rna-mediated regulation: how many functions does it serve? bioinformatics seminar series kriton...
TRANSCRIPT
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