Regolazione della stabilità degli mRNA: - deadenilazione, decapping e degradazione esonucleolitica (Pab1 polyA binding protein, Dcp1 decaping enzyme, Xrn1 esonuclaesi 5’-3’, esosoma esonucleasi 3’-5’) - taglio endonucleolitico e degradazione esonucleolitica (mRNA per recettore transferrina, siRNA) Regolazione della traduzione degli mRNA: - sequenze regolative al 5’ UTR (mRNA per ferritina) - sequenze regolative al 3’ UTR (interazione tra miRNA e mRNA - lin4/lin14, let7/lin-41)
1997 - 100,000 human genes.
2001 - 75,000 - 84,000
Human Gene Index of the Institute for Genomic Research
Unigene database of the National Center for Biotechnology
2004 - 25.000 protein coding genes
2005 - FANTOM 3 project - 62% of the mouse genome is
181,000 independent transcripts,.
- many have alternative promoters and polyadenylation sites
- half are noncoding RNAs
- 70% of the mapped transcription units overlap to some extent with a transcript from the
opposite strand -
large - rRNA
small - snRNAs
A complex family of microscopic (21-23 nt long) non-coding RNAs
Model for a common pathway
in which miRNAs direct
and siRNAs direct target
RNA destruction (RNAi)
The overexpression of the CHS (Chalcone synthase) gene in petunia
leads to lack of flower pigmentation instead of increasing it.
Napoli et al., (1990)
Jorgensen et al., (1996)
The transgene causes the suppression of both the exogenous and
RNAi correlates with the production of small RNAs
•In plants, during PTGS, small
RNA of 25 nt are found. They
are absent in control plants.
•Such RNAs are
complementary to both the
sense and antisense sequences
of the silenced gene
Hamilton e Baulcombe (1999)
siRNAs have a well defined structure
19 nt duplex
2 nt 3’ overhangs
Dicer has two RNase III domains
Mutants in the Dicer gene abolished RNAi
A model of dsRNA processing by Dicer
• Dicer functions as a monomer (i.e., intra-molecular y-dimer)
• PAZ domain recognizes the end
• Dicer has a single processing center, with two independent
• Each RNase III domain cuts one RNA strand in a polar way
Family of Argonaute proteins
Ago proteins in different
Carmell & Hannon, 2002
Role in RNAi/miRNAs: • components of RISC/miRNPs
• bind siRNAs/miRNAs
• 4 Argonautes (Ago1-4)
• Ago2 is a “Slicer” (Piwi ~ RNaseH)
Meccanismo dell! RNAi
Lee RC, Feinbaum RL, Ambros V.
Cell. 1993 75:843-54.
The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense
complementarity to lin-14.
lin-4 is essential for the normal temporal control of diverse postembryonic developmental events in
C. elegans. lin-4 acts by negatively regulating the level of LIN-14 protein, creating a temporal
decrease in LIN-14 protein starting in the first larval stage (L1). We have cloned the C. elegans
lin-4 locus by chromosomal walking and transformation rescue. We used the C. elegans clone to
isolate the gene from three other Caenorhabditis species; all four Caenorhabditis clones functionally
rescue the lin-4 null allele of C. elegans. Comparison of the lin-4 genomic sequence from these four
species and site-directed mutagenesis of potential open reading frames indicated that lin-4 does
not encode a protein. Two small lin-4 transcripts of approximately 22 and 61 nt were identified
in C. elegans and found to contain sequences complementary to a repeated sequence element in
the 3' untranslated region (UTR) of lin-14 mRNA, suggesting that lin-4 regulates lin-14
translation via an antisense RNA-RNA interaction.
Wightman B, Ha I, Ruvkun G.
Cell. 1993 75:855-62.
Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates
temporal pattern formation in C. elegans.
During C. elegans development, the temporal pattern of many cell lineages is specified by graded activity of the
heterochronic gene Lin-14. Here we demonstrate that a temporal gradient in Lin-14 protein is generated
posttranscriptionally by multiple elements in the lin-14 3'UTR that are regulated by the heterochronic gene Lin-4.
The lin-14 3'UTR is both necessary and sufficient to confer lin-4-mediated posttranscriptional temporal regulation. The
function of the lin-14 3'UTR is conserved between C. elegans and C. briggsae. Among the conserved sequences are
seven elements that are each complement… Espandi »ary to the lin-4 RNAs. A reporter gene bearing three of these elements
shows partial temporal gradient activity. These data suggest a molecular mechanism for Lin-14p temporal gradient
formation: the lin-4 RNAs base pair to sites in the lin-14 3'UTR to form multiple RNA duplexes that downregulate lin-14 translation.
lin-4 loss-of-function mutations display reiterations of early fates at inappropriately late
developmental stages; cell lineage patterns normally specific for the L1 are reiterated
at later stages. The consequences of these heterochronic developmental patterns include
the absence of adult structures (such as adult cuticle and the vulve) and the prevention of
lin-14 null mutations cause a phenotype opposite to that of lin-4 and are completely epistatic
to lin-4, which is consistent with lin-4 acting as a negative regulator of lin-14.
lin-14 mutants skip the expression of L1-specific events and precociously execute programs
normally specific for L2. L3, L4 and adult stages.
Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie
AE, Horvitz HR, Ruvkun G.
Nature. 2000 403:901-6.
The 21-nucleotide let-7 RNA regulates developmental timing in
The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that
are temporally controlled to specify the timing of developmental events. Mutations in
heterochronic genes cause temporal transformations in cell fates in which stage-specific
events are omitted or reiterated. Here we show that let-7 is a heterochronic switch gene.
Loss of let-7 gene activity causes reiteration of larval cell fates during the adult
stage, whereas increased let-7 gene dosage causes precocious expression of adult
fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is
complementary to elements in the 3' untranslated regions of the heterochronic genes lin-14,
lin-28, lin-41, lin-42 and daf-12, indicating that expression of these genes may be directly
controlled by let-7. A reporter gene bearing the lin-41 3' untranslated region is temporally
regulated in a let-7-dependent manner. A second regulatory RNA, lin-4, negatively regulates
lin-14 and lin-28 through RNA-RNA interactions with their 3' untranslated regions. We
propose that the sequential stage-specific expression of the lin-4 and let-7 regulatory
RNAs triggers transitions in the complement of heterochronic regulatory proteins to
coordinate developmental timing.
the first microRNA
Cell. 1993 75:843-54, 855-862
Nature. 2000 403:901-6
C. elegans and vertebrates
both involved in developmental regulation
A model for the successive regulation of heterochronic gene activities by the lin-4 and let7 RNAs. LIN-14 and LIN-28 expression levels are decreased by lin-4 RNA expression at
the end of the first larval stage to allow progression to late larval stages. In late larval
stages, the expression of LIN-41 and other genes may be similarly downregulated by the
let-7 RNA, relieving their repression of LIN-29 protein expression and allowing
progression to the adult stage. Because the lin-29 mRNA does not contain sites
complementary to the let-7 RNA, lin-29 is not likely to be a direct target of let-7.
lin-4 and let-7 displayed partial complementarity
to the 3’ UTR of lin-41 and lin-14 mRNAs
lin-4 and let-7 were described as translational
repressors of their target mRNAs
Small RNA binding modes
Examples of the imprecise base pairing of
animal miRNAs with their targets. The lin-4
miRNA is shown with its complementary sites
in lin-14 (a) and lin-28 (b). There are several
further complementary sites of imprecise base
pairing in the 3' UTR of lin-14; only one site
is predicted for lin-4 in the lin-28 3' UTR. c,
During larval development of C. elegans, lin4 coordinates the downregulation of LIN-14
and LIN-28 protein concentrations, which in
turn regulates the expression of stage-specific
Approaches to miRNA gene
discovery and the functional
characterization of miRNA
approaches to the study of
developmental timing in C.
elegans identified lin-4 and let7; and genetic analysis of the
specification of C. elegans
neuronal cell type identified lsy6. Genetic analysis of mutations
affecting programmed cell death
in Drosophila led to the cloning
of bantam25, 26 and mir-14
miRNA genes. Examples of
miRNAs that were identified by
genomics, and whose functions
were subsequently analysed
using reverse genetics are mouse
miR-181 and mir-273.
hundreds of microRNAs!
“Identification of novel genes coding for small expressed RNAs”
(Lagos-Quintana et al., 2001)
In questo lavoro,vennero
individuati dei piccoli RNA
non codificanti in cellule
umane HeLa ed i « Comprimi
Regolazione dell' mRNA
Materiale didattico per il corso di Biologia Molecolare II della Prof.ssa Irene Bozzoni, all'interno del quale sono affrontati i seguenti argomenti: la regolazione della stabilità degli mRNA e la regolazione della traduzione degli mRNA; RNA interference (RNAi); il meccanismo dell'interferenza dell'RNA.