Precisión en la elaboración de conjeturas

For an individual nascent mRNA, introns must be removed and exons have to be precisely joined together to maintain the reading frame. Constitutive exons are constantly included in mature transcripts, while there are several types of variable exons (Figure 1.5.1a). A cassette exon is one that is either included or excluded (skipped) from mature mRNA. As opposed to exon skipping, some introns can be variably retained. Two neighbouring exons can be included in a mutually exclusive pattern. Differential splice sites at the 5’ or 3’ end can change the exon length. Transcript can have alternative initiations and first exons or termination and 3’ exon (Smith, C.W. and Valcárcel, J. 2000; Xing, Y. and Lee, C. 2006; Hu, A. and Fu, X.D. 2007). Exon skipping is the most frequent alternative splicing events in mammals (Sugnet, C.W. et al. 2004).

Figure 1.5.1a: pre-mRNA splicing patterns and functional complexity

Splicing regulators, e.g. SR proteins and heterogeneous nuclear ribonucleoproteins, modulate alternative splicing of nascent messenger RNAs in different patterns in a gene specific, tissue dependent, or cell signal dependent manner, which alters the structure of the mature mRNA transcripts as well as the function of proteins isoforms (Hu, A. and Fu, XD. 2007).

Figure 1.5.1b: Splicing machinery

Stepwise assemble of spliceosome, a multiple snRNP complex, to the target exon-intron boundary to cleave the intron promoted by SR protein binding to the exonic splice enhancer (Li, Q. et al. 2007).

Alternative splicing is directed by a complex set of splicing codes consisting of consensus sequences of splicing sites at exon-intron boundaries and exonic or intronic regulatory elements called exonic splice enhancers (ESE) or intronic splice enhancers (ISE), and exonic or intronic splice silencers (ESS/ISS) (Black, D.L. 2003). The

splicing sites are critical for all exons and were identified from alignment of intron- exon boundary sequences (Berget, S.M. 1995). The splice site sequences are recognized by small nuclear RNAs (snRNAs) or small nuclear ribonucleoprotein particles (snRNPs) through specific base pair interaction (Jurica, M.S. & Moore, M.J. 2003). During splicing, five snRNA / snRNPs assemble in a stepwise manner to form the splicing machinery referred to as the spliceosome. U1 snRNP binds to 5’ splice site at a conserved GU dinucleotide, U2AF (U2 snRNP auxiliary factor) recognizes the 3’ splice site polypyrimidine tract and a conserved AG dinucleotide with its 65 and 35 kD subunits respectively (E complex) (Lim, S.R. & Hertel, K.J. 2004; Chiara, M.D. & Reed, R. 1995). U2AF65 then recruits U2 which is specific for the branch point sequence at the 3’ splice site (A complex). Subsequently the U4/U6-U5 complex joins (B complex) and remodelling of RNA and protein components in the B complex forms an enzymatically active C complex to catalyse the RNA-RNA trans- esterification reactions to cleave the intron and connect the exons (Martin, A.J. et al. 2005) (Figure 1.5.1b).

Conserved sequences at about 10 nucleotides serve as splicing enhancers and / or silencers, and occur near the exon/intron boundaries. This directs the spliceosome machinery to the right splice site rather than potential cryptic sites of the pre-mRNA. The action of two classes of splicing regulatory proteins, Serine/Arginine rich proteins (SR proteins) and heterogeneous nuclear ribonucleoproteins (hnRNPs) are best studied (Zhu, J. et al. 2001). SR proteins bind to splicing enhancers to initiate exon recognition and inclusion (Fairbrother, W.G. 2002; Reddy, A.S. 2007), although they may inhibit splicing as well (Barnard, D.C. 2002). SR proteins have two common domains: the RNA recognition motif (RRM) and the RS domain with repeated

arginine/serine dipeptides. The RRM (also referred to as the RNA binding domain, RBD) targets the protein to a particular RNA sequence, whereas the RS domain modifies the affinity of RNA binding. SR proteins bind both ESE and ISE to positively regulate alternative splicing through recruiting the spliceosome to the adjacent exon (Black, D.L. 2003; Graveley, B.R. 2000). SR protein binding sites are found in both constitutive and variable exons suggesting their roles in both constitutive and alternative splicing (Mayeda A, et al. 1999; Stark, J.M. et al. 1999).

The hnRNP proteins are a large group of proteins associated with mRNA precursors that were termed heterogeneous nuclear RNAs (hnRNAs) to describe their size heterogeneity and cellular location (Dreyfuss, G. et al. 1993). hnRNPs are highly conserved among vertebrates. Most hnRNP proteins have one or more RRMs which are also found in many other RNA binding proteins. The RRM domain comprises approximately 90 amino acids with two consensus sequences termed, RNP1 and RNP2 submotifs. RNP1 is the most conserved octapeptide segment in RRM that is comprised of Lys/Arg-Gly-Phe/Tyr-Gly/Ala-Phe-Val-XPhe/Tyr. RNP2 is a less conserved hexapeptide sequence of Ile/Val/Leu-Phe/Tyr-Ile/Val/Leu-X-Asn-Leu. The aromatic amino acids Phe/Tyr in RNP1 and RNP2 are essential for RNA binding (Dreyfuss, G. et al. 1993; Birney, E. et al. 1993). From studies on the best characterised hnRNP A1 and PTB (Polypyrimidine Tract Binding protein, or hnRNP I), it has been shown that hnRNPs repress spliceosomal assembly either by multimerization along exons or by blocking the recruitment of snRNPs to loop out exons (Zhu, J. et al. 2001; Tange, T.O. et al. 2001; Sharma, S. 2005; Martinez- Contreras, R. 2006). Roles of some hnRNPs in alternative exon splicing have been established, as well as evidence that hnRNPs regulate constitutive splicing events

(Pozzoli, U. and Sironi, M. 2005). Major splice regulators identified so far are listed in table 1.4.

Name Domains Binding Sites targets

9G8/Sfrs7 RRM, RS, C2HC Znf (GAC)n Tau, GnRH, 9G8 SC35/Sfrs2 RRM, RS UGCUGUU AChE SRp55/Sfrs6 RRM, RS GGCAGCACCUG cTnT, CD44 SRp75/Sfrs4 RRM, RS GAAGGA FN1, E1A, CD45 SR / SR related proteins SRm160/Srm1 RS, PWI AUGAAGAGGA CD44

hnRNP A1 RRM, RGG UAGGGA/U Hipk3, Smn2, c-H-ras

hnRNP H

RRM, RGG,

GYR, GY GGGA, G-rich PLP, HIV tat, Bcl-x

hnRNP I / PTB RRM UCUU, CUCUCU nPTB, c-SRC, Fas, cTnt, hnRNP A1 etc hnRNP L RRM C/A-rich eNOS, CD45 hnRNPs hnRNP LL RRM ? CD45, Nalp1, etc

TIA1 RRM U-rich Fas, MYPT1, IL-8, etc

others

Sam68 KH A/U-rich Bcl-x

Table 1.4: Some key alternative splicing regulators (Gabut, M. et al. 2008)

The three dimensional structure of the RRM of many RNA binding proteins has been solved by X-ray diffraction and NMR. Most RRM structures consist of similar four strands and two helices arranged in an alpha/beta sandwich (β1-α1-β2-β3-α2-β4), although in some cases there is a third helix present. The RNP1 and RNP2 submotifs

are located in the central β3 and β1 strands with conserved aromatic residues contacting RNA (Dreyfuss, G. et al. 1993; Birney, E. et al. 1993).

RRM structure and its binding studies revealed that it can interact with target RNA or protein via its β sheet surface; it also interacts with proteins via α2 helix on the other side of RRM. The α3 helix of U2AF65’s interaction with the β sheet surface of RRM can occlude RNA binding. The α3 helix swings away upon RNA binding, facilitating protein dimerization and RNA binding in U1A RRM (Singh, R. and Valcárcel, J. 2005)

It has been an interesting issue how hnRNPs and SR proteins specifically bind to degenerate nucleic acid sequence and regulate target nascent mRNA splicing (reviewed by Singh, R. and Valcárcel, J. 2005). Splicing enhancer and silencer sequences have been defined for recognition by major SR proteins and hnRNPs respectively. However, these specific regulatory elements are wide spread throughout the genome and still provide complex potential high-affinity binding sites for alternative usage. Additional protein factors are required to build up the specificity of splicing including tissue or cell-type specific expression of RNA binding proteins and/or their intracellular localization; effects of their cofactors or interaction with other snRNPs; the splicing isoforms of hnRNPs and SR proteins themselves and the sensitivities to the specific context comprised of other cofactors; usage of multiple RRM domains and KH (hnRNP K homolog), another abundant RNA binding motif, and domain tethering; and alternative splicing in responding to specific cell signals (Singh, R. and Valcárcel,, J. 2005; Lareau, L.F. et al. 2007; Shin, C. and Manley, J.L. 2004; Rothrock, C. et al. 2003).

Dysregulation of alternative splicing resulting from mutations in splice sites or regulatory elements has been implicated in a wide spectrum of human diseases including cancer and autoimmune diseases. More than 15% of mutations causing genetic diseases affect pre-mRNA alternative splicing (Krawczak, M. 1992). There is increasing attention being given to developing therapeutic approaches that modulate alternative splicing in target cells.