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
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
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.)
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
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…