1. Classical and auxiliary splicing signals (n = G, A, U, or C; y = pyrimidine; r = purine). (A) Classical splice sites: The classical splicing signals found in the major class (>99%) of human introns are required for recognition of all exons. There is also a minor class of introns using different classical sequences and different spliceosome components (Tarn and Steitz 1997). (B) Classical and auxiliary splicing elements and binding factors: Factors that bind classical and auxiliary splicing elements. Auxiliary elements within exons (ESEs and ESSs) and introns (ISEs and ISSs) are commonly required for efficient splicing of constitutive and alternative exons. Intronic elements also serve to modulate cell-specific use of alternative exons by binding multicomponent regulatory complexes. (C) Cis-acting splicing mutations. Mutations that disrupt cis-acting elements required for premRNA splicing can result in defective splicing that causes disease.

2. Splicing del gene Dscam

3. 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

4. Comparative genomics of splicing  levels in several well-studied metazoans
Humanb
Mouseb
Flyc
Wormc
Genome  size
3,300 MB
165 MB
100 MB
Protein-coding genes
22,180
22,740
13,937
20,541
Multiexonic genes (percentage with 2+
isoforms)
21,144 (88%)
19,654 (63%)
11,767 (45%)
20,008 (25%)
Isoforms (average number per gene)
215,170 (3.4)
94,929 (2.4)
29,173 (1.9)
56,820 (1.2)
Average number of unique exons per gene(median)
33 (26)
22 (15)
7.5 (4)
8.6 (6)
Average number of unique introns per multiexonic gene (median)
28 (21)
19 (12)
8.7 (5)
7.2 (5)
Average exon length (median length)
320 bp (145 bp)
323 bp (141 bp)
494 bp (272 bp)
222 bp (157 bp)
Average intron length (median length)
7,563 bp (1,964 bp)
6,063 bp (1,693 bp)
2,068 bp (642 bp)
561 bp (354 bp)
Genes (all)
63,677
39,179
15,682
46,726
Isoforms (all) (average number per gene)
aOn the basis of both initial (92, 93) and more recent deep (5, 6) RNA-sequencing (RNA-seq) data, 95% (92, 93) to 100% (5, 6) of human genes may
encode two or more (2+) isoforms, and other vertebrates, especially primates, may be similar in that most of those genes also encode 2+ isoforms (5, 6).
Relevant Drosophila RNA-seq data are from References 255 and 256, and relevant Caenorhabditis elegans RNA-seq data are from Reference 257.
bThe numbers are based on annotations from Ensembl (which does not use RNA-seq data for annotations). For current Ensembl versions of human and
mouse gene/transcriptome annotations, see and
musculus/Info/Annotation.
cThe Drosophila and C. elegans gene/transcriptome annotations were imported from FlyBASE andWormBASE, respectively; see

5. Splicing alternativo

6. 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.

7. Regolazione dello splicing

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

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

10. Regolazione positiva dello splicing

11. Regolazione negativa dello splicing

12. Alternative splicing of the Fas receptor pre-mRNA

13. 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

14. 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)

15. Poliadenilazione alternativa

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

17. Ruolo della poliadenilazione alternativa

19. 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.

20. Scelta del sito di splicing/poliadenilazione

22. 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

26. Struttura del poro nucleare

28. 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

30. Substrato
Carrier

33. 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.

34. Segnali e recettori per il trasporto verso il nucleo

35. Segnali e recettori per il trasporto verso il citoplasma

36. Segnali e recettori per lo “shuttling”

37. Studio del trasporto citoplasma-nucleo

39. 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.

40. Trasporto dell'mRNA
 Figure 1 | Steps of mRNA export from nuclear transcription to the cytoplasm. The life cycle of a messenger ribonucleoprotein particle (mRNP) is shown, from its biogenesis and maturation into an export-competent form to its subsequent transport from sites of transcription and processing in the nucleus to the cytoplasm, where it is extensively remodelled. These steps are all coupled; for example, transcription-export complex TREX is recruited to the nascent mRNP by the splicing machinery. After a mature mRNP has been generated, the conserved nuclear RNA export factor 1 (NXF1) is recruited to the mRNP through direct interactions with several TREX components and SR splicing factors. Cargo mRNAs from both TREX and TREX-2 are transferred to NXF1 and its cofactor p15 for transit through the nuclear pore by interacting directly with the nucleoporins that line the pore. The kinetics of translocation of mRNAs from sites of transcription through nuclear pore complexes (NPCs) in mammalian cells suggest that transport to NPCs can take several minutes, whereas translocation through NPCs takes milliseconds. It is unknown whether TREX and TREX-2 cooperate to export the same transcripts or mediate alternative export routes and whether TREX-2 is recruited to transcripts in the nuclear interior as in the case of TREX. CBC, cap-binding complex; eIF4E, eukaryotic translation initiation factor 4E; EJC, exon–junction complex; InsP6, inositol hexakisphosphate; PABP, poly(A)-binding protein; Pol II, RNA polymerase II. 

41. Trasporto dell'mRNA
Figure 3 | Examples of biological pathways that are regulated by selective mRNA export. Components of transcription-export complex TREX, such as ALY, THO complex subunit 2 (THOC2) and THOC5, contribute to the selective export of a subset of mRNAs and thus to regulation of specific biological processes including maintenance of pluripotency, haematopoiesis, heat shock and safeguarding of genome integrity. TREX-2, through germinal centre-associated nuclear protein (GANP), also functions in the export of transcripts that are required for gene expression. Only the components of TREX and TREX-2 complexes that have been shown to contribute to selectivity are indicated. It is unknown whether the complete TREX and TREX-2 complexes contribute to selectivity, and thus they are represented as transparent in the figure. Although most mRNAs use TREX, TREX-2 and nuclear RNA export factor 1 (NXF1) receptors to transit through nuclear pore complexes (NPCs), a subset of mRNAs use chromosome region maintenance 1 protein homologue (CRM1), which is the main protein export receptor. Eukaryotic translation initiation factor 4E (eIF4E) and CRM1 preferentially export a subset of mRNAs that encode proteins involved in proliferation, survival, metastasis and invasion. Nucleoporins such as nuclear pore complex protein NUP96, which is a constituent of the NUP107–NUP160 complex, may contribute to the export of specific subsets of transcripts, such as those encoding cell cycle regulators and immune response factors. Whether NUP96 achieves this by modulating interactions of mRNA export factors at the NPC or in the nuclear interior, where a proportion of NUP96 is thought to localize, is unknown. Each of these potential pathways, together with the functional subset of transcripts that they export, is shown schematically. One important unresolved question is whether TREX and TREX-2 mediate alternative export routes, or whether they cooperate to export the same transcripts. IPMK, inositol polyphosphate multikinase; LRPPRC, leucine-rich PPR motif-containing protein; PtdIns(3,4,5)P3, phosphatidylinositol‑3,4,5‑trisphosphate. 

44. 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).

45. 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.

46. 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.