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Coupling between transcriptional and
post-transcriptional regulations.
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Two alternative translation initiation mechanisms
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IRESes Viral and tightly controlled cellular
genes mRNAs
Cellular mRNAs
http//ifr31w3.toulouse.inserm.fr/iresdatabase/
Vagner et al., EMBO reports, 2001
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IRESes Regulations ?
Do IRESes have cell type specificities ?
Are there cell type specific or limiting IRES
trans-acting factors (ITAFs)? ( Involvement of
transcriptional level and mRNA accumulation )
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IRES identification strategy  Lucky Luke 
bicistronic vector
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Comparison of IRES activities in different
cell-type
LucF/LucR
Transient transfection with CMV promoter
IRES activities are cell type specific
Involvement of particular Proteins or RNPs ?
Weak Activities of cellular IRESes due to large
amount of RNA ?
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Comparison of IRES activities in transient and in
permanent transfection
Important decrease of EMCV IRES, but not of
cellular IRESs activities,
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Effect of mRNA expression level on IRES activities
Comparison of IRES activities with two distinct
promoters
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Effect of mRNA expression level on IRES
activities CMV or PGK-1 Promoter
LucF LucR
Hek 293 Cells
PGK1 promoter Decrease of EMCV IRES activity
Activation of FGF-2, VEGF, PDGF and Mulv IRES
activities
No effect of mRNA decrase on Bip and Myc IRES
activities
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Effect of mRNA expression level on IRES
activities CMV / PGK-1 Promoter
PGK1 promoter decrease of EMCV IRES activity in
all cell types
Cell-type dependent activation of c-myc and FGF-2
IRESs
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Effect of mRNA expression level on IRES
activities CMV / PGK-1 Promoter
LucF LucR
Low efficiencies of all IRESs with CMV Promoter
All IRESs are activated with PGK-1 Promoter,
except for EMCV IRES
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Effect of mRNA expression level on IRES
activities inducible bicistronic vector
Transient transfection assay, in MEF-3T3
cell-line expressing tetracycline transactivator
(tTA)
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Effect of mRNA accumulation inducible
bicistronic vector
Myc and FGF-2 IRES activities are independent of
transcription level
EMCV IRES activity is dependent on the
transcription level
ITAF able to inhibit EMCV IRES at low
transcription level?
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Conclusion Cellular IRESs activities are
influenced by the promoter In a cell type
specific manner.
Hypotheses

• Cell-type specific limiting ITAF?

• Specific ITAF whose activity would be
• coupled to a transcriptional event?

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Cell-type specific limiting ITAF or coupling to a
transcriptional event ?
CHO cells
LucF/LucR
Limiting ITAF hypothesis increase of FGF-2 IRES
RNA level in cells should down-regulate IRES
activity.
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Cellular IRES activities are coupled to a
transcriptional event
LucF/LucR
CHO cells
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INSERM U397, Endocrinologie et Communication
Cellulaire Directeur Francis Bayard Institut
Fédératif de Recherche Louis Bugnard, CHU
Rangueil, Toulouse, France.
Anne-Catherine Prats Hervé Prats, Yvan
Martineau, Laurent Monbrun, Camille
Allera, Caroline Castano, Laurent Creancier,
Christine Le Bec, Olivier Danos
Genethon III , Groupe de Vectorologie EVRY, FRANCE