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)