GenomesSizegenes (Nb)ORF (%) Bacteria E. Coli4,6428988 Archaebacteria P. abyssi1,76206591 Eucaryotes S. cerevisiae13640068 A. thaliana1252550029 C. elegans1001800027.

Genomes Size genes (Nb) ORF (%)

Bacteria E. Coli 4,6 4289 88

ArchaebacteriaP. abyssi 1,76 2065 91

EucaryotesS. cerevisiae 13 6400 68A. thaliana 125 25500 29C. elegans 100 18000 27D. melanogaster 180 13600 13H. sapiens 3000 24500 1,4

sRNA 1-2%

Intron encoded RNAs miRNA…

Thanks to in silico analysis and genome sequencing

How to generate diversity?Non coding regions increased with complexity

Function of novel RNAs Phylogeny, RNA structure

Essential functionCommon RNA structure

Activity- Structure

Multiple roles of RNA in gene regulation

Regulation of translation (and degradation) in prokaryotes

A rapid way to adapt the bacteria to the environment…

ribosomes

DNA

mRNA

ribosome

peptidemRNA

Transcription-translation-degradation coupling

Kinetic aspect (RNA folding, association of trans-acting ligands..)

Yusupov et al. 2001. Science 92:883

Initiation of translation in procaryotes : a key step for regulation

IF1IF3 fMet-tRNA

mRNA

IF2

30S/IF1/IF3

Dissociation ofsubunits

Codon-anticodoninteraction

1

2

From Laursen et al. 2005 Microbiol. Mol Biol Rev. 69:101

Assembly of the30S initiation complex(rate in the sec range)

Formation of thepre-initiation complex

mRNA binding

mRNA structure modulates the efficiency of translation

Transcriptiontermination- Riboswitch

- sRNA- Protein

Translation- Riboswitch- sRNA binding site- Protein binding site

Translational coupling

Degradation

Protein-mediated translational control

E. coli ThrRS E. coli S15

Feedback regulation

tRNA

Ribosome

ThrRS

Threonyl-tRNA synthetase regulates its own expression

Romby & Springer (2003) TIG 19:155

G

U

C

Sankaranarayanan et al. 1999. Cell 97: 371Torres-Larios et al. 2002 NSB 9: 343

Operator-ThrRS

Repression of translationDegradation of mRNA

ThrRS

30S

Translation

Ternary complex

Mimicry and translational control

Aminoacylation

tRNA-ThrRS

ARNm

ARNt

Romby & Springer (2003) TIG 19:155

The ribosome accomodates secondary structure upstream from the SD sequence of mRNA

Jenner et al. (2005) Science 308:120

Conserved operator in eubacteria suggests a conserved regulatory mechanism

Other tRNA-like structure in mRNA ?

Thrstarvation

B. Subtilis uncharged tRNA induced antitermination of transcription of the cognate aaRS gene

Putzer et al. 2002 NARes. 30:3026 and ref therein

Protein-mediated translational control

E. coli ThrRS E. coli S15

Feedback regulation

Site 2

Site 1

Nikulin et al. 2000 NSB 7:273Serganov et al. 2002 JMB 320:963

Ribosomal S15 protein entraps the 30S subunit into a dead-end initiation complex

GGGACGCUGA

UCCUGCGGCU

AGAGAUU

UCUCUAA

U

L1

L2

S2

S1

5'

3'

(Thr7, Thr4)

Thr21

Arg57

His41Ser51

Asp48

Gln27

Gly22

A

-30

-40

-57

+10

SD

Diversity of S15-dependent regulatory mechanisms

5'

3'

5'3'

5'

3'

H20

H21

H22

Site 2

Site 1

SD

SD

16S rRNAbinding site

E. coli rpsO Entrapment

T. thermophilus rpsO Competition

Serganov et al. 2002 JMB 320:963Serganov et al. 2003 EMBO J 22:1898Mathy et al. 2004 Mol. Microbiol. 52:661

sRNAs - Regulatory RNAs

An heterogeneous class of RNADiversity of regulatory mechanismsRegulate directly or indirectly multiple genes Coupling the structure and the regulatory activity

OxyS

Chromosomally encoded sRNAs - biological rolesChromosomally encoded sRNAs - biological rolesChromosomally encoded sRNAs - biological rolesChromosomally encoded sRNAs - biological roles

MicF

MicC

MicA

DsrA

RprA

RyhB

IstR

6S RNA

CsrBCsrC

GcvB

Spot42

sRNAs target(s) response/ biological roleompF mRNA

ompC mRNA

ompA mRNA

hns mRNA

rpoS mRNA

fhlA mRNA

encodes

membrane stress

membrane stress

membrane stress?

oxidative stresstranscriptional activator

thermoregulation

iron-storage proteins

SOS response

stationary phase survival

carbon metabolism, virulence

–

CsrA protein

70

tisAB mRNA

porin

porin

porin

transcriptional regulator

stress response S

oppA+dppA mRNA? peptide transportperiplasmic bind. proteins

ldrD mRNARdlD killing peptide

toxin

purine metabolism?

sodB mRNA etc

galK mRNA etc

ftsZ mRNADicF cell division protein cell division

gal operon enzyme sugar metabolism

Sigma factor

regulator

iron homeostasis

general stress+

–

–

–

–

–

–

–

–

–

–

?

–

+

GadY gadX mRNA transcriptional activatoracid stress+

(from Wagner EGH et al.)

How do regulatory RNAs regulate?

Wagner et al. (2002) Adv in Genetics 46, 361-396

One sRNA can regulatemultiple target--> pleiotropic effects

How to defineregulatory networks ?

Gottesman (2004) Annu Rev Microbiol 58:303

Helix BC3’5’5’3’

E3’5’3’5’()3’Helix BHelix B’Helix C5’3’5’

Inhibition of translation initiation

RNase III cleavage site

Atap SD5’CopACopT5’3’3’3’5’B5’3’

The binding pathway of the antisense RNA involves different steps in a hierarchical way

Kolb et al. 2000 RNA 6: 311. EMBO J 19: 5905Kolb et al. 2001 JMolBiol 309: 605. NAR 29: 3145

Homologous antisense and target RNA structure and sequences highlight similar topology for the resulting complexes

CCCCGaUAAUCUU-C UUCAAC UUUGGC GAGUACGAA -AAGAUUAcCGGGGCCCCGaUAAUCUU-C GUCAAG UUUGGC GACUGCGAA GAAGAUUAcCGGGGaCCCGaUAAUCUU-C AUCUAG UUUGGC GACGAGGA- GAAGAUUAcCGGGgCCCCGaUAAUCUU-C UCUAAC UUUGGC GAGUGCAGA -AAGAUUAcCGGGGCCCCGaUAAUCUU-C UUUAAC UUUGGC GAGUGCAGA -AAGAUUAUCGGGGCCCCGaUAAaCUU-C CUCAUC UUUGGC GAGGCGAG- -AACgUUAcCGGGGCCCCGaUAAUCUU-C UCAUUU CUUGGC GGGAACGA- -AAGAUUAaCGGGGCCCCGaUAAUCU--U UCAAUG UUUGGC GACGUAGA- --AGAUUAcCGGGGCCCCGaUAAUCUU-C UUCAAU CUUGGC GGAAGGAA- -AAGAUUAaCGGGGCCCCGaUAAUCUU-C UUUAAC UUUGGC GAGUACAGA AAAGAUUAcCGGGGCCCCGaUAAUCUU-C AGAAAG UUUGGC GACUGAG-- -AAGAUUAcCGGGG

CCCCACUAUCUUUCU UACGAA CUUGGC GGAACGAC- GAAAGAUAGUGGGGCCCCAcUAUUUUU-C CUCGAA CUUGGC GGAACGCA- GAAAAAUAaUGGGGCCCCAcUAUUUUU-C CUCGAA CUUGGC GGAACGCA- GAAA-AUAaUGGGGCCCCAUaAUCUUCCU GUCGAA CUUGGC GGAACGCAC AAAGAUaGUAGGGGCCCCACUAUCUUUCC UCGAAC UUUGGC GGGCUCGUG AAAGAUAGUAGGGGCCCCUGAUcCUAUUU CAGAAC UUUGGC CGGCUCGGA AUAGa-AUCAGGGG

R1/R100colV2-K94

P307pSU212pSU316

pYVE439-90/pCD1pFM82139pLV1402pGSH500

pB171pO157

(IncFII)(IncFI)(IncFIc)(IncFVI)(IncFIII)

col1b-P9pMU720pMU707pIE545

R387pMU407

(IncI1)(IncB)(IncB)(IncZ)(IncK)(IncL/M)

6-7 nts loop

Presence of bulged

nts

Stable helix

Sensory mRNAs - Riboswitches (alternative conformers...)

RepressionActivation

Narberhaus F. (2002) Arch. Microbio. 178, 404Mandal M. & Breaker R. (2003) Cell 113, 577Nudler E. & Mironov A. (2004) TBS 29, 11

Self-induced mRNA degradation

Translational control in

eukaryotes

more widespread than expected…

Formation of the Initiation complex -> Rate-limiting step

Global control:Initiation factor as target(Phosphorylation..)

Specific control:mRNA as target

Initiation of translation in eukaryotes

From Gebauer & Hentze (2004) Nature Rev Moll Cell Biol. 5:827

43S Pre-initiation complex

- cap structure and the polyA tails : canonical motifs- Secondary structures close to the 5’ end block translation initiation- IRES: ribosome entry site mediates cap-independant translation, shunt mechanism…-short ORF reduced translation of the main ORF - binding sites for trans-acting regulatory factors (protein, miRNA…)

Elements that influence translation of mRNA

mRNP-specific regulation of the initiation complex assembly

Steric blockage

Gebauer & Hentze (2004) Nature Rev Moll Cell Biol. 5:827

(iron metabolism)

Sequestration ofeIF-4E

(CPEB-Maskin : early development in xenopus Bicoid: anteroposterioraxis in drosophila)

Cap-independent inhibitionInterference with ribosomescanning

(SXL prevents X-chromosome dosage compensation in drosophila)

Eukaryotic post-transcriptional operons ?

Iron regulatory protein

One regulatory protein targets functionally related mRNAs (Keene 2003 Mol Cell 12: 1347)

FerritinTransferrin receptore-ALASMitochondrial aconitase…

Dandekar & Hentze (1995 TIG 11:45)

IRP and iron metabolism

Fragile X-syndrome Protein (associated in large mRNPs)

FMRP

AAAA

m7Gm7G mRNA

FMR1*PP2Ac (Rac1 pathway)**MAP1bFGF-2***?…

FMRP and neuronal function (actin cytoskeleton remodeling…)

*Schaeffer et al. 2001 EMBO J 20:4803**Castets et al. 2005 Hum Mol Gen. 14:835***Bonnal et al. 2003 JBC 278:39330Darnell et al. 2001 Cell 107:489; 2005 Genes Dev

Eukaryotic miRNAs regulate translation

RISC…

Target of miRNA? (20% of mRNA…)(complementarity, stability, structure…)How miRNA inhibits translation?Identify regulatory networks…

C. elegans lin-4

C. elegans let-7

C. elegans lsy-6

D. melanogaster bantam

Developmental timing (lin-14, lin-28)

Developmental timing (lin-14, lin-28)

Neuronal cell fate (cog-1)

Cell death (hld)

Ambros (2004) Nature 431:351

RISC

- ARN-dependent regulatory networks

Some remarks…

- Multiple roles of RNA in gene regulation : all steps of the mRNA are submitted to control (splicing, transport, localization, translation, degradation…)

- Characterization of mRNP, expression pattern of the small RNAs (miRNAsRNA…

- Search for structured elements (conserved or not..) in leader regions of mRNA (involved in function dependent of environmental cues, metabolism, riboswitches, target for miRNA, sRNA…)

- Annotation of the known regulatory elements in mRNA (riboswitches, translational operators)

- Regulation and kinetics : kinetic folding of the RNA…