Nivel de desarrollo del lenguaje

__________________________ Homology Domain-structure References

SRrps from humans U l 70K U2AF65 U2AF35 H C Cl H RH l CIk-1 Clk-2 Clk-3 hPRP16 HEL117 B1C8-160 kDa B4A11-300 kDa Sipl HsSWAP U2AF65 Pep 22 Clk-1 Clk-1 P rp l6 RRM-RS RS-3 RRMs RS RS-3 RRMs DEAH-RS SWAP SWAP RS-DEAH DEAD-RS SURP-RS SURP-RS (Spritz et ak, 1987) (Query et ak, 1989) (Zamore & Green, 1989) (Zhang et ak, 1992) (Imai et ak, 1993) (One et ak, 1994)

(Johnson & Smith, 1991) (Hanes et ak, 1994) (Hanes et ak, 1994) (Schwer&Guthrie, 1992) (Sukegawa&Blobel, 1995) (Blencowe et ak, 1995) (Blencowe et ak, 1995) (Zhang and Wu, 1998) (Denhez & Lafyatis, 1994) SRrps from other species

Tra {Drosophila) RS (Boggs et ak, 1987)

Tra-2 {Drosophila) RS (Amrein et ak, 1988)

SWAP {Drosophila) SURP-RS

(Goralski et ak, 1989) (Chou et ak, 1987) xU 1 70k {Xenopus) = U1 70k (Etzerodt et ak, 1988) mU2AF65 (mouse) = U2AF65 RS-3 RRMs (Sailer et ak, 1992) U 2afbp-rsl (mouse) - U2AF35 RS (Hayashizaki et ak, 1994) U2afbp-rs2 (mouse) = U2AF65 RS-3 RRMs (Yamaoka et ak, 1995) dU2AF50 {Drosophila) = U2AF65 RS-3 RRMs (Kanaar et ak, 1993) dU2AF38 {Drosophila) = U2AF35 RS (Rudner et ak, 1998) Prp2 {S. pombe) - U2AF65 RS-3 RRMs (Potashkin et ak, 1993) Prp22 {S. pombe) «H R H 1 RS-DEAH (Company et ak, 1991) Y C L llc {S. cerevisiae) - U2AF65 RS-3 RRMs (Birney et ak, 1992) MUD2 {S. cerevisiae) = U2AF65 RS-3 RRMs (Abovich et ak, 1994)

mClk-1/Sty (mouse) = Clk-1 (Ben David et ak, 1991)

RNA recognition motifs (RRM)

The most common RNA-binding motif in proteins binding to hnRNA (or pre-mRNA) is the canonical RNP consensus RNA-binding domain (CS-RBD, RNP or RRM motif, as it is called from now on (Bandziulis et ah, 1989; Dreyfuss et ah, 1993; Dreyfuss et ah, 1988; Query et ah, 1989). Phylogenetic analysis and sequence alignments revealed that this region of around 80 amino acids is highly conserved between different species in which current diversity arose through gene duplication with retention of its tertiary structure (Bimey et ah, 1993; Kenan et ah, 1991). This domain represents an essential part within a large family of many pre-mRNA and pre-rRNA processing factors including snRNP polypeptides, hnRNP proteins, and poly- (A) binding proteins.

The most conserved residues are located within two submotifs, the RNP-1 octamer and the RNP-2 hexamer, located about 30 amino acids apart. A structural model of two RRMs o f U l- snRNP A polypeptide as well as of hnRNP C1/C2 has been derived on the basis of X-ray crystallography and NMR spectroscopy (Hoffman et ah, 1991; W ittekind et ah, 1992), revealing a p i - a l - p 2 - |3 3 - a 2 - p 4 structure, with the RNP-1 and RNP-2 segments lying juxtaposed on the adjacent central antiparallel p3 and p i strands, respectively. Experiments employing protein-RNA UV-crosslinking (Kenan et ah, 1991) conforms with mutagenesis in conjunction with RNA binding studies (lessen et ah, 1991) providing evidence that the RNA stretches across the p-sheet to make contact with specific residues.

New evidence suggests that several aromatic residues within RNP-1 and RNP-2 of U l-snR N P A mediate ring-stacking interactions with single-stranded RNA bases (Oubridge et ah, 1994). Substitution o f these amongst other conserved residues by mutagenesis affect the binding to U1 snRN A (Scherly et ah, 1989). The atypical RRM , w hich contains an invariant heptapeptide, is less conserved between its RNP-1 and RNP-2 subm otives, although its tertiary fold is thought to be of similar structure to that of canonical RRMs (Birney et ah,

1993), suggesting that this region may bind to specific pre-m RNA sequence motifs not commonly shared.

The function of SR proteins

SR proteins have been shown to act both as specific regulators and as essential splicing factors in vitro and in vivo (Fu et ah, 1992; Ge and Manley, 1990; Krainer et ah, 1990;

Caceres et ah, 1994; Zahler et ah, 1992; Zahler et ah, 1993). Individual members of the SR protein family are able to complement splicing deficient cytoplasmic S I 00 extracts that were depleted in all SR proteins (Cavaloc et ah, 1994; Fu et ah, 1992; Ge et ah, 1991; Kim et ah, 1992; Krainer et ah, 1990; Krainer et ah, 1991; Zahler et ah, 1992). Furthermore, SR proteins can be co-purified using monoclonal antibodies that preferentially immunoprecipitate early

spliceosomal-pre-mRNA complexes and inhibit the first transestérification reactions during constitutive splicing (Blencowe et al., 1994).

Consequently, interaction of SR proteins to pre-mRNA is believed to be one of the earliest steps in spliceosome assembly, enhancing the binding of U1 snRNP to the 5' splice site to form a commitment complex (Eperon et al., 1993; Fu, 1993; Staknis and Reed, 1994) and occurs presumably by the bridging between specific splice sites and the U1-70K domain (Eperon et al., 1993; Fu, 1993; Kohtz et al., 1994; Staknis and Reed, 1994).

The use of the yeast two-hybrid system to examine protein-protein interactions has extended these studies, suggesting that cooperation of SRps with snRNP particles in binding to pre- mRNA is possibly mediated by interactions between arginine/serine rich domains present in both SR proteins and U1-70K (Amrein et al., 1994; Kohtz et al., 1994; Wu and M aniatis,

1993). Since the binding of snRNP-U2, and U1 snRNP to the 3' splice site is also enhanced in the presence of SR proteins - in this case by interaction with the RS domain of U2AF^^ (Staknis and Reed, 1994) it has been postulated that donor and acceptor sites are linked together via a network of protein-protein interactions involving U1 snRNP, U2AF and SR proteins (Amrein et al., 1994; Kohtz et al., 1994; Wu and Maniatis, 1993; Staknis and Reed,

1994).

Thus SR proteins may function as bridging factors facilitating or stabilising interactions between donor and acceptor splice sites with components of the spliceosome in the earliest steps of pre-mRNA binding.

SR proteins excert non-redundant functions in vivo

Even though members of the SR protein family are functionally similar with respect to restoring splicing in SR-depleted lysates in in vitro (Fu et al., 1992; Krainer et al., 1990; M ayeda et al., 1992; Zahler et al., 1992), there is accumulating evidence that each member displays a distinct spectrum of mRNA substrate specificity (Fu, 1993; Kim et al., 1992; Tian and Maniatis, 1993; Watakabe et al., 1993; Yeakley et al., 1996; Zahler et al., 1993).

This data is supported by a number of studies in different organisms in which individual SR proteins were disrupted. D rosophila B52, the homolog of human SRp55, is essential for development in embryonic and larval stages, as a B52 null mutant indicates (Peng and Mount, 1995; Ring and Lis, 1994). Furthermore, overexpression of this protein also leads to various developm ental abnormalities (Kraus and Lis, 1994). O verexpression of X I 6, the mouse homolog of human SRp20, appears to be lethal in cell transfectants and transgenic mice (Jumaa et al., 1997). More recently targeted disruption of the SR protein SF2/ASF in a chicken B-cell line showed that this SR protein is essential for cell viability and could not be

substituted by other SR proteins, supporting the notion that individual splicing factors are not functionally redundant (Wang et al., 1996).

Activity o f SR proteins in alternative splicing

Many SR proteins such as SRpSOa were shown to shift the splice site selection to proximal and distal 5' splice sites of reporter genes (Caceres et al., 1994; M ayeda and Krainer, 1992). SRp30b shifts alternative splicing exclusively to the proximal 5' splice site in an E l A pre- mRNA in vitro, whereas SRp40, SRp55, and SRp75 showed can also promote the use of the distal 5' splice site (Zahler et al., 1992) and similar differences were observed in vivo using co-transfection assays (Screaton et al., 1995).

In alternative splicing the activity of some SR proteins can be antagonised by the splicing factor hnRNP A1 in a dose dependent fashion (Fu et al., 1992; M ayeda and Krainer, 1992) although this effect is dependent on the size of the alternative exon and the strength o f its proceeding polypyrimidine tract (Mayeda et al., 1993). Other hnRNFs, such as A2 and B1 have even stronger antagonizing effects to SRp30a (Mayeda et al., 1994). An excess of these hnRNFs promotes the use of distal 5' splice sites, wheras high levels of SRp30a favours the use of proximal 5' splice sites.

This effect may be mediated by recruitment of U1 snRNF to 5' splice sites as discussed earlier, suggesting a model to explain the mechanism of SRp30a action in alternative splicing. According to this, specific SR proteins, such as SRp30a, may act on the pre-mRNA to overpower alternative splice sites by selecting normally weak splice sites, and enhancing their affinity in U1 snRNF binding. In contrast, hnRNFs may decrease binding to U1 snRNF, which results in use of the highest affinity splice site. Therefore, variations in the intracellular levels of antagonistic splicing factors could influence different modes o f alternative splicing

in vivo.

Alternative splicing enhancers

Although SR proteins have been shown to exert distinct functions in vitro, much less is known about their substrate specificities in vivo and the structural basis o f their observed splicing specificities. An important step towards understanding how alternative splicing processes may be regulated was the finding that specific exonic sequences interact with members of a family of SR proteins. Experiments using the D rosophila doublesex (dsx) enhancer have shown that SR proteins SC35 and SRp55 bind cooperatively to exonic recognition elements (EREs) in the presence of the specific splicing regulators tra and tra-2 (Lynch and Maniatis, 1995; Tian and Maniatis, 1993). In mammalian cells members of the SR protein family were shown to interact with similar EREs (Lavigueur et al., 1993).

Interactions between SR proteins and certain sequences adjacent to donor and acceptor sites as well as exonic splicing enhancer elements have been documented (Fu and Maniatis, 1992; Staknis and Reed, 1994; Sun et al., 1993; Tian and Maniatis, 1993; Zuo and Manley, 1994). One class of purine-rich cis-elem ents com m only found associated w ith m am m alian alternatively spliced exons are referred to as exonic recognition enhancers (EREs) or exon recognition sequences (ERSs). EREs have been defined as exonic elements that are capable of interacting w ith trans-acting factors in a sequence specific manner, thereby facilitating splicing o f adjacent introns. Since the discovery o f the first ER E in vertebrate immunoglobulin |i heavy chain pre-mRNA (W atakabe et al., 1993), numerous splicing enhancers have been identified in a variety of alternatively spliced genes including the bovine growth hormone gene pre-mRNA (Dirksen et al., 1994), fibronectin ED I exon (Lavigueur et al., 1993) chicken cardiac troponin T exon 5 (Xu et al., 1993), and calcitonin/CGRP exon 4 (van Oers et al., 1994).

W hether this interaction is direct or relies on additional factors was addressed more recently by studies using calcitonin/CGRP transcripts containing GAA repeats. The results provided strong evidence for the requirement of an auxiliary factor in order to recruit SRp40, a member of the SR protein family of splicing factors (Yeakley et al., 1996). In contrast to this study, some SRps but not others, were shown to interact directly with enhancers of bovine growth hormone (Sun et al., 1993) as well as cardiac troponin pre-mRNA (Ramchatesingh et al.,

1995). SR protein may therefore mediate interactions between EREs and the 3' splice site via R N A -proteins interactions, by their RRM binding specificities and via protein-protein interactions with their RS domain and similar domains present in com ponents o f the spliceosome.

Specific functions of RRM and SR domains

The requirem ent for different domains was investigated in various studies using deletion mutants o f SR proteins. It became evident that the RS domain is absolutely essential for constitutive splicing, whereas it seems to be dispensable in alternative splicing activity of SRp30a. A protein with a mutated or even deleted SR domain can still promote alternative splicing in a dose dependent manner if minimal amounts of wild-type SRp30a are present (Caceres and Krainer, 1993; Zuo and Manley, 1993) whereas both RRM s of SRp30a are required for constitutive as well as alternative splicing in vitro. Recent studies have shown that at least one RRM and the RS domain are both sufficient and essential nuclear localisation signals of SR proteins (Caceres et al., 1997). Each of the RRMs are able to bind RNA with distinct specificity (Tacke and M anley, 1995), but both together appear to be acting synergistically (Caceres and Krainer, 1993; Zuo and Manley, 1993).

Evidence for direct interaction of the RRM with RNA comes from several approaches, but specific binding sites for RRM proteins have been defined in only a few cases. Specific RNA sites are recognised by U 1 and U2 snRNPs and the Sex-lethal (Sxl) protein o f Drosophila,

which binds to the transformer (tra) pre-mRNA. Relatively little sequence specificity is required for the binding of most of the other RRMs i.e. the binding o f helix-destabilising proteins (U P l, UP2, HOP and SSBl that recognise ssDNA and ssRNA (Broitman et al., 1987; H errick and Alberts, 1976; Jong et al., 1987; Planck and W ilson, 1980). hnR N P-A 1 was shown to bind single stranded DNA and RNA (Herrick et al., 1976) and nucleolin can interact with processed 18-S and 28-S RNA in a Northwestern assay (Bugler et al., 1987). Another example is PAB which recognises the homopolymer poly(A) (Sachs et al., 1986).

Regulation o f SR protein expression

Regulation of alternative splice site choice in different tissues and stages of cell activity and cell cycle may be determined by the unique combination between SR proteins, hnRNPs and components of the constitutive splicing machinery. Consequently variations in the alternative splicing pattern are reflected in differences in tissue distribution and abundances as a result of corresponding changes in the ratio of these splicing factors. This certainly holds true for a number of SR proteins as well as hnRNPs as different total and relative amounts are found in a wide range o f different cell types and tissues (Ayane et al., 1991; Zahler et al., 1993). However little is known about the regulation of SR protein expression levels.

The promoter regions of murine SRp20 has recently been characterised, and found to be rich in GC nucleotides, frequently seen in many housekeeping genes, contains a TATA box, which is often associated with tissue specific genes, and in addition two E2F consensus motifs were described. As E2F transfection can induce promoter activity, the possibility was raised that SRp20 expression is coupled to the cell cycle (Jumaa et al., 1997). The promoter of another SR protein, SRpSOb revealed the presence of several myb-recognition elements, suggesting that its expression may be modulated by the nuclear proto oncogene c-myb, which is highly expressed in immature hematopoietic cells (Sureau et al., 1992), whereas the expression of rat HRS/SRp40 has been reported to be induced by insulin (Diamond et al., 1993). Changes in the expression pattern of lymphocytes in response to mitogenic stim ulation have been reported for human SRp40, which was dramatically downregulated, but also for SRp30c which was upregulated by up to 5- fold for a few days during activation (Screaton et al.,

1995).

The expression of non SR-splicing factors such as hnRNP A1 appear to be dependent on the activation state of certain cell types and a number of sites for transcriptional regulators were identified, including GC-rich elements, Spl-binding sites, a CAAT box and putative sites for USF/M LTF and myc gene products (Biamonti et al., 1993). Alterations in the pattern of

alternative splicing as a result of oncogenic transformation have been described for a number of genes including CD44 (Gunthert et ah, 1991); the fibronectin gene (M agnuson et ah,

1991), the tropomyosin gene family (Matsumura et ah, 1983), and many other important genes. The change in isoform expression may be reflected by alterations in the ratio between alternative splicing factors, which are associated with, or may even be responsible for the establishment of the transformed phenotype.

Localisation o f splicing and transcription

Expression of eucaryotic genes is a multistep mechanism that includes transcription, splicing, polyadenylation and transport o f the processed mRNA out o f the nucleus into the com partm ents of the endoplasmic reticulum. It is becoming increasingly clear that these processes take place in highly organised compartments of the nucleus and that the various steps are tightly linked with each other. The nucleus is organised into different nuclear subcompartments: the nucleolus in which predominantly ribosomal RNAs are synthesised and processed (Strouboulis and Wolffe, 1996) and the nuclear pore complex (NPC), via which all nuclear-cytoplasmic trafficking of RNA as well as nuclear proteins occurs. Early examination of these compartments using using electron microscopy showed that the majority of introns are spliced cotranscriptionally from pre-mRNA (Beyer and Osheim, 1988) and this was confirmed later using more sophisticated microdisection methods (Bauren and W ieslander,

1994).

Strong evidence has accumulated since the discovery at the beginning of this decade that eucaryotic splicing takes place in discrete compartments in highly ordered compartments within the nucleus (Fu and Maniatis, 1990; Spector, 1996). Immunofluorescent staining using antibodies directed to nuclear antigens, typically splicing factors, have visualised 20-50 bright spots which are referred to as nuclear speckles (Spector, 1993). Therefore it was suggested that these speckles were the sites at which spliceosome assembly takes place (Spector et ah,

1991).

In situ hybridisation using antisense 2' 0-m ethyl RNA oligonucleotides has provided insights into the localisation o f snRNA-containing organelles (Huang and Spector, 1991; Huang and Spector, 1992; Carmo Fonseca et al., 1991). From electron microscopy studies, it was proposed that these foci like compartments may correspond to coiled bodies (Carmo Fonseca et al., 1992). New techniques including in situ hybridisation to detect specific gene transcripts indicated that both synthesis and splicing of RNA polymerase 11 transcripts colocalise in dicrete foci that can be associated with nuclear speckles enriched with splicing factors (Huang and Spector, 1996; Huang and Spector, 1991; Jimenez and Spector, 1993; Xing et al., 1993; Xing et al., 1995).

Although a direct role for speckles in the splicing of RNA pol II transcripts was suggested, there is accumulating evidence that these compartments represent storage sites for splicing factors (Singer and Green, 1997). However the nuclear distribution of splicing factors appears to be strongly dependent on the physiological state of the cell, as their distribution is more diffuse during high transcriptional activity (Zeng et al., 1997). Recent studies indicate that the sites of viral transcription of transfected adenovirus constructs were random ly distributed throughout the nucleoplasm , often distant from the speckles. H ow ever, no unspliced transcripts were detected.

When transcriptional activity was increased the distribution of the speckles became coincident with the localisation o f viral genomes, suggesting that speckles represent reservoirs, which can supply splicing factors to sites of transcriptional activity (Bridge et al., 1995; Jimenez and Spector, 1993). This notion is in line with recent evidence that splicing factors can migrate between the speckles towards sites of active transcription, and that inhibition of transcription resulted in an increase of speckle density (Misteli et al., 1997)

Shuttling o f SR proteins

Tim e-lapse fluorescence microscopy of green fluorescent protein SR (GFP-SR) fusion proteins provide strong indications that speckles are highly dynamic structures between which splicing factors shuttle and they subsequently accumulate at sites of transcription as a result of RS domain phosphorylation (Misteli et al., 1997). This supports the view that nuclear speckles represent storage sites for alternative and constitutive splicing factors and function to supply these factors to sites of active transcription (Dirks et al., 1997; Huang and Spector,

1996; Jimenez and Spector, 1993; Pombo et al., 1994).

These dynamic events are sensitive to inhibitors of kinases and Ser/Thr phosphatases, indicating that phosphorylation of the RS domain represents an important regulatory function in this process (M isteli et al., 1997). Recently a num ber o f kinases that specifically phosphorylate SR proteins have been implicated in the regulation o f subnuclear localisation of splicing factors: SRPK-1 is cell cycle regulated and able to induce disassembly of nuclear speckles as well as inhibition of splicing (Gui et al., 1994). Another kinase CLK/STY, a prototype o f the LAM M ER kinases containing an RS domain, was isolated by screening