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The genomic revolution of non-coding RNAs  Class 4 part 2 Bacterial small RNAs ( sRNAs )

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Large scale experimental support  and finding the direction of regulation

RyhB  - an sRNA induced  under iron limitation dmsA yncE hybO hemN ? ? ? ?

Type of regulation – repression or activation?

Get support to targets by RNA- seq  experiments

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Comparative RNA- seq WT versus sRNA deletion WT versus sRNA over-expression

Log 2 FC    -4.3                                                                                                         6.8

Effect only on translation False positives Other, yet unknown,        regulation mechanisms

Example : Change in target expression under upregulation of the sRNA  RyhB , an sRNA induced under iron limitation

Log 2 FC: log 2  fold change in expression  levels between  the two strains

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Testing effect on translation by ribosome profiling

Ceti type
Of interest
'n vivo capture Of translati
a Ribosorne profiting
Nuclease treatment
footprints
ribosornes and rnRNAs- I sis
b rnRNA—seq
Libr
en era tion
Deep seqtænci
region
Stop
Genornic position
UTR
Random f
neration
Deep sequencin
rnapping
posit ion
5' trÄtsc:ript end
indicates
rnentation
indicates
Stop

Brar  and  Weissman  (2015) Nat. Rev. Mol. Cell Biol .

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Change in ribosome profiling under over-expression of RyhB Log2FC RNA- seq         -4.3                                                                                                         6.8

Wang et al., (2015)  Nuc . Acids Res. 43:  10308–10320

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Supporting the targets by small-scale experiments

Proving the base pairing by compensatory mutations Supporting the target by reporter assays

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Plasmid cloning of target gfp fusion
and sRNA under control of
constitutive promoter
Combine plasmids
in E. coli recA
target 5' UTR
Low-copy,
CmR
sRNA gene
High-copy,
AmpR

Urban, JH and Vogel, J.,  Nuc . Acids Res. 2007,  35:1018- 10 37

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From the target information and expression patterns it was inferred that many sRNAs are involved in response to stress Examples RyhB   –  iron limitation OxyS   –  oxidative stress MicF   –  high  osmolarity

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VNUS+
VNES-
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81
sgu
uonelsuell ou
YNES+
sgu
uonelsue'l
VNUS-
sgu
YN81-u le6ue1

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YNYS+
YNYS-
3 eseN8
YNEW
eseN8
YNYS+
YNYS-
YNEII-I le6ue1
81

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Revealing regulation mode by intersection  with cleavage data of a RNase

Targets of  RNase III Targets of  sRNA Common targets

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Mapping the cleavage targets of RNase III

WT Mutant Read start count Comparing read start counts  per position

Wild Type 5’PPP 5’P 5’OH    Adapter ligation 5’PPP 5’OH  Library  preparation and sequencing Mapping reads to genome

RNase III deletion mutant 5’PPP 5’P 5’OH  5’PPP 5’OH

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The  ceRNA  mechanism operates in prokaryotes

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In prokaryotes the  ceRNA  mechanism was shown to play a role in normal physiology

Uninduced (no chitosugars)
ChiX
chi? mRNA
ChiP translation repressed.
ChiP mRNA destabilized
Induced by chitosugars
chbBCARFC mRNA
Sequestration of ChiX.
chlP mRNA translated
os
chi? mRNA

The sRNA  ChiX  represses the gene  chiP , encoding  ChiP In presence of  chitosugars   ChiP  is needed In presence of  chitosugars  the operon  chbB  is transcribed An intergenic region in  chbB   contains  a binding site of  ChiX ChiX  is sequestered by  chbB  and the repression of  chiP  is relieved

Example of  ceRNA  mechanism involving  sRNAs

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