1. Effetto di mutazioni silentiwt
samesense mutation
Exonic point mutations that cause altered
splicing. (a) Nonsense mutations (red circles)
can result in both degradation of spliced
mRNAs (nonsense-mediated decay) and
abnormal splicing, often manifested as
skipping of the mutant exon. The skipping of
exons containing nonsense mutations may
result from presumptive nuclear reading frame
scanning or disruption of secondary structure.
(b) Exon skipping can also result from the
disruption of a splicing enhancer sequence
within the skipped exon. In the case of ESE
abrogation, aberrant exon skipping may be
associated with any type of base substitution
in the exon (black circle).
protein
protein

2. Mutazioni negli esoni possono alterare lo splicing
samesense mutation
Exonic point mutations that cause altered
splicing. (a) Nonsense mutations (red circles)
can result in both degradation of spliced
mRNAs (nonsense-mediated decay) and
abnormal splicing, often manifested as
skipping of the mutant exon. The skipping of
exons containing nonsense mutations may
result from presumptive nuclear reading frame
scanning or disruption of secondary structure.
(b) Exon skipping can also result from the
disruption of a splicing enhancer sequence
within the skipped exon. In the case of ESE
abrogation, aberrant exon skipping may be
associated with any type of base substitution
in the exon (black circle).

3. Il caso della SMA (Spinal Muscolar Atrophy)
C>T (silent)

4. Mutazioni negli esoni possono alterare lo splicing

5. Proteine SR
Schematic diagram of human SR proteins and SR related proteins+
A: The domain structures of the known members of the human SR protein family are depicted+
RRM: RNA recognition motif; RRMH: RRM homology; Z: zinc knuckle, RS: arginine/ serine-rich domain+
B: The domain structures for some of the human SRrps that participate in pre-mRNAsplicing are depicted+
All proteins, with the exception of SRm300, are drawn to scale+
RRM: RNA recognition motif; RS: arginine/serine-rich domain; Zn: zinc finger; DEXD/H Box: motif characteristic of RNA helicases+

6. Ruolo dele proteine SR
RS domain-dependent and -independent activities of SR proteins. (a) The RS domain of SF2/ASF is needed to mediate splicing that depends on intron-definition interactions. The RS domain of SF2/ASF may interact with the RS domains of U1-70K and U2AF35 to facilitate cross-intron bridging. (b) Splicing of some introns with a weak Py tract requires U2AF35 binding at the 3′ss, as well as the RS domain of SF2/ASF. (c) The RS domain and U2AF35 are dispensable for splicing of some introns with strong splice sites. The RS domain may not be required for mediating the activity of certain ESEs. In this case, two possible mechanisms are envisaged: first, other portions of the protein, besides the RS domain, may mediate critical protein–protein interactions; second, binding of SF2/ASF to the pre-mRNA via the RRMs may be sufficient to promote splicing by competing with inhibitory factors (I), such as hnRNP proteins, which have antagonistic activities in splice-site selection.

7. Proteine SR
Famiglia di fattori di splicing che hanno uno o due RRM seguito da un dominio RS
Possono legare gli enhancer di splicing (ESE) e interagire con altre componenti dell’apparato di splicing

8. 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.

9. Il caso della SMA (Spinal Muscolar Atrophy)

10. Definizione dell'esone
Exon recognition. The correct 5' (GU) and 3' (AG) splice sites are recognized by the splicing machinery on the basis of their proximity to exons. The exons contain exonic splicing enhancers (ESEs) that are binding sites for SR proteins. When bound to an ESE, the SR proteins recruit U1 snRNP to the downstream 5' splice site, and the splicing factor U2AF (65 and 35 kDa subunits) to the pyrimidine tract (YYYY) and the AG dinucleotide of the upstream 3' splice site, respectively. In turn, U2AF recruits U2 snRNP to the branchpoint sequence (A). Thus, the bound SR proteins recruit splicing factors to form a ‘cross-exon’ recognition complex. SR proteins also function in ‘cross-intron’ recognition by facilitating the interactions between U1 snRNP bound to the upstream 58 splice site and U2 snRNP bound to the branchpoint sequence.

11. Transcription termination
RNA polymerase I terminates transcription at an 18 base terminator sequence.
RNA polymerase III terminates transcription in poly(U)4 sequence embedded in a G·C-rich sequence.
In pol II transcripts the sequence AAUAAA is a signal for cleavage to generate a 3 end of mRNA that is polyadenylated.

13. Complesso proteico per la formazione del 3'
CPSF riconosce seq. AAUAAA
CstF lega GU, stimola
CFI, CFII endonucleasi
PAP Poli(A) Polimerasi
PABP Poly(A) Binding Protein

16. RNA polymerasi II termination
The reaction requires a protein complex that contains a specificity factor, an endonuclease, and poly(A) polymerase.
The specificity factor and endonuclease cleave RNA downstream of AAUAAA.
The specificity factor and poly(A) polymerase add ~200 A residues processively to the 3 end.

22. Messenger RNA factory dynamics
Messenger RNA factory dynamics. The pol II CTD is phosphorylated predominantly on Ser5 shortly after initiation of the RNA chain. During elongation, there is a net loss of Ser5 phosphates (P5) and a net gain of Ser2 phosphates (P2). Capping enzymes guanylyltransferase (GT) and methyltransferase (MT), as well as cleavage/polyadenylation factors (C/P), are recruited at the 5′ end of the gene. During elongation, GT is selectively released and more C/Ps are progressively recruited.

24. Fosforilazione della coda CTD
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

25. Fosforilazione della coda CTD
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

26. “Fabbrica dell’RNA” (RNA factory)
Interdipendenza tra trascrizione e modificazioni post-trascrizionali
Pol II senza CTD inibisce splicing, terminazione e poliadenilazione
CPSF e CstF legano il CTD
CPSF si trova associato a TFIID

27. Splicing del gene Dscam

28. Complessità genoma-proteoma
Percentuale di geni con splicing alternativo varia tra 35 e 59
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

29. Splicing alternativo

30. 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.

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

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

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

34. 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

35. Alternative splicing
Specific exons may be excluded or included in the RNA product by using or failing to use a pair of splicing junctions.
Exons may be extended by changing one of the splice junctions to use an alternative junction.
Alternative splicing may depend on SR proteins or specific factors

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

39. Figure 2 . Regulation of Immunoglobulin Expression Low affinity binding of CstF to the upstream ms site is indicated by a hatched pattern.

42. 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

48. 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

50. Substrato
Carrier

59. 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.

62. 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).

63. 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.

64. 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.