1. Complessità genoma-proteoma
La percentuale di geni con splicing alternativo arriva al 95%
Il gene Dscam di Drosofila (recettore per la guida degli assoni) contiene 95 possibili esoni che fanno splicing alternativo per un totale di isoforme proteiche possibili
Il 15% delle mutazioni che causano malattie genetiche provocano difetti di splicing

2. Splicing alternativo

3. Maturazione alternativa e capacità codificante
Figure 2. Alternative splicing generates variable segments within mRNAs. Alternative promoters: Selection of one of multiple first exons results in variability at the 5_ terminus of the mRNA (1). The determinative regulatory step is selection of a promoter rather than splice-site selection. The effect on the coding potential depends on the location of the translation initiation codon. If translation initiates in at least one of the first exons, the encoded proteins will contain different N termini. Alternatively, if translation initiates in the common exon, the different mRNAs will contain different 5_ untranslated regions but encode identical proteins. Red indicates variable regions within the mRNA and encoded protein. Alternative splicing of internal exons: Alternative splicing patterns for internal exons include cassette (2), alternative 5_ splice sites (3), alternative 3_ splice sites (4), intron retention (5), and mutually exclusive (6). The variable segment within the mRNA results from insertion/deletion, or a mutually exclusive swap. The effects on coding potential are an in-frame insertion or deletion, a readingframe shift, or introduction of a stop codon. mRNAs containing a stop codon >50 nt upstream of the position of the terminal intron are degraded by nonsensemediated decay (see text). Therefore, introduction of a premature termination codon into an mRNA by alternative splicing can be a mechanism to down-regulate expression of a gene. Alternative terminal exons: The 3_ end of an mRNA is determined by a directed cleavage event followed by addition of the poly(A) tail (Proudfoot et al. 2002). Selection of one of multiple terminal exons (7) results from a competition between cleavage at the upstream poly(A) site or splicing to the downstream 3_ splice site. There are also examples of competition between a 5_ splice site and a poly(A) site within an upstream terminal exon (8). Variability at the 3_ end of the mRNA produces either proteins with different C termini or mRNAs with different 3_-UTRs.

4. Regolazione dello splicing
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

5. Controllo negativo
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

6. Controllo positivo enhancer attivatore
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

7. Regolazione positiva dello splicing

8. Regolazione negativa dello splicing

9. Alternative splicing of the Fas receptor pre-mRNA

10. tra
sxl
Figure 2 Regulation of alternative pre-mRNA splicing in the Drosophila sex-determination pathway. a, Alternative selection of 3’ splice sites preceding exon 2 of tra pre-mRNA is regulated by the SXL protein. In males, the splicing factor U2AF binds to the proximal 3’ splice site, leading to an mRNA containing a premature translational stop codon (UAG). In females, SXL binds to the proximal 3’ splice site, thus preventing the binding of U2AF. Instead, U2AF binds to the distal 3’ splice site, leading to an mRNA that encodes functional TRA protein. In all panels, the exons are indicated by coloured rectangles, while introns are shown as pale grey lines. b, Alternative inclusion of exon 3 of sxl pre-mRNA is regulated by SXL protein. In both males and females, the first step of the splicing reaction results in lariat formation at the branchpoint sequence upstream from the 3’ splice site preceding exon 3. Subsequently, the second-step splicing factor SPF45 binds to the AG dinucleotide of this splice site. In males, SPF45 promotes the second step of the splicing reaction, leading to the inclusion of exon 3. In females, SXL binds to a sequence upstream of the AG dinucleotide, interacts with SPF45 and inhibits its activity. This prevents the second step of the splicing reaction, leading to the exclusion of exon 3 and splicing of exon 2 to exon 4. Seven constitutively spliced exons are not shown. c, Alternative splicing of dsx pre-mRNA is regulated by the assembly of heterotrimeric protein complexes on female-specific ESEs. The first three exons are constitutively spliced in both sexes. In males, the 3’ splice site preceding exon 4 is not recognized by the splicing machinery, resulting in the exclusion of this exon, and splicing of exon 3 to exon 5. In females, the female-specific TRA protein promotes the binding of the SR protein RBP1, and the SR-like protein TRA2 to six copies of an ESE (indicated by green rectangles). These splicing enhancer complexes then recruit the splicing machinery to the 3’ splice site preceding exon 4, leading to its inclusion in the mRNA. In females, polyadenylation (pA) occurs downstream of exon 4, whereas in males it occurs downstream of exon 6. ‘S’ designates the splicing machinery.
dsx

11. Splicing alternativo
Esoni specifici possono essere inclusi o esclusi a seconda dell’uso dei siti di splicing
Alcuni esoni possono essere estesi o ridotti a seconda dell’uso dei siti di splicing
Splicing alternativo può dipendere da fattori positivi (proteine SR) o negativi (hnRNP)

12. Poliadenilazione alternativa

13. Ruolo della poliadenilazione alternativa
stabilità dell'mRNA
localizzazione dell'mRNA
espressione di proteine diverse
regolazione della traduzione

14. Ruolo della poliadenilazione alternativa

16. Scelta del sito di poliadenilazione
Figure 2 . Regulation of Immunoglobulin Expression Low affinity binding of CstF to the upstream ms site is indicated by a hatched pattern.

17. Scelta del sito di splicing/poliadenilazione

19. Scelta del sito di splicing/poliadenilazione
Sequenze cis-agenti che definiscono e/o favoriscono la scelta del sito di maturazione
Presenza (attività) di fattori specifici
Concentrazione (attività) di fattori costitutivi

23. Struttura del poro nucleare

25. Poro nucleare
Il complesso del poro nucleare (NPC) è grande 125 MDa (66 MDa in lievito)
2000 NPC nei vertebrati (200 in lievito)
NPC è composto da circa 1000 proteine di tipi diversi (di ciascuna almeno 8 copie)
Molecole fino a 9 nm (30-40 kDa) diffondono liberamente attraverso l’NPC
Molecole fino a 25 nm vengono attivamente trasportate attraverso l’NPC

27. Substrato
Carrier

30. Componenti del trasporto nucleare
Figure 1. Nuclear Transport Receptors and Adaptors unclear composition and function. The cytoplasmic filaments (pur-
Most known soluble transport receptors are large (90–130 kDa), ple) and nuclear basket (orange) are involved in the initial and termiacidic
proteins that are related by amino acid sequence. They consti- nal stages of translocation. The locations of some nucleoporins
tute a protein family referred to here as the nuclear transport recep- (Nups), as determined by immuno–electron microscopy, are inditor
family. Importin b and transportin, the first receptors to be de- cated. The gray portion represents the double membrane of the
scribed, mediate the import of basic NLS-bearing cargos and nuclear envelope. (This figure was adapted from Pante and Aebi,
M9-bearing cargos, respectively. The figure depicts the domain 1996.)
structure of the receptors (A), and of the adaptor proteins that mediate
the interaction of several cargos with importin b (B). IBB is the
importin b–binding domain. association of receptor–cargo complexeswith particular
The structures of an adaptor, importin a, and two import receptors, Nups triggers dissociation of a Nup–receptor–cargo
importin b and transportin, were recently reported (Mattaj and Conti, complex that moves through the pore as a unit (Nakielny
1999). The ARM repeats of importin a and the HEAT repeats of et al., 1999; Zolotukhin and Felber, 1999).
receptor proteins form nonglobular, superhelical structures, pre- It is unclear whether cargos move through an individsenting
extended surfaces that are perfectly designed for making ual NPC in both directions at the same time, or whether multiple contacts with cargos and regulatory molecules.

31. Segnali e recettori per il trasporto verso il nucleo

32. Segnali e recettori per il trasporto verso il citoplasma

33. Segnali e recettori per lo “shuttling”

34. Studio del trasporto citoplasma-nucleo

36. Trasporto dell'mRNA
Nuclear export of mRNA. mRNAs undergo several ordered processing steps before export to the cytoplasm. These maturation events are important for correct packaging of the mRNA. A key step is the deposition of the exon–exon junction complex (EJC) onto the mRNA. The EJC consists of multiple mRNA binding proteins including Y14, RNPS1, SRm160, DEK, Mago and Upf3 [39–48]. The EJC is dynamic and some components remain associated with the mRNA even after export to cytoplasm (EJC*). The EJC therefore links mRNA processing and transport to cytoplasmic events such as cytoplasmic localization and nonsense-mediated decay (NMD). In NMD, a premature stop codon is recognised if it is upstream of a splice junction. The EJC* is able to communicate the relative position of the splice junction to the NMD machinery. A translating ribosome encountering a stop codon is depicted in grey. An alternative exit route was described for ARE-containing mRNAs [64]. ARE-containing mRNAs can gain access to transportin 2 (Trn2) or Crm1 via the ARE-interacting protein HuR, either directly or through the additional adaptors pp32/April.

39. Figure 3. The Ran GTPase Cycle
Ran is maintained as RanGTP in the nucleus by the activity of the Ran GTP-GDP exchange factor (RanGEF or RCC1) and as RanGDPin the cytoplasm by the RanGTPase activating protein (RanGAP). RanBP1 in the cytoplasm and RanBP1-like domains on the cytoplasmic fibrils of the NPC are coactivators of RanGAP (not shown).
The structures of RanGDP, RanGEF, RanGAP and a RanGTP-RanBP1 domain complex have been solved, providing structural explanations for some of the biochemical properties of Ran and its regulators, and highlighting the importance of the C-terminal extension of Ran, which is unique among small GTPases. This C-terminal region functions as a novel molecular switch (Macara, 1999).

40. Figure 4. The Differential Effects of RanGTP on Nuclear Import and Nuclear Export Receptor- Cargo Complexes Import receptors bind their cargos in a RanGTP-independent manner and RanGTP causes dissociation of these complexes. They are thus permitted to form in the cytoplasm and dissociate in the RanGTP-rich nucleus. Export receptors form stable complexes with their cargos only in the presence of RanGTP. These ternary complexes are thought to be the export unit, and dissociate in the cytoplasm and/or on the cytoplasmic filaments of the pore where RanGAP activity converts the RanGTP to RanGDP. Crystal structures of import receptors bound to cargo (importin b–IBB complex) and bound to Ran (transportin-RanGppNHp and importin b–RanGppNHp complexes) suggest how Ran may mediate cargo unloading (Macara, 1999; Mattaj and Conti, 1999). Ran contacts an acidic loop in the central region of the receptor molecules, and this interaction is probably responsible, at least in part, for cargo displacement. Structures of export receptors have not yet been reported.

41. Transport through the nuclear pores
The NLS and NES consist of short sequences that are necessary and sufficient for proteins to be transported through the nuclear pores.
Transport receptors have the dual properties of recognizing NLS or NES sequences and binding to the nuclear pore.
The direction of transport is controlled by the state of the monomeric G protein, Ran.
The nucleus contains Ran-GTP, which stabilizes export complexes, while the cytosol contains Ran-GDP, which stabilizes import complexes.
The mechanism of movement does not involve a motor.