Dirección de ventas

Although mechanisms of alternative splicing regulation are diverse and largely uncharacterized, PTB, Nova, and Fox splicing factors have been extensively studied and serve as good examples for regulation. PTB is a widely expressed RNA binding protein that has been well studied for its functions to silence neural- and muscle-specific exons in a variety of cell types (Zhang et al., 1999). PTB and PTB isoforms bind to pyrimidine-rich motifs related to the core

sequence, UCUU, through four RRMs to silence alternative exons (Wagner and Garcia-Blanco, 2001; Spellman and Smith, 2006). PTB is best characterized for its role in silencing the neural-specific NI exon of the tyrosine kinase, c-src, in nonneuronal cells (Black and Grabowski, 2003).

PTB silences the NI exon by binding to motifs in the flanking introns to loop out the exon and block access of the splicing machinery to the splice sites. PTB can also bind near the 3´ splice site of an alternative exon to antagonize U2AF⁶⁵ binding (Singh et al., 1995) or to sequester the branch site (Ashiya and Grabowski, 1997). It can also silence an exon by propagating across the RNA to antagonize binding of splicing enhancers and the general splicing machinery (Wagner and Garcia-Blanco, 2001). Furthermore, two recent studies have demonstrated that PTB can silence alternative exons by inhibiting exon and intron definition events (Izquierdo et al., 2005; Sharma et al., 2005). Thus, PTB is a remarkably versatile splicing silencer.

Mechanisms of splicing silencing and enhancement by Nova proteins have also been studied at the level of spliceosome assembly. Nova1 and Nova2 are brain-specific RNA binding proteins that were first identified as target antigens in the neurological disorder, paraneoplastic opsoclonus-myoclonus ataxia (Buckanovich et al., 1993). Nova proteins have 3 KH-type RBDs, which recognize consensus YCAY motifs (Y, pyrimidine) to enhance or silence splicing of alternative exons (Dredge and Darnell, 2003; Dredge et al., 2005). The interaction of Nova with an ESS inhibits binding of U1 snRNP to the adjacent 5´ splice site (Ule et al., 2006). However, when Nova binds to an ISE downstream of an alternative exon, this interaction promotes assembly with U2 snRNP. It is likely that the positioning of SREs in the pre-mRNA influences the functions of splicing factors (Dredge et al., 2005).

The mammalian Fox proteins are homologs of the Caenorhabditis elegans feminizing gene on X protein. Mammalian Fox family members, Fox1 and Fox2, are expressed in neurons of the adult brain and function as enhancers of brain-specific exons that have Fox binding sites in their downstream intron. Fox1 and Fox2 bind to a short but specific consensus motif, UGCAUG, and function to activate splicing of an alternative exon (Underwood et al., 2005).

Fox1 has been characterized for its function as a splicing enhancer of the NI exon of the c-src transcript (Underwood et al., 2005). Fox1 binds downstream of the NI exon at a conserved UGCAUG hexamer in a way that is thought to antagonize the splicing silencer, PTB. Like Nova, Fox proteins can also act as splicing silencers when bound to the intron upstream of an alternatively spliced exon (Fukumura et al., 2007).

Taken together, alternative splicing factors are remarkably versatile proteins that contact SREs to tune splicing patterns in a tissue-specific manner. These factors often have different effects on spliceosome assembly depending on the position and sequence context of their cognate RNA binding sites. Furthermore, competition and cooperation of alternative splicing factors acting on the same pre-mRNA creates a tug-of-war for splicing regulation. Still a common characteristic of these RNA binding proteins is that they recognize short and degenerate sequence motifs, which once again raises the question of how splicing factors can accurately select the correct target exons for regulation.

1.4.5 Regulation of tissue- and developmental-specific expression of splicing factors

In recent years, several alternative splicing factors have been characterized for having unique features that contribute to their tissue- and developmental-specific expression and functions. It is becoming clear that these features are more common than once thought and may actually be characteristic of most alternative splicing factors. These characteristics include alternative splicing within the splicing factor transcript, autoregulation and NMD, cross-regulation of family members, and regulation by micro RNAs (miRNAs). Several examples are described below.

Many alternative splicing factors are themselves alternatively spliced to generate several protein isoforms that carryout distinct functions in specific cell types. For example, mammalian Fox1 and Fox2 transcripts are extensively alternatively spliced to generate multiple protein isoforms (Underwood et al., 2005). Skipping of one exon within Fox transcripts from both genes

deletes essential amino acid residues in the protein to eliminate RNA binding activity (Nakahata and Kawamoto, 2005). In this way, alternative splicing of splicing factor transcripts can be used to regulate their function.

Furthermore, many alternative splicing factors regulate their own expression and activity by controlling alternative splicing of exons within their own transcript. For example, Nova1 has the ability to silence an exon within its own transcript (Dredge et al., 2005). The peptide segment corresponding to this exon is phosphorylated in vivo and may serve as a localization signal or to modulate protein function. Furthermore, many factors from the SR family of splicing enhancers have been shown to increase inclusion of an exon within their own transcript to cause a shift in the reading frame revealing a premature termination codon (PTC) that targets the transcript for NMD (Ni et al., 2007). NMD is a quality control process that selectively degrades mRNAs containing PTCs to control for genetic and splicing errors and regulate protein expression (Chang et al., 2007). Conversely, several splicing silencers have been shown to function by the opposite mechanism to cause skipping of an exon within their own transcript to expose a PTC (Wollerton et al., 2004; Baraniak et al., 2006). Therefore, NMD is a process utilized by splicing factors to control their own protein levels in the cell.

Additionally, cross-regulation of closely related family members has been shown for several splicing factors. In the nervous system, cross-regulation between PTB family members drives mutually exclusive isoform expression in different cell types. In the mouse brain, neural PTB (nPTB) is exclusively expressed in neurons, while PTB expression is restricted to glial cells (Boutz et al., 2007b). One explanation for this mutually exclusive pattern of expression is that PTB causes skipping of an exon in the nPTB transcript by a mechanism similar to autoregulation to target the transcript for NMD. PTB and nPTB regulate distinct, yet overlapping sets of exons and their cell-specific expression is associated with the differentiation of neuronal and glial cells during brain development.

The expression of PTB is also regulated through post-transcriptional events involving miRNAs. miRNAs are a well studied group of ~22 nucleotide RNAs that bear complimentarity to sequences in the 3´ untranslated regions (UTRs) of certain mRNAs where their interactions either repress protein synthesis or promote mRNA decay (He and Hannon, 2004). The neuron-specific miRNA, miR-124, inhibits expression of PTB during neuronal differentiation (Makeyev et al., 2007). Decrease in PTB expression leads to an increase of nPTB expression, which is associated with a global switch from non-neuronal to neuronal splicing patterns. Furthermore, a role for miR-133 in the regulation of nPTB expression has been demonstrated in differentiating muscle cells (Boutz et al., 2007a).

Taken together, these characteristics suggest that global patterns of alternative splicing can be controlled in different cell types and during development by several different quality control mechanisms. How other splicing factors are targets of such regulation is still to be investigated.