Discovered about 20 years ago, microRNA are small non-coding RNA (21-23 nt) involved in the post-transcriptional regulation of gene expression. They are involved in numerous regulatory pathways. However, the analysis of their function and their expression level is a bit tricky, explaining that they were only integrated in the molecular pathways during the last decade.
Here, we present a brief summary of the events that lead to the discovery of microRNAs, as well as the molecular tools used for their analysis and their role in the molecular control of flowering time.
Of course, feel free to leave any constructive comment about those slides !
microRNAs in Arabidopsis : discovery, functions & involvment in the control of flowering time.
1. microRNAs in Arabidopsis : discovery, functions &
implication in the control of flowering time.
April 2014
Frédéric Bouché
Plant
Physiology
ULg
2. 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 ?
3. 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 ?
6. Plant
Physiology
ULg
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.
7. Plant
Physiology
ULg
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.
8. Plant
Physiology
ULg
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.
9. Plant
Physiology
ULg
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
10. Plant
Physiology
ULg
Wightman et al., 1993 Cell.
Lee et al., 1993 Cell.
lin-4 encodes a small untranslated RNA
How were the microRNAs discovered ?
1993
11. Plant
Physiology
ULg
Reinhart et al., 2000 Science
Hutvagner 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
12. Plant
Physiology
ULg
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.
2001
2002
How were the microRNAs discovered ?
16. PMRD database
Plant
Physiology
ULg
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
17. PMRD database
miRBase
Plant
Physiology
ULg
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
18. PMRD database
miRBase
Plant
Physiology
ULg
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
19. 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 ?
22. Plant
Physiology
ULg
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
microRNA
microRNA
microRNA*
microRNA*
23. Plant
Physiology
ULg
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.
Thermodynamic
instability
24. Plant
Physiology
ULg
Ossowski et al., 2008 The Plant Journal.How are they produced ?
mRNA
microRNA
Percentage
of observed
mismatch
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
25. 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 ?
31. Plant
Physiology
ULg
Northern blot
PCR
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 ?
32. Plant
Physiology
ULg
Northern blot
PCR
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 ?
36. Plant
Physiology
ULg
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
37. Plant
Physiology
ULg
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 ?
39. Plant
Physiology
ULg
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
GENETIC
ENGINEERING
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 ?
40. Plant
Physiology
ULg
Artificial microRNAs
Schwab et al., 2006 The Plant Cell.
Ossowski et al., 2008 The Plant Journal.
www.wmd3.weigelworld.org
GENETIC
ENGINEERING
Artificial microRNA are produced using directed mutagenesis and subsequent Agrobacterium
tumefasciens-mediated Arabidopsis transformation
How to study miRNAs and use them in genetic engineering ?
41. 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 ?
46. Plant
Physiology
ULg
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
47. Plant
Physiology
ULg
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
48. Plant
Physiology
ULg
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
49. Plant
Physiology
ULg
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
50. Plant
Physiology
ULg
Schwab et al., 2005 Developmental Cell.
Wu and Poethig, 2006 Development.Roles of microRNAs in the control of flowering time
51. Plant
Physiology
ULg
Schwab et al., 2005 Developmental Cell.
Wu and Poethig, 2006 Development.Roles of microRNAs in the control of flowering time
52. Plant
Physiology
ULg
Schwab et al., 2005 Developmental Cell.
Wu and Poethig, 2006 Development.
Wild type
JUVENILE ADULT
Roles of microRNAs in the control of flowering time
53. Plant
Physiology
ULg
Schwab et al., 2005 Developmental Cell.
Wu and Poethig, 2006 Development.
FLOWERING
TIME
DELAYED
35S:miR156
Wild type
JUVENILE ADULT
JUVENILE ADULT
Roles of microRNAs in the control of flowering time
54. Plant
Physiology
ULg
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
55. Plant
Physiology
ULg
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
56. Plant
Physiology
ULg
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
58. Plant
Physiology
ULg
Fahlgren et al., 2006 Current Biology.
Rubio-Somoza and Weigel, 2011 Tr. in. Pl. Sci.
Montgomery et al., 2008 Cell.
35S::miR390
Late flowering
Floral meristem
identity genes
SHOOTAPICALMERISTEMLEAVES
AGING
AGING
Roles of microRNAs in the control of flowering time
59. Plant
Physiology
ULg
Ho and Weigel, 2014 The Plant Cell.
Sarvepalli and Nath, 2011 The Plant Journal.
Weigel et al., 2000 Plant Physiol.
35S::miR319
Late flowering
Schommer et al., 2012 Sign. Com. in Plants
Floral meristem
identity genes
SHOOTAPICALMERISTEMLEAVES
AGING
AGING
Roles of microRNAs in the control of flowering time
60. Plant
Physiology
ULg
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