micrornas in arabidopsis : discovery, functions & implication in the control of flowering time
TRANSCRIPT
microRNAs in Arabidopsis : discovery, functions & implication in the control of flowering time.
April 2014
Frédéric Bouché
Plant PhysiologyULg
How microRNAs were discovered ?
How are they produced ?
How to study miRNAs and use them in genetic engineering ?
What is their role in the control of flowering time ?
How are they produced ?
How microRNAs were discovered ?
How to study miRNAs and use them in genetic engineering ?
What is their role in the control of flowering time ?
Plant PhysiologyULg
Caenorhabditis elegans
How were the microRNAs discovered ?
1993
Plant PhysiologyULg
Ambros & Horvitz, 1987 Genes Dev.
Ambros, 1989 Cell. Wightman et al, 1989 Genes Dev.
1993
How were the microRNAs discovered ?
Plant PhysiologyULg
Aging factors
Ambros & Horvitz, 1987 Genes Dev.
Ambros, 1989 Cell. Wightman et al, 1989 Genes Dev.
1993
How were the microRNAs discovered ?
adapted from http://www.mun.ca/biology/desmid/brian/BIOL3530/DEVO_06/ch06f01.jpg
The development of C. elegans implies aging factors.
Plant PhysiologyULg
Aging factors
Ambros & Horvitz, 1987 Genes Dev.
Ambros, 1989 Cell. Wightman et al, 1989 Genes Dev.
1993
How were the microRNAs discovered ?
adapted from http://www.mun.ca/biology/desmid/brian/BIOL3530/DEVO_06/ch06f01.jpg
The development of C. elegans implies aging factors.
Plant PhysiologyULg
Aging factors
Ambros & Horvitz, 1987 Genes Dev.
Ambros, 1989 Cell. Wightman et al, 1989 Genes Dev.
1993
How were the microRNAs discovered ?
adapted from http://www.mun.ca/biology/desmid/brian/BIOL3530/DEVO_06/ch06f01.jpg
The development of C. elegans implies aging factors.
Plant PhysiologyULg
Ambros & Horvitz, 1987 Genes Dev.
Ambros, 1989 Cell. Wightman et al, 1989 Genes Dev. How were the microRNAs discovered ?
1993
adapted from http://www.mun.ca/biology/desmid/brian/BIOL3530/DEVO_06/ch06f01.jpg
lin-4 prevents the translation of lin-14 through an unknown mechanism
Plant PhysiologyULg
Wightman et al., 1993 Cell.Lee et al., 1993 Cell.
lin-4 encodes a small untranslated RNA
How were the microRNAs discovered ?
1993
Plant PhysiologyULg
Reinhart et al., 2000 ScienceHutvagner and Zamore, 2002 Science
Olsen & Ambros 1999 Dev. Biol.
2002
How were the microRNAs discovered ?
The level of complementarity between a microRNA and its target determine its mode of action.
miRNA
miRNA
Plant PhysiologyULg
Lagos-Quintana et al., 2001 Science.
In the early 2000’s, microRNAs were identified in several species.
Llave et al., 2002 The Plant Cell.Rhoades et al., 2002 Cell.
20012002
How were the microRNAs discovered ?
Plant PhysiologyULg
How were the microRNAs discovered ? Bentwich et al., 2005 Nature Genetics.Lewis et al., 2005 Cell.
2005
Plant PhysiologyULg
Adapted from http://www.mirbase.org/blog/
2014 MicroRNAs & literature
How were the microRNAs discovered ?
Plant PhysiologyULg
Adapted from http://www.mirbase.org/blog/
2014
How were the microRNAs discovered ?
MicroRNAs & literature
PMRD database
Plant PhysiologyULg
Data from http://www.mirbase.org/ & http://bioinformatics.cau.edu.cn/PMRD/
Kozomara and Griffiths-Jones, 2014 Nucleic Acids Res.
2014
How were the microRNAs discovered ?
Number of microRNAs in Arabidopsis thaliana
PMRD database
miRBase
Plant PhysiologyULg
Data from http://www.mirbase.org/ & http://bioinformatics.cau.edu.cn/PMRD/
Kozomara and Griffiths-Jones, 2014 Nucleic Acids Res.
2014
How were the microRNAs discovered ?
Number of microRNAs in Arabidopsis thaliana
PMRD database
miRBase
Plant PhysiologyULg
Data from http://www.mirbase.org/ & http://bioinformatics.cau.edu.cn/PMRD/
miRBase High confidence
Kozomara and Griffiths-Jones, 2014 Nucleic Acids Res.
2014
How were the microRNAs discovered ?
Number of microRNAs in Arabidopsis thaliana
How are they produced ?
How microRNAs were discovered ?
How to study miRNAs and use them in genetic engineering ?
What is their role in the control of flowering time ?
Plant PhysiologyULg
How are they produced ? Reviewed in Spanudakis & Jackson, 2014 J Exp. Bot.
Plant PhysiologyULg
Reviewed in Spanudakis & Jackson, 2014 J Exp. Bot.How are they produced ?
Plant PhysiologyULg
How are they produced ?
The length of the pre-miRNA loop varies among species
Length of the pre-miRNA loop (nt)
Plants
Metazoans
Roberts et al., 2013 Mobile Genetic Elements
mic
roR
NA
mic
roR
NA
microR
NA
* microR
NA
*
Plant PhysiologyULg
Khvorova et al, 2003 Cell. Rajagopalan et al., 2006 Genes & Dev.Eamens et al., 2009 RNA.
microRNA
microRNA* (Degraded in most cases)
How are they produced ?
DR B1
The selection of the strand used as the microRNA is base on thermodynamic parameters.
Thermodynamicinstability
Plant PhysiologyULg
Ossowski et al., 2008 The Plant Journal.How are they produced ?
mRNA
microRNA
Percentageof observedmismatch
3’ END Clivage region 5’ END
The complementarity between the microRNA and its target is not always perfect but the clivage region is highly conserved
How are they produced ?
How microRNAs were discovered ?
How to study miRNAs and use them in genetic engineering ?
How to study miRNAs and use them in genetic engineering ?
What is their role in the control of flowering time ?
Plant PhysiologyULg
VISUALIZATION FUNCTIONALSTUDIES
GENETICENGINEERING
How to study miRNAs and use them in genetic engineering ?
Plant PhysiologyULg
VISUALIZATION FUNCTIONALSTUDIES
GENETICENGINEERING
How to study miRNAs and use them in genetic engineering ?
Plant PhysiologyULg
RNA extraction
VISUALIZATION
RNAsRNAs
Columns
How to study miRNAs and use them in genetic engineering ?
Plant PhysiologyULg
Northern blotPCR
Transcriptomics
RNA extraction
VISUALIZATION
microRNAs
Hybrid method
How to study miRNAs and use them in genetic engineering ?
Plant PhysiologyULg
Northern blotPCR
RNA extraction
Transcriptomics
VISUALIZATION
Labeled probe
How to study miRNAs and use them in genetic engineering ?
Plant PhysiologyULg
Northern blotPCR
Chen et al., 2005 Nucleic Acids Res.
Shi and Chiang, 2005 BioTechniques.Varkonyi-Gasic et al., 2007 Plant Methods.
Busk, 2014 BMC Bioinformatics
cDNA cDNA
RNA extraction
Transcriptomics
VISUALIZATION
mRNA
Classical RT-PCR on mRNA Stem-loop RT-PCR on miRNA
How to study miRNAs and use them in genetic engineering ?
Plant PhysiologyULg
Northern blotPCR
Transcriptomics
Thomson et al., 2004 Nature Methods.
Reviewed in Wang et al., 2009 Nature Rev. Gen.Liu et al., 2008 RNA.
Microarrays Next generation sequencing
RNA extraction
VISUALIZATION
How to study miRNAs and use them in genetic engineering ?
Plant PhysiologyULg
VISUALIZATION FUNCTIONALSTUDIES
GENETICENGINEERING
How to study miRNAs and use them in genetic engineering ?
Plant PhysiologyULg
Over-expressionKnock down
FUNCTIONALSTUDIES
Target mutagenesis
How to study miRNAs and use them in genetic engineering ?
Plant PhysiologyULg
UBIQUITOUS OR TISSUE-SPECIFIC PROMOTER TERMINATOR
OVER-EXPRESSION
How to study miRNAs and use them in genetic engineering ?
microRNA
Plant PhysiologyULg
Franco-Zorrilla et al., 2007 Nature Genetics
Se
microRNA
mRNA
RISC COMPLEX
The knock-down of microRNA can be done using modified targets that prevent microRNA from degrading their regular targets.
How to study miRNAs and use them in genetic engineering ?
microRNA
Plant PhysiologyULg
microRNA
To assess the effect of a microRNA on a specific gene, it can be useful to modify the targeted region of this gene using silent mutations
How to study miRNAs and use them in genetic engineering ?
Plant PhysiologyULg
VISUALIZATION FUNCTIONALSTUDIES
GENETICENGINEERING
How to study miRNAs and use them in genetic engineering ?
Plant PhysiologyULg
Artificial microRNAs
Schwab et al., 2006 The Plant Cell.Ossowski et al., 2008 The Plant Journal.
Promoter of interest
Conserved sequence
www.wmd3.weigelworld.org
GENETICENGINEERING
Directed mutagenesis
The design of artificial microRNAs allows to knock-down the expression of one or several genes.
How to study miRNAs and use them in genetic engineering ?
Plant PhysiologyULg
Artificial microRNAs
Schwab et al., 2006 The Plant Cell.Ossowski et al., 2008 The Plant Journal.www.wmd3.weigelworld.org
GENETICENGINEERING
Artificial microRNA are produced using directed mutagenesis and subsequent Agrobacterium tumefasciens-mediated Arabidopsis transformation
How to study miRNAs and use them in genetic engineering ?
How are they produced ?
What is their role in the control of flowering time ?
How microRNAs were discovered ?
How to study miRNAs and use them in genetic engineering ?
Plant PhysiologyULg
FLORAL TRANSITION
JUVENILE TO ADULT TRANSITION
Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
ENVIRONMENTAL FACTORS
ENDOGENOUS FACTORS
Flowering time is controlled by different factors
PHOTOPERIOD VERNALIZATION
AMBIENT TEMPERATURE
SUGARS
HORMONES
AGEING
CIRCADIAN CLOCK
Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Roles of microRNAs in the control of flowering time
?Plant
PhysiologyULg
WHAT ABOUTmicroRNAs
Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Aukerman & Sakai, 2003 The Plant Cell.
Todesco et al., 2010 PLoS genetics.Mathieu et al., 2009 PLoS biology.Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Aukerman & Sakai, 2003 The Plant Cell.
Todesco et al., 2010 PLoS genetics.Mathieu et al., 2009 PLoS biology.Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Aukerman & Sakai, 2003 The Plant Cell.
Todesco et al., 2010 PLoS genetics.Mathieu et al., 2009 PLoS biology.Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Aukerman & Sakai, 2003 The Plant Cell.
Todesco et al., 2010 PLoS genetics.Mathieu et al., 2009 PLoS biology.Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Schwab et al., 2005 Developmental Cell.
Wu and Poethig, 2006 Development.Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Schwab et al., 2005 Developmental Cell.
Wu and Poethig, 2006 Development.Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Schwab et al., 2005 Developmental Cell.
Wu and Poethig, 2006 Development.
Wild type
JUVENILE ADULT
Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Schwab et al., 2005 Developmental Cell.
Wu and Poethig, 2006 Development.
FLOWERING TIME
DELAYED35S:miR156
Wild type
JUVENILE ADULT
JUVENILE ADULT
Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Schwab et al., 2005 Developmental Cell.
Wu and Poethig, 2006 Development.
FLOWERING TIME
DELAYED
ACCELERATED
rSPL3
35S:miR156
Wild type
JUVENILE ADULT
JUVENILE
JUVENILE
ADULT
ADULT
Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Yu et al., 2013 Elife.Yang et al., 2013 Elife.
SUCROSE
miR156 expression
level
Sucrose content
The diminution in miR156 through the Arabidopsis development is mediated by an increase in the sucrose content.
Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Wu et al., 2009 Cell.Zhu and Helliwell, 2010 J. Exp. Bot.
SUCROSE
Flowering
FLORAL TRANSITION
JUVENILE TO ADULT TRANSITION
The balance between miR156 and miR172 is a key component of the endogenous flowering pathway
Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Floral meristem identity genes
SHO
OT
API
CAL
MER
ISTE
MLE
AVES
AGING
AGING
Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Fahlgren et al., 2006 Current Biology.Rubio-Somoza and Weigel, 2011 Tr. in. Pl. Sci.
Montgomery et al., 2008 Cell.
35S::miR390Late flowering
Floral meristem identity genes
SHO
OT
API
CAL
MER
ISTE
MLE
AVES
AGING
AGING
Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Ho and Weigel, 2014 The Plant Cell.Sarvepalli and Nath, 2011 The Plant Journal.Weigel et al., 2000 Plant Physiol.
35S::miR319Late flowering
Schommer et al., 2012 Sign. Com. in Plants
Floral meristem identity genes
SHO
OT
API
CAL
MER
ISTE
MLE
AVES
AGING
AGING
Roles of microRNAs in the control of flowering time
Plant PhysiologyULg
Weng et al., 2005 BMC Genomics Willmann & Poethig, 2007 Curr Opin Plant Biol.
microRNAs conservation during evolution
Roles of microRNAs in the control of flowering time
Plant PhysiologyULg