RNA-mediated regulation:How many functions does it

serve?

BioInformatics Seminar Series

Kriton Kalantidis IMBB and Dept of Biology, UoC

van der Krol et al., Plant Cell 2:291 (1990) + Plant Mol Biol 14:457 (1990).

Effects of expression of CHS sense and antisense RNA on flower pigmentation in

Petunia

RNA silencing

• RNA mediated impairment of transcription- TGS-methylation (shRNAs, rasiRNAs and siRNAs?)

• RNA mediated impairment of translation-PTGS (siRNAs and some miRNAs) and -translational arrest (miRNAs)

small RNA may impair gene expression

small RNAs

specific degradation of

target RNA

RNA interference (RNAi)

short interfering RNA (siRNA)

changes in chromatin structure

regulation of “epigenome”

short heterochromatic RNA (shRNA)

Processed from endogenous transcripts

micro RNA (miRNA)

regulation of genesparticularly in development

and more…

The processing pathway of siRNA & miRNA

from Dykxhoorn et al., (2003) Nature Reviews 4, 457-466.

short interfering RNA (siRNA)short interfering RNA (siRNA)

NN

ca 21 Nucleotides

NNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNN

3’

3’

- N N N

- N

N -

N N N -

two 3’ protrudingunpaired nucleotides

5’ P -

- P 5’

5’ terminal phosphates

RNA mediated regulationKey Players

• The Dicers, one or more members

1- S. pombe, C. elegans, Mammals

2- Drosophila

4- Dicots (plants, e.g. Arabidopsis)

5- Monocots (plants e.g. Rice)

At least two diversified functions: miRNA processing and siRNA generation

• Argonaute proteins (Piwi and Paz domains responsible for the slicing of the target molecule), often part of the RISC complex, very large family of genes

• RdRP, present only in some organisms (plants, worms, fungi)

• Other complexes (Drosha/Pasha, RITS etc)

RNA mediated regulationKey Players 2.

Biological role of silencing

Protection against invasive RNAsespecially RNA viruses

Silencing of transposable elements

Regulation of expression of endogenous genes

MicroRNAs

• short (20-25nt) RNA molecules

• translated as precursor RNA molecule

• post transcriptional gene regulation

• target mRNA for cleavage or translational repression

• very abundant class of genes

miRNAs are typical RNA-polII transcripts

Open Questions1. RNA silencing

• Initiation: (plants, worm, fly)What is aberrant RNA?

Is there a threshold?

• Maintenance: (plants, worm, fly?)Is there an endogenous “cooling system”

“Degradive PCR”? Needs methylation?

• Systemic Spread: (plants, worm?)A threshold needed?What is the silencing signal?How does it move?

Integrated Model for RNA silencing

Hutvagner and Zamore, Curr. Op. Genet. & Dev. 12, 225-232 (2002)

Open Questions1. RNA silencing

• Initiation: (plants, worm, fly)What is aberrant RNA?

Is there a threshold?

• Maintenance: (plants, worm, fly?)Is there an endogenous “cooling system”

“Degradive PCR”? Needs methylation?

• Systemic Spread: (plants, worm?)A threshold needed?What is the silencing signal?How does it move?

Open Questions2. miRNAs

• How many genes do they regulate?

• What is the importance of miRNA families?

• What gives miRNAs their expression specificities?

• What is the mode of their function, translational arrest, and at what stage?

RNA silencing related questions

current answears• Initiation:

What is aberrant RNA?- ? Must be qualitative specificityIs there a threshold?- seems to be / We have evidence

• Maintenance:“Degradive PCR”? It involves RdRP activity, not proven

Is there an endogenous “cooling system”, maybe Eri involved

Needs methylation? Most people seem to think so (?)

• Systemic Spread:A threshold needed?-seems to/ we have some evidenceWhat is the silencing signal? RNA but of what sort?How does it move? Together with metabolites (we say)

Transgenic GFP expressing N.benthamiana is our system

Multiple thresholds seem to regulate initiation and spread of

silencing

a b

Short Range Silencing:A type of silencing that does not spread

Short Range Silencing is (likely): •A post transcriptional phenomenon •Caused by small amounts of siRNAs

Inducer of RNAi

Threshold 1 silencing Threshold 2

Short range

Local & systemic

Ignoreif

below

if

above

If below

If above

A simple model for the “red spots”:

ERI-1:

An RNase that deactivates siRNAs

N

N

N

N

N

N

N

N

N

3’ N

N 3'

N - P 5’

P -5’

ca 21 Nucleotides

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N 3’

N- P 5’

P -5’

ca 21 Nucleotides

N

3’ N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

ERI-1

pART27 antiEri15840bp

SalI (1)NotI (962)

KpnI (2362)

SalI (3012)

•AccI (4452)

NotI (6002)

SalI (8222)

RB

CaMV 35S

Eri 600bp fragment

intron

Eri 600bp fragment

•ocs 3'

Tn7 Spr/Str

nptII

Eri1 was suppressed with the use of hairpin constructs, one using the Arabidopsis and the other the tobacco ortholog

Nt Nb 3Nt Nb 3rdrd d 6 d 6thth d 10 d 10thth d transgenic d transgenic

Analysis of Eri Expression, following agroinfiltration with Eri-hairpin, using RT-PCR

Suppression of Eri1 in N. benhtamiana transforms short range silencing to

local and systemic

Some Conclusions

• The short range signal is likely siRNA and moves faster but not solely through cytoplasmic canals

• SRS can be induced by silencing inducers at concentrations below an arbitrary threshold.

• Suppression of ERI causes the transition from short range to systemic silencing

The silencing signal moves from metabolic source to

metabolic sink

Systemic silencing can happen spontaneously or can be induced

Spontaneous systemic silencing

Induced systemic silencing

Grafting experiments to monitor PTGS spreadGrafting experiments to monitor PTGS spread

Non silencedNon silencedsilencedsilenced

transgenetransgenetransgenetransgene

Grafting experiments to monitor PTGS spreadGrafting experiments to monitor PTGS spread

Non-silencedNon-silencedsilencedsilenced

transgenetransgenetransgenetransgene

Silencing spreads from root-stock to scionSilencing spreads from root-stock to scion

silencedNon-silenced

TransgeneTransgene

Systemic spread,The questions addressed:

• What determines the movement of the silencing signal?

• What influences the efficiency of silencing transmission?

Silencing does not necessarily move from stock to scion

32Pi can be used to track the flow of phloem sap

The silencing signal recapitulates the pattern of phloem flow

Conclusions?

• The silencing signal seems to follow the phloem flow

• The plant architecture influences the movement of the signal

• There may be a threshold of signal that needs to be exceeded to establish silencing

Current answers on miRNAs

• How many –and which- genes do they regulate? – hundreds to thousands (estimated up to 20% of the total) found some

• What is the importance of miRNA families? Multiple levels of regulation?

• What gives miRNAs their expression specificities?polII promoters, any specificities?

• What is the mode of their function, translational arrest, and at what stage? Both arrest and degradation have been found

Identifying miRNA regulated genes related to cancer

Various ways of identifying miRNA targets:

•Bioinformatics

•Experimental

In both cases the interaction has to be experimentally validated!

The Microinspector program

A software to detect

miRNA binding sites

in a given mRNA target

A software to detect

miRNA binding sites

in a given mRNA target

A service for

those interested in

finding whether a

particular gene has

miRNA binding sites

A service for

those interested in

finding whether a

particular gene has

miRNA binding sites

currently accesible at:

www.imbb.forth.gr/

webtools.html

currently accesible at:

www.imbb.forth.gr/

webtools.html

miRNAs and oncogenes

We got all the human UTR region’s sequences (

http://bighost.area.ba.cnr.it/BIG/UTRHome/)

and extracted all the oncogenes’ UTRs from them

• We ran the dataset with the modified version of microInspector which can grab a whole set of sequences and analyze them for all miRNAs in a particular organism (in this case – Human miRNAs)

• From the results we selected manually only the best structures (binding sites) - about 60 onco and onco-related genes.

miRNAs and oncogenes

393 CC159364 3'UTR in Homo sapiens, v-raf-1 murine leukemia viral oncogene homolog 1, clone MGC:9026 IMAGE:3904404, mRNA, complete cds.

atcagcttctggaggaatgcatgtcacaggcg

hsa-miR-346 (((((...((((((...((((((&))))))..))))))...)))))..........

-25.1

2500 CC003805 3'UTR in Human B-cell leukemia/lymphoma 2 (bcl-2) proto-oncogene mRNA encoding bcl-2-alpha protein, complete cds.

atgaatatggaatatccaatcctgtgctgcta

hsa-miR-16 ..(((((.....((((((((((&))))))))))..)))))...............

-23.3

2500 CC003805 3'UTR in Human B-cell leukemia/lymphoma 2 (bcl-2) proto-oncogene mRNA encoding bcl-2-alpha protein, complete cds.

atgaatatggaatatccaatcctgtgctgcta

hsa-miR-195 .(((((....(((((((((((&))))))))))).)))))...............

-26.5

2123 CC011534 3'UTR in Homo sapiens proto-oncogene (Wnt-5a) mRNA, complete cds.

atgatatccacatcagccaactgtggctcttt

hsa-miR-28 .((((...((((((.((((((.&.))))))))))))...))))............

-23.5

699 CC095437 3'UTR in Homo sapiens Pim-2h, hUGT2, hUGT1, genes for pim-2 protooncogene homolog, UDP-galactose transporter 1, UDP-galactose transporter 2, complete cds.

atggatggttttttgggggatgggctagggga

hsa-miR-331 ((((((((..((.(((((((.&.))))))).))...))))))))..........

-31.3

4919 3HSA015779 'UTRin Human K-ras oncogene protein mRNA,complete cds.

atgttaatgccagtcaccagcaggctatttca

hsa-let-7b (..(((.((((..(((((((((&)))))))))..)))))))..)...........

-24.8

Example - 6 results from the result table around Bcl2/miR16 row

miRNAs and oncogenes

• Johnson et al. 2005. RAS is regulated by the let-7 micro RNA family; Cell, 120(5):635-47.

• Cimino A. et al. 2005. miR15 and 16 induce apoptosis by targeting Bcl-2. PNAS Sept15.

mir15 cDNA name

c-myc binding protein MYCBP- regulation of transcription, DNA-dependent NM_012333 AMY1

zinc finger protein NM_014415

VENT-like homeobox 2 - transcription factor activity NM_014468 VENTX2

ankyrin 3, node of Ranvier (ankyrin G) - isoform 2 NM_001149 Ankyrin-G

Common mir15+16

Homo sapiens eferin (Rab11-FIP3), mRNA NM_014700 Rab11-FIP3

Additional gene ► Bcl2, oncogene,anti-apoptotic, correlation with CLL,

possible regulation by mir16 found with microInspector

Candidate genesCandidate genes

miRNAs and oncogenes

Binding of miRNA 16 and 15 to Bcl-2 and MYCBD respectively

miR16/Bcl-2 miR15/MYCBP

Bste II cleavage sites in vectorG/ G T G A C C ....... G/ G T T A C C

C C A C T G/ G ...... C C A A T G /G

BsteII Overhangs5’ G G T T A C C 3’

C C A C T G G

Cassete endsG G T G A C ....... G T T A C C

C C A C T G .... C A A T G G

New vector: pHRL-BsteIINew vector: pHRL-BsteII

• Principle: Creating vector that doesn’t re-ligate

• Created from pHRL-TK, Promega, by insertion of BsteII binding sites

• Enables insertion of microRNA binding site only, in Luciferase 3’UTR (screened with microInspector for other micro-RNA binding)

Synthetic cassette design and analysis

mutation tgcacaatggcctttctgtgctattt Wild type tgcacaatggcctttctgtgctgttt

mir15cacaaaccauuaugugcugcua

MYCBP mir-binding site

ccaatcctgtgctgcta ccaatcctgtgctgata

Bcl2 mir-binding site

perfect match

mir16cgccaatatttacgtgctgcta

Luciferase analysis – Luciferase analysis – normalisation by normalisation by β-β-galactosidasegalactosidase

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

control wtBcl2 mutBcl2 PM16

RL

Us

0

100000

200000

300000

400000

500000

600000

cont

rol

wtMYCBP

mut

MYCBP

PM15

RL

Us

Acknowledgements

FOSRAK (STREP) MC Training Site

Current and new Members:

N. Vrettos

S. Tzortzakaki

E. Koscianska

A. Dalakouras

T. Alexiadis

MC students:

A. Dumitru

V. Baev

V. Russinov

H. Schumacher

Recent Members:

K. Skreka

B. Tournier

A. Boutla

Collaborations:

P. Rombi- CNRS, Str, FR

A. Hatzigeorgiou-Penn-State, Ph, USA

J. Burgyan- Godolo, HU

C. Delidakis- IMBB, Forth

N. Tavernarakis- IMBB, Forth

K. Kotsabasis, Dept. of Biology, UoC, GR

M. Tsagris, Dept. of Biology, UoC, GR

Martin Tabler