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.
1997 - 100,000 human genes. Incyte Genomics 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 transcribed. 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 -
Eukaryotic RNAs coding RNAs large - rRNA Xist
small - snRNAs snoRNAs scRNAs gRNAs miRNAs siRNAs rasiRNAs …?….
splicing modification transl. control editing transl.control RNA stability chromatin
A complex family of microscopic (21-23 nt long) non-coding RNAs RNA cleavage
Model for a common pathway in which miRNAs direct translational repression and siRNAs direct target RNA destruction (RNAi)
Cosuppression 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 endogenous genes
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 dsRNA
Mutants in the Dicer gene abolished RNAi
A model of dsRNA processing by Dicer Dicer
ATPase/Helicase, Helicase, DUF 283 DUF 283
RIIIa D1320, E1364
, 5 , 3
• 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 catalytic sites • Each RNase III domain cuts one RNA strand in a polar way
Family of Argonaute proteins Piwi
Ago proteins in different organisms
Mammals Drosophila C. elegans
Carmell & Hannon, 2002
Role in RNAi/miRNAs: • components of RISC/miRNPs • bind siRNAs/miRNAs
• 4 Argonautes (Ago1-4) • Ago2 is a “Slicer” (Piwi ~ RNaseH)
8 5 27
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 egg laying. 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 Caenorhabditis elegans. !
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.
…1993… 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 developmental events.
Approaches to miRNA gene discovery and the functional characterization of miRNA genes. Forward genetics 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.
…after 2000 hundreds of microRNAs! all organisms and viruses
“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