sRNAs : small non coding RNAs

- Regulate genes involved in fast adaptative processes (environment, stress-related response)- Diverse in size- Structure of sRNA and the targets essential for efficient regulation- Act as activator or repressor - Regulate either at the transcriptional or at the post-transcriptional levels

- Sequestration of regulatory proteins- through base pairings with mRNA : antisense RNAs

sRNA gene in inverted orientation of the target --> full complementaritywith the target (plasmid, transposon, bacteriophage)

DNAmRNA

Antisense RNA

sRNA gene different from the target gene --> partial complementarity(adaptation to stress…)

DNAmRNA

sRNAGuillier et al. 2006 Genes &Dev 20:2338Romby et al. 2006 Curr Opin Microbiol. 9:229Wagner et al. 2002 Adv Genet 46:361

Rom

Antisense RNAs in extra-chromosomal elements

Control essential functions of Plasmids : replication, conjugation, post-segregational killing Transposon : transpositionBacteriophages: lysis/lysogenyWagner et al. 2002 Adv Genet 46:361

CopT

tap repA+

Translational coupling--> synthesis of RepA

SD

-

tap repACopT

CopA

CopA inhibits tap translation --> indirect inhibitionof RepA synthesis

Antisense RNA regulation of plasmid R1 replication

Blomberg et al. 1994 MolMicrobiol 12:49Malmgren et al 2006 RNA 2:1022

A multistep binding pathway

Prevents ribosomeBinding at tap RBS

RNase III cleavage

Kolb et al. 2000 EMBO 19:5905Kolb et al. 2001 Nucl Acids Res 29:3145

Antisense RNA regulate plasmid maintenance

Frank & Gerdes (1996) Mol Microbiol 21:1049-1060

sRNA encoded by the bacterial chromosome

- Discovered in the early 1970s by direct labeling of total cellularRNAs (RNase P, tmRNA, 6S RNA, Spot 42…)

- 1990’s: sRNAs (small non coding RNAs) from chromosomes(Functional screens)

- 2001+: Systematic screens find >70 sRNAs in E. coli Argaman et al. 2001 Curr Biol 11: 941 (14)

Wassarman et al. 2001 Genes & Dev 15: 1637 (18)Rivas et al. 2001 Curr Biol 11: 1369 (11)Vogel & Wagner 2005 Handbook of RNA biochemistry 595-642

autonomoussRNA geneWhat’s an sRNA?

Protein coding region5’ UTR 3’ UTR

?Attenuation,Processing. Ribosome cleavage

at STOP codon.

Are they regulators (RNA, protein targets)?Are they sensed in the cell ?

Mattick & Gagen (2001) The evolution of controlled multitasked gene networks:the role of introns and other noncoding RNAs in the development of complex organisms. Mol Biol Evol

mRNA

Small RNA

Overview of the sRNA screening approaches

Direct labeling and cloning- For abundant sRNA- Do not distinguish between primary transcripts and processed sRNAs- Detection of species-specific sRNAs

Functional screens (genetic screens)- give immediate clues about functional involvement- difficult if sRNA essential, or if the sRNA is acting under special conditions

Biocomputational screens- Rapid but generates many candidates to be tested- fruitful if phylogenetic comparisons are available

Microarrays- Transcriptional profiles for all genes (expensive)- fast profiling of conditions dependent sRNA expression patterns- detection of species-specific sRNAs- Not always consistent with northern analysis

Shotgun cloning & protein coimmunoprecipitation- Detection of processed and primary transcripts- Detection of species-specific sRNAs- Sequencing (technology 454- Margulies et al. 2005 Nature 437:376)

Vogel & Wagner 2005 Handbook of RNA biochemistry 595-642

Biocomputational approachintegrated features of ten known E.coli small RNAs (Argaman et al. 2001 Current Biology)

Located in “empty ” intergenic regions (IGRs).

sRNA

• Genomic arrangement

UUUU

3-8 nt

5-10 bp>60% GC

>4 U-residues

Rho-independent terminatorsσ 70-promoters

+1TTGACA

-35 box -10 box

15-19 nt spacer

• Prediction of transcription signals in IGRs

• Conservation in IGRs

BLASTN: E.coli vs. Salmonella, Yersinia, Klebsiella.

TATAAT

sRNA gene

E.c.Shig.

SalmonellaYersinia.

Intergenicconservation

Promoter &Terminator

RNAstructure

Microarraydetection

+ - -

Hershberg 2003 NAR 55 sRNA loci and ~ 1000 candidates

Argaman 2001 Current Biology

Wassarman 2001 Genes & Dev

Rivas 2001 Current Biology

Carter 2001 NAR

Chen 2002 Biosystems

Tjaden 2002 NAR

RNomics

- -+ - - - -

++ +- -+- -+- -+- -- - - -+-- - - -+-

Vogel 2003 NAR

Zhang 2003 Mol Micro

Kawano 2004 NAR

- - - -+- -Hfq- -- -+- -

-

-

Systematic screens for new sRNAs in Escherichia coli since 2001

E. coli, Salmonella, Yersinia, ... : ~ 4.5 Mb genome, ~ 4500 genes, 200–300 sRNAs (or 5 %)

Argaman et al. 2001 Current Biology

RyhB

CsrC

MicA

Many bacterial sRNA genes

are highly growth- and

stress-regulated

(and many are upregulated in

stationary phase).

sRNA stability

What is the signature of a sRNA?

sRNA often referred to asstable RNAs (20-60 min half-life).

sroB

sroC

copA control

gcvB

sraJ

0’ 1’ 2’ 4’ 8’ 16’ 32’

20 4 6 8

10

12

14

16

18

20

22

24

26

28

30

32

sraD

sraC

gcvB

sraJ

sroG

sroA

copA

sroH

csrC

sroF

sraH

sroE

sroC

sroB

rygB

sraE

sroD

rybB

QUAD

Approximate half-life:

exponential,stationary phase

Half-lives of 18 new sRNAs.

De J. Vogel

Argaman et al. 2001 Current Biology

Small noncoding RNAs (sRNAs) in Escherichia coli

0 50 100 150 200 250 300 350 400 nt

tmRNA

4.5 S

RydC CsrB

Spot 42

MicF

OxyS

DsrA6 S

MicA

MicC IstR2

IstR1SgrS

RyhB

M1

CsrC

RprA

GcvB

InvR

RseX

OmrAB

J. Vogel 2005 Biol Chem 386:1219

Ribosomerescue

SRP RNARNase P

tRNA 5’ ends

0 50 100 150 200 250 300 350 400 nt

tmRNA

Functional diversity of E. coli sRNAs

Specialized housekeeping RNAs.

4.5 S

tmRNA

4.5 S

M1

J. Vogel 2005 Biol Chem 386:1219

Regulates RNApolymerase.

0 50 100 150 200 250 300 350 400 nt

Antagonists of globalregulatory protein, CsrA.

Functional diversity of E. coli sRNAs

Specialized housekeeping RNAs.Regulators of protein activity (RNA – protein interactions).

4.5 S

M1

tmRNA

CsrB6 SCsrC

J. Vogel 2005 Biol Chem 386:1219

CsrA « Carbon storage regulator »

mRNA targets

CsrA, a global regulator whichinhibits glucogenesis, glycogen biosynthesis and catabolism,biofilm formationactivates glycolysis, acetate metabolism

Romeo 1998 Mol. Microbiol. 29:1321Guttierrez et al. 2005 Jbact 187: 3496

CsrB inhibits the action of a global regulator CsrA in enterobacteria

Oxidative stress

Modulatorsugar operons

0 50 100 150 200 250 300 350 400 nt

4.5 S

Spot 42

OxyS

RyhB

Ironhomeostasis

Phosposhugarstress

Functional diversity of E. coli sRNAs

Specialized housekeeping RNAs.Regulators of protein activity (RNA – protein interactions).sRNAs that target mRNAs (RNA – RNA interactions).

Acid stress,cold shock

Cellsurfacestress

SOS response

Periplasmicproteins

4.5 S

M1

tmRNA

CsrB6 SCsrC

RydC

Spot 42

OxyS

DsrAMicC IstR2

IstR1SgrS

RyhB

RprA

GcvB

OmrAB

J. Vogel 2005 Biol Chem 386:1219

0 50 100 150 200 250 300 350 400 nt

4.5 S

Spot 42

OxyS

Functional diversity of E. coli sRNAs

Specialized housekeeping RNAs.Regulators of protein activity.sRNAs that target mRNAs (RNA – RNA interactions).

… that regulate outer membrane proteins.

OmpD

4.5 S

Spot 42

OxyS

RyhB

4.5 S

M1

tmRNA

CsrB6 SCsrC

RydC

Spot 42

MicF

OxyS

DsrA

MicA

MicC IstR2

IstR1SgrS

RyhB

RprA

GcvB

InvR

RseX

OmrABOmpA

OmpT

OmpF

OmpC

Guillier et al. 2006 Genes & Dev

MicA

luxSAI-2 quorum sensing

micA

Regulates ompA mRNA translation in stationary phase.Udekwu et al. 2005 Genes & Dev 19:2935

log stat

gshA

70 nt

Model for micA/ompA system (enterobacteria)

Hfq

Udekwu et al. 2005 Genes & Dev 19:2935

Contrôle du métabolisme du fer (E. coli)

Le fer est essentiel au métabolisme cellulaireUn excès de fer peut conduire à la formation de radicaux libres (oxydation Fe2+)--> dommages cellulaires --> essentiel de contrôler la concentration intracellulaire du fer

Carence en Fe2+ Fur RyhB sodB (superoxyde dismutase)

bfr (bacterioferritine)ftn (ferritine)…

RyhB

Protéine HfqSm-like

SD

RNase E

Dégradation par des exoribonucléases(pnpase, RNase II) de 3’ vers 5’

1- Blocage du site de reconnaissancedu ribosome2- La protéine Hfq associée à l’ARN RyhBguide la RNase E pour initier la dégradationde l’ARNm

Coupure initiale

sodB

RepressionActivation

Masse et al. 2003 Genes & Dev 17:2374Masse et al. 2005 J Bact 187: 6962Geissmann et al. 2004 EMBO J 23:396

Regulation in LstR/ tisAB system in E. coli SOS response-DNA damage

Vogel et al. 2004 Curr Biol.14:2471

DsrA

Stationary phase-stressSigma38 factor

sRNA dependent regulatory pathways

Repoila et al. 2003 Mol Microbiol 48:855Romby et al. 2006 Curr. Opin Microbiol 9:229

Iron limitation

Fur

RhyB

Target mRNAs(iron bindingProteins)

1 sRNA target multiple mRNAs1 mRNA can be regulated by several sRNAs

Integration of many signals

sRNA control of quorum sensing in vibrio fisheri

Bassler et al. 2006 Cell 125:237

Quorum sensing induced sRNAs regulate virulence in v. cholerae

Lenz et al. 2004 Cell 118:69Bassler et al 2006 Cell 125:237

- Numerous adaptive/accessory genes22 exoenzymes, 40 toxines, 20 Adhesins> 40 other putative virulence factors

- Regulation of virulence genes involves a complex interplaybetween several two-component systems, transcriptionalfactors, and regulatory RNA…

- Major cause of nosocomial infections; antibiotic multiresistance- Foodborne pathogenic bacteria

Regulation of virulence genes in Staphylococcus aureus

ExoproteinsHemolysins..

-

AIP

AgrDp AgrA

p

ARNII

AgrAResponseregulator

Cytoplasmic membrane

AgrBreceptor

COOHNH2

AgrC

H

hld

Two-componentsystem

P2P3

agrCagrA D agrB

Quorum sensingcassette

ARNIII

Adhesins(Protein A..)

Novick, 2003 Mol Microbio 48:1429

Quorum-sensing dependent regulation and RNAIII

Activation

Repression

Hld translation

A A multifunctional multifunctional RNARNA

Morfeldt et al. 1995 EMBO J 14, 4569-4577Novick et al. 1993 EMBO J 12:3967Huntzinger et al. 2005 EMBO J 24:824

RNAs and virulence

- Novel sRNAs in S. aureus, L. monocytogenes, S. pyogenesPichon & Felden 2005 PNAS 102:14249 (S. aureus)Christiansen et al. 2006 RNA 12:1383 (L. monocytogenes)Livny et al. 2005 NARes 34: 3486 (Streptococcus, Pseudomonas..)

- How to find their targets ? Helper proteins (Hfq…) ?

- Relation host-pathogen

- Are bacterial sRNAs expressed within the host?- sRNA in defense mechanism against the host?- RNAi-like mechanism in bacteria? Makarova et al. 2006 Bioldirect 1:7

- Other type of sRNAs ?

functionally related enzymes Functionally related sRNAtargeting operon

A B C D E