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.
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. 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 …… …?… non-coding RNAs 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 siRNA miRNA 7m G AA A P Translational repression miRNA 7m AAA G Chromatin modification rasiRNA nuc nuc Model for a common pathway in which miRNAs direct translational repression and siRNAs direct target RNA destruction (RNAi) RNA interference 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 siRNAs Mutants in the Dicer gene abolished RNAi A model of dsRNA processing by Dicer Dicer DUF283 ATPase/helicase PAZ RIIIa RIIIb *dsRBD D1709, E1813 ATPase/Helicase, Helicase, DUF 283 DUF 283 D dsRB RIIIa D1320, E1364 , 5 , 3 RIIIb PAZ • 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 PAZ ~900aa Hiwi/Piwi Ago proteins in different organisms Ago Mammals Drosophila C. elegans Carmell & Hannon, 2002 Role in RNAi/miRNAs: • components of RISC/miRNPs • bind siRNAs/miRNAs Mammals: • 4 Argonautes (Ago1-4) • Ago2 is a “Slicer” (Piwi ~ RNaseH) 8 5 27 RNAi mechanism 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. Her… Espandi »e 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 complementary 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 lin-4 5’-ucccugagaccucaaguguga-3’ …2000… C. elegans the second Nature. 2000 403:901-6 5’-ugagguaguagguuguauaguu-3’ let-7 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. stRNAs lin-4 and let-7 displayed partial complementarity to the 3’ UTR of lin-41 and lin-14 mRNAs lin-41 3’UTR let-7 lin-4 lin-14 3’UTR lin-4 and let-7 were described as translational repressors of their target mRNAs Small RNA binding modes degradation translational repression. 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 devel « Comprimi