut

ant /

WT

trans

latio

n ra

te

model

Conclusions

• Initiation might be the origin of the ribosome density/

mRNA length dependence

• Elongation can be used to regulate gene expression

through the tuning of single-codon elongation rates

• Both initiation and elongation can be limiting steps

of translation

Further perspectives

• How to independently study and modulate initiation and elongation?

…undergoing experiments…

Acknowledgements

Thank you

Ian StansfieldBarbara GorgoniIMS, University of Aberdeen

Jean-Charles WalterL2C, University of Montpellier

soutien UM Défi InPhyNiTi (Interfaces physiques Numérique et Théorique)

Sarah GuiziouJerome BonnetCBS, University of Montpellier

Yoann MeriguetDIMNP, University of Montpellier

Lucas Dias FernandesAlessandro de MouraMaria C RomanoICSMB, University of Aberdeen

The relative positioning of codons (elongation) affects translational efficiency

Shuffling synonymous codons does not affect the final product (aa-sequence) and keeps the same codon usage

(codon used in a sequence).

original

synonymous



We are going to design sequences with high, medium and small translation efficiencies and test them

The limited amount of resources constrains translation efficiency*

*Translation efficiency = overall rate of translation per mRNAScott et al., Science 2010

There is evidence that the amount of ribosomes restrains translation efficiency (see works by Scott, Klumpp, Hwa; Shah et al. from Plotkin-Kudla groups).

Competition for translational resources affects translation

The limited amount of resources constrains translation efficiency*

*Translation efficiency = overall rate of translation per mRNAScott et al., Science 2010

There is evidence that the amount of ribosomes restrains translation efficiency (see works by Scott, Klumpp, Hwa; Shah et al. from Plotkin-Kudla groups).

Competition for translational resources affects translation

Can competition for resources be exploited to regulate gene expression?

Conclusions

• Finite-size effects are relevant in translation

• The three-dimensional conformation of the polysome might enhance initiation (ribosome

“recycling”)

• The selfish mRNA: is recycling a way to build up a

personal pool of ribosomes?

2. Use strep-tag at the 5‘end to detect the

queue

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

SUP70wild-type

multicopytRNACUG

sup70-65

Fold

-cha

nge

inlu

cife

rase

expr

essi

on

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

Mod

elpr

edic

tion:

fold

-cha

nge

inlu

cife

rase

expr

essi

on

B

C

A

luc

luc

luc

luc

5 x CAA

10 x CAA

5 x CAG

10 x CAG

*** *********

*** *********

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

0.0 0.5 1.0 1.5 2.0 6 9 12 150.00

0.02

0.04

0.06

0.08

0.10



luci

fera

seex

pres

sion

rate

10 20 30 40 500.00

0.05

0.10

0.15

codon number

ribo

som

ald

en

sity

0 2 4 6 8 100.020

0.040

0.060

0.080


Luci

fera

seex

pres

sion

rate

D

F

E



5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

SUP70wild-type

multicopytRNACUG

sup70-65

Fold

-cha

nge

inlu

cife

rase

expr

essi

on

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

Mod

elpr

edic

tion:

fold

-cha

nge

inlu

cife

rase

expr

essi

on

B

C

A

luc

luc

luc

luc

5 x CAA

10 x CAA

5 x CAG

10 x CAG

*** *********

*** *********

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

0.0 0.5 1.0 1.5 2.0 6 9 12 150.00

0.02

0.04

0.06

0.08

0.10



luci

fera

seex

pres

sion

rate

10 20 30 40 500.00

0.05

0.10

0.15

codon number

ribo

som

ald

en

sity

0 2 4 6 8 100.020

0.040

0.060

0.080


Luci

fera

seex

pres

sion

rate

D

F

E



5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

SUP70wild-type

multicopytRNACUG

sup70-65

Fold

-cha

nge

inlu

cife

rase

expr

essi

on5 x

CAA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

Mod

elpr

edic

tion:

fold

-cha

nge

inlu

cife

rase

expr

essi

on

B

C

A

luc

luc

luc

luc

5 x CAA

10 x CAA

5 x CAG

10 x CAG

*** *********

*** *********

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

0.0 0.5 1.0 1.5 2.0 6 9 12 150.00

0.02

0.04

0.06

0.08

0.10



luci

fera

seex

pres

sion

rate

10 20 30 40 500.00

0.05

0.10

0.15

codon number

rib

oso

ma

lde

nsi

ty

0 2 4 6 8 100.020

0.040

0.060

0.080


Luci

fera

seex

pres

sion

rate

D

F

E



5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

SUP70wild-type

multicopytRNACUG

sup70-65

Fold

-cha

nge

inlu

cife

rase

expr

essi

on

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

Mod

elpr

edic

tion:

fold

-cha

nge

inlu

cife

rase

expr

essi

on

B

C

A

luc

luc

luc

luc

5 x CAA

10 x CAA

5 x CAG

10 x CAG

*** *********

*** *********

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

0.0 0.5 1.0 1.5 2.0 6 9 12 150.00

0.02

0.04

0.06

0.08

0.10



luci

fera

seex

pres

sion

rate

10 20 30 40 500.00

0.05

0.10

0.15

codon number

rib

oso

ma

lde

nsi

ty

0 2 4 6 8 100.020

0.040

0.060

0.080


Luci

fera

seex

pres

sion

rate

D

F

E



A.J. Kemp, R. Betney, L. Ciandrini, A.C.M. Schwenger, M.C. Romano, I. Stansfield, Molecular Microbiology (2013)

Good agreement with theory

2. Use strep-tag at the 5‘end to detect the

queueCAG causes ribosomes

queue upstream the

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

SUP70wild-type

multicopytRNACUG

sup70-65

Fold

-cha

nge

inlu

cife

rase

expr

essi

on

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG

5 x C

AA

10 x

CAA

5 x C

AG

10 x

CAG0.0

0.2

0.4

0.6

0.8

1.0

1.2

Mod

elpr

edic

tion:

fold

-cha

nge

inlu

cife

rase

expr

essi

on

B

C

A

luc

luc

luc

luc

5 x CAA

10 x CAA

5 x CAG

10 x CAG

*** *********

*** *********

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

pSUP70 (CEN)

pSUP70-[2µ]

psup70-65 (CEN)

0.0 0.5 1.0 1.5 2.0 6 9 12 150.00

0.02

0.04

0.06

0.08

0.10



luci

fera

seex

pres

sion

rate

10 20 30 40 500.00

0.05

0.10

0.15

codon number

rib

oso

ma

lde

nsi

ty

0 2 4 6 8 100.020

0.040

0.060

0.080


Luci

fera

seex

pres

sion

rate

D

F

E



CAG repeats

CAA repeats

mRNA-dependent estimates of the initiation rates

density from experiments

initiation rates

Integration between model and experimental data

=

=

1 site = 1 codon

mRNA 1D-lattice

ribosomeparticle

initiation elongationtermination

α βγ

γ

mRNA-dependent estimates of the initiation rates

Important to discern the interplay between initiation and elongation

Gene Ontology: (i) regulatory proteins

(ii) cytoplasmic translation (iii) constituents of

ribosomes (iv) respiratory chains -

unannotated


�: mRNA-dependent estimates of the initiation ratesProtein production rates
