ARAUD Tanguy Hors Thèse

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• Langue: English

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Département de Microbiologie et Médecine Moléculaire
Centre Médicale Universitaire
Overview of the miRNA
pathways
by Tanguy ARAUD
Hors Thèse Manuscript
Co-directeurs de thèse: Dr J. CURRAN (CMU)
Pr J.D. ROCHAIX (Science III)
Examinateur: Pr D. BELIN (CMU)
Pr W. REITH (CMU)
Novembre 2008Overview of the miRNA pathways
Index
Once upon a time… 3
What’s a microRNA? 4
Universal nomenclature 5
Genetic organisation and transcription 7
miRNA Biogenesis 8
Nuclear processing: Cleavage by the Drosha-DGCR8 Complex 8
Nuclear export 10
Cytoplasmic procy DICER 10
Incorporation into RISC and activation 11
Biological activity 12
Translational inhibition at the elongation step. 13
Translational inhibition at the initiation step 15
Indirect degradation 15
Translational activation 16
Regulation of microRNA activity 16
Disease 17
miRNAs and cancer 17
Viral infection and host defence 18
Heart disease 20
Conclusion 20
Acknowledgements 21
Bibliographie 21
Hors-Thèse Tanguy ARAUD Page 2Overview of the miRNA pathways
Once upon a time…
The micro RNA (miRNA) story started in the mid 1970’s with the isolation of a
mutation (e912) in Caenorhabditis elegans (C. elegans) (Hodgkin, 1974). This mutation
affected the lin-4 gene. It blocked ventral hypodermal cell division during development
and generated a “Vulvaless” phenotype (Horvitz and Sulston, 1980) (figure1). It appeared
that the e912 mutation was causing a failure of temporal development, indicating that
lin-4 might encode a master regulator of developmental timing (Chalfie et al., 1981).
A B
Figure 1: Caenorhabditis elegans Vulvaless phenotype.
Those images are extracted from the online review of C.elegans biology WormBooK (http://www.
wormbook.org). A- WT phenotype. The vulva is present in the adult female worm. It is necessary for egg-
laying and for copulation with males. B- Vulvaless phenotype. Mutation e912 induces the loss of the lin-4
gene, and loss of the vulva in the female worm.
Later, during amplification of these mutated nematodes the Horvitz group isolated
revertants showing normal development. lin-14 null alleles were identified in these
animals. Surprisingly, the developmental timing defects of lin-14 were opposite to those
of lin-4 (Ambros and Horvitz, 1984), suggesting that lin-4 might encode a trans-acting
negative regulator of lin-14. Fortunately, two new nematode mutants, showing the same
phenotype as e912 were isolated (n355 and n536). These gain-of-function mutations
affected the lin-14 gene (Ruvkun et al., 1989). Deletions in the 3′ untranslated region
(UTR) of the lin-14 mRNA were characterized in both n355 and n536. This was correlated
with an inappropriately high level of LIN-14 expression during development (Wightman
et al., 1991). Despite these observations the unusual characteristics of miRNA delayed
there discovery. A size <700bp and no well characterized ORF (Open Reading Frame) were
not compatible with the definition of a gene at that time. Furthermore, two transcripts
of about 60 bp and 20 bp were detected using the RNase Protection Assay (RPA), with the
shorter form being the most abundant. It was only with the appearance of bioinformatic
software, that the antisense theory was elaborated. The complementary sequence of the
shorter lin-4 transcript was found to be repeated seven times within the 3’UTR of lin-14.
Hors-Thèse Tanguy ARAUD Page 3Overview of the miRNA pathways
The formation of this RNA:RNA duplex with a temporal gradient, down regulated the
expression of lin-14 during development (Lee et al., 1993; Wightman et al., 1993).
However, the discovery of the world’s first miRNA did not trigger a gold rush until
the discovery of a second tiny RNA, let-7, in C. elegans. This 21 nt RNA was complementary
to regions within the 3’UTR of lin-14, lin-28, lin-41, lin-42 and daf-12, indicating that
expression of these genes might be directly controlled by let-7 (Reinhart et al., 2000).
Nonetheless, the term “microRNA” was only introduced in 2001 in a set of three articles
(Lau et al., 2001; Lee and Ambros, 2001; Ruvkun, 2001).
The generation of full genome databases for different species, demonstrated that
let-7 was conserved across most of the animal phylogeny, with a temporal regulation also
conserved in a wide range of species (Pasquinelli et al., 2000). Computational methods
predicted more than 6000 potential miRNA genes covering 58 species (Artzi et al., 2008;
Lim et al., 2003a). Finally, the number of repository miRNA databases and prediction
tools also increased (miBase (Griffiths-Jones et al., 2008), miRNApath (Chiromatzo et al.,
2007), miRGen (Megraw et al., 2007), miRNAMap (Hsu et al., 2006)), providing resources
for further studies (figure 2).
600 Figure 2: Increasing numbers of
publication in the miRNA field.500
Since the 70’s the number of
400 publications has increased with the
exponential growth in the last five 300
years, indicating that this field is
200 very active. Data used to create the
charts was extracted from the web 100
site “ISI web of Knowledge” (http://
0 apps.isiknowledge.com).
Year of publication
What’s a microRNA?
MiRNAs are a class of non-coding RNA genes. They belong to the RNA interference
familly (RNAi family) along with small interfering RNA (siRNA) and PIWI interacting RNA
(piRNA). There final products are small single strand RNA molecules, about 19-21
nucleotides (nt) long with 3’ hydroxyl and 5’ phosphate ends. piRNAs are a little bit longer,
about 24-27 nt. It is largely accepted that these highly conserved RNAs generally regulate
the expression of genes. They acts as guide molecules by base-pairing with the 3’-UTR (or
5’UTR) of specific mRNAs leading to translational repression and/or mRNA cleavage (see
below).
Hors-Thèse Tanguy ARAUD Page 4
1993
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2006
2007
Number of publicationsOverview of the miRNA pathways
Ambros and coworkers defined a small RNA molecule as a miRNA using five
criteria (Ambros et al., 2003; Griffiths-Jones et al., 2006):
• Detection of a distinct 22-nt RNA transcript.
• Identification of the 22-nt sequence ina library of cDNAs madefrom size-fractionated
RNA. Such sequences must precisely match the genomic sequence of the organism
from which they were cloned.
• Prediction of a hairpin precursor (usually about 60–80 nt) containing the 22-nt
miRNA sequence within one arm.
• Phylogenetic conservation of the 22-nt miRNA sequence and its predicted precursor
secondary structure.
• Detection of increased precursor accumulation in organisms with reduced Dicer
function (see later).
Those five criteria are necessary, but nowadays it seems that they are not sufficient
to unambiguously define a small RNA as a miRNA. Indeed siRNA are very similar to miRNA
and the distinction between them is increasingly becomingobscure. The most important
requirement for a miRNA is a contiguous and perfect base pairing with its target of 6
nucleotides (nucleotide 2 to 8 from the 5’ extremity). This is called the “seed” region
(figure 3). It nucleates the interaction of the miRNA with its mRNA target. Another
important feature is the presence of a bulge in the middle of the sequence. Both miRNA
and siRNA are produced via similar pathways and have similar mechanisms of action
(Ambros, 2004; Bartel, 2004). That’s why most of the discoveries about siRNA processing
were simply extended to miRNAs and vice versa. Recent observations have shown that
both can suppress translation of mRNAs (in the case of an imperfect match, miRNAs) or
can cleave target RNAs (in the case of a perfect match, siRNAs), and that they can be
interchangeable (Cullen, 2004; Yekta et al., 2004; Zeng et al., 2003). siRNAs were originally
proposed to act mainly as an antiviral defence and transposon repression system via the
phenomenon of RNAi (Hunter, 2000), but recent findings indicate that such RNAs may
play a much broader role in gene and genome regulation. Finally, we can ask if the same
small RNA can act as a miRNA on one mRNA target and as a siRNA on another?
Universal nomenclature
Because the number of miRNAs discovered is growing rapidly, 2909 in 36 genomes
in June 2005 to 5071 in 58 genomes in August 2007 (Griffiths-Jones et al., 2008), an
universal nomenclature became necessary.
The primary transcript, which is the result of transcription (see below), is named
pri-miR, while the product of Drosha is called pre-mir (figure 5). Finally, miRNAs are
named using the “mir” abbreviation surrounded by a three or four letter species prefix
Hors-Thèse Tanguy ARAUD Page 5Overview of the miRNA pathways
primary transcript of lin4A
5’
UA G U U U C A U C A
UGCUU CCG CCUG CC C GAGA CUC A GUGUGAGGUA
U
ACGAG GGC GGAC GGG CUCU GGGU C A C A CUUCGU
G U U A C U C C C A UUA G3’ U A
pre-cel-lin4B
U5’ U U C A U C A
CC C GAGA CUC A GUGUGAGGUA
U
GGG CUCU GGGU C A C A CUUCGU
UU C C C A G3’ C A
Guide strand lin-4 duplexC
5’ U U C A
3’CC C GAGA CUC A GUGUGA
GGG CUCU GGGU C A C A 5’U C C C
3’ C A
passenger st