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The genomic revolution of non-coding RNAs  Class 4 – part 1 Bacterial small RNAs (sRNAs)

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Similarities and differences between  bacterial small RNAs ( sRNAs ) and miRNAs

miRNA                                                             sRNA Length 		~22nt				 50-400nt Genomic location 	intergenic, intronic			intergenic, 3’ UTR Conservation 	conserved 			conserved Highly abundant        >2000 in human 			>100 in  E. coli Mechanism 	base-pairing with mRNA 		base-pairing with mRNA  Binding location	 usually at 3’ UTR			usually at 5’ UTR                  Regulatory role 	negative 				negative or positive  						 Impact  on  	translation, mRNA stability		translation, mRNA stability Protein chaperon 	  Argonaute  			 Hfq 				 Involved in central  development,  immune response,	response to stress,	 virulence	 processes 		cancer…..

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VNUS+
VNES-
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81
sgu
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sgu
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VNUS-
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The complete genome sequence of  Escherichia coli  K-12

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Only  a few sRNAs were known in  E. coli  when the genome sequencing was completed

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Computational (followed by experimental verification in some studies) Argaman  et al.     (2001)	Transcription signals in intergenic regions and conservation Wassarman  et al. (2001) 	Conservation Rivas and Eddy   (2001)  	Conservation of secondary structure (SS) Carter et al.          (2001) 	Machine learning approach Chen et al.           (2002) 	Transcription signals Yachie  et al.         (2003) 	Hidden Markov Models Coventry et al.      (2004) 	MSA and conservation of SS Washietl  et al.      (2005) 	SS conservation and thermodynamic stability Sridhar and Rafi   (2007) 	Comparative genomics   Experimental Wassarman  et al. (2001) 	Microarrays Tjaden  et al.         (2002) 	Microarrays Zhang et al.          (2003) 	 Coimmunoprecipitation  with RNA-binding 				proteins Vogel et al.           (2003)	 RNomics , shotgun cloning Kawano et al.       (2005) 	Cloning Sittka  et al.           (2008)        co-IP followed by RNA- seq

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By 2007 ~100 sRNA-encoding genes were identified in the intergenic regions of  E. coli

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total RNA
Microarray
RNA-Seq
RNA extracted from
Hfq.colP
size-selection/
rRNA & tRNA depletion
Shotgun cloning &
Sanger sequencing
RNA extracted from
RsmA-Hiss colP

Experimental screens for bacterial small RNAs

From Sharma and Vogel,  2009, Current Opinion in Microbiology, 12:536–546

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(c)
wild cells
hfq cells
cop
cop
make extract
a-FLAG antibody,
extract RNA
convert to 5? RNA,
polyadenylation,
5' linker ligation,
cDNA synthesis
Pyrosequencing
1000
Itoc
Imc
i 50
aso
150
InvR 89 nt
(3236 113 cDNAs)
SroB 84 nt
27 cDNAs)
SraH 120+58 nt
(2292 j 55 cDNAs)
6S RNA 183 nt
(451 1836 cDNAs)
Current Opinbn in Microbiology

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Dynamic sRNA profiles over bacterial growth

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What is the function of specific  sRNAs in the bacterial cell?

Study function through target identification

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CopraRNA  (Comparative Prediction Algorithm  for sRNA Targets)

Features Detect  mRNAs with complementary sequences to the sRNA Stability of sRNA-mRNA binding  Conservation of targets (but not necessarily binding sites)

Wright, Richter,  Papenfort , Mann, Vogel, Hess,  Backofen , Georg  (2013), PNAS 110,  E3487 – E3496

New targets based on CopraRNA predictions: tested 23, confirmed 17 by small-scale experiments (74%)

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Experimental identification of sRNA targets

Overexpression or knockdown/knockout  of an sRNA       and following change in gene expression by       microarray or RNA- seq IP-based technologies to determine  Hfq -bound RNAs      - Single: CLIP- seq      - RNA pairs: RIL- seq  ( Hfq ) CLIP – Crosslinking and IP RIL-  RNA Interaction by Ligation

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RIL-seq   R NA  I nteraction by  L igation and  seq uencing

RNA extraction, library preparation, and sequencing

Mapping sequenced fragment to  E. coli  genome

In vivo  protein-RNA crosslinking by UV irradiation

Melamed  et al. (2016) Mol. Cell,  63:884- 8 97

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Finding complementary sequences in sRNA and target mRNAs Large-scale experiments under sRNA overexpression or deletion Small-scale experiments under sRNA overexpression or deletion

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yciW ...CGCGACTTACGCCGCTCCCCGGGCCTGATTC CACCAGGGAAA GTAATCATCGGTTGGCACAAATCACG.. pgi ...AACCAGGCACTAACGGTCAGCACGCGTTCTACCAGCTGATC CACCAGGGAAC CAAAATGGTACCGTGC.. bamE ...TTACCGTCCTGACATC AACCAGGGGAA CTATCTGACCGCTAACGACGTATCCAAAATACGTGTTGGCA.. ompC ...GTCTGGTTGACGGCCTGAACTTTGCTGTTCAG TACCAGGGTAA AAACGGCAACCCATCTGGTGAAGGC.. spa ...TCAACTTCACCGCTGGCGGATGTTTCTAT CACCAGGGCAC TGCCGCCGGAAGCGCAGCTGATGATGCA..

yciW 	TGATTC  CACCAGGGAAA   GTAATC pgi 	CTGATC  CACCAGGGAAC   CAAAAT bamE 	GACATC  AACCAGGGGAA   CTATCT ompC 	GTTCAG  TACCAGGGTAA   AAACGG spa 	TTCTAT  CACCAGGGCAC   TGCCGC

ArcZ  target set revealed by RIL- seq  (partial list)

Sequence analysis of the targets For each sRNA, we search the sequences of a putative target set for a common motif:

Do target sequences of a sRNA  share a common motif?

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