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PERSPECTIVE
1 Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, USA; 2 College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China
Neuronal differentiation involves extensive reprogramming of gene expression. Many neuronal-specific genes are actively repressed in nonneuronal cells, while many others are induced in response to cell differentiation cues. Together these constitute the transcriptome in neurons to instruct specific neuronal functions (Rosenfeld et al. 2006
). The transcriptome in neurons is further diversified by alternative splicing, arising from the expression of a number of neuronal-specific RNA-binding splicing regulators (Black and Grabowski 2003). In this issue of Genes & Development, Boutz et al. (2007b) report a novel switch in the expression of a pair of related splicing regulators that occurs during neuronal differentiation. These proteins, known as polypyrimidine tract-binding proteins (PTB) and neural PTB (nPTB), are structurally and functionally similar, but PTB is widely expressed in nonneuronal cells, and nPTB is restricted to neurons. Remarkably, 
25% of neuronally induced alternative splicing events detected by mRNA isoform-sensitive splicing arrays are estimated to result from the down-regulation of PTB, and the up-regulation of its neuronal-specific cousin nPTB.
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Splicing control by PTB |
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 Top Splicing control by PTB PTB regulation of nPTB...Splicing reprogramming induced...Transcriptional and post...The road ahead: linking...References |
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), which are frequently part of the 3' splice site in most constitutively spliced genes. Consequently, PTB binding often antagonizes the function of the essential splicing factor U2AF in the recognition of the 3' splice site (Singh et al. 1995). PTB-binding sites are also present in many discrete intronic locations to function as cis-acting splicing silencer elements, and PTB has been identified as a negative splicing regulator of many alternative splicing events (Lin and Patton 1995; Chan and Black 1997; Wagner and Garcia-Blanco 2001). Recent biochemical analyses further revealed that PTB could alter the recognition of splicing signals that are far away from its binding sites, indicating an extended competition between the splicing machinery and the PTB-induced splicing silencing complexes (Izquierdo et al. 2005; Sharma et al. 2005; Spellman and Smith 2006).
While the role of PTB in regulated splicing has been well documented on specific minigene models, the breadth of its involvement in regulated splicing has remained undetermined until now. Using RNA interference (RNAi) in combination with splicing arrays, Boutz et al. (2007b) found that PTB knockdown in N2A neuroblastoma cells indeed altered the splicing pattern of many genes, identifying PTB as a key splicing regulator in mammalian cells. A large percentage (83%) of the splicing changes triggered by PTB RNAi were also detected in differentiating P19 cells upon neuronal induction by retinoic acid, consistent with the strong suppression of PTB expression during the induction. Strikingly, judging from the common changes from PTB RNAi in N2A cells, altered splicing in response to PTB inactivation could account for 25% of the induced alternative splicing in differentiated P19 cells. This suggests that PTB down-regulation is a major factor in establishing the splicing-mediated neuronal differentiation program. Mechanistically, many of the neuronally induced switches in isoform expression appear to be directly mediated by PTB in nonneuronal cells, because of the presence of the expected CU-rich motifs near the alternative exons. Other splicing events may be indirectly induced by PTB-regulated splicing of other splicing regulators. Another recent splicing array study revealed that systems where one alternative splicing factor is controlled by another splicing regulator appear to be common, and may create a variety of feedback and feed forward regulation at the splicing level (Ni et al. 2007).
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PTB regulation of nPTB expression |
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TopSplicing control by PTBPTB regulation of nPTB...Splicing reprogramming induced...Transcriptional and post...The road ahead: linking...References |
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; Kikuchi et al. 2000; Markovtsov et al. 2000; Polydorides et al. 2000). In the current study, detailed immunocytochemistry further showed in brain sections that PTB is mainly expressed in cells of nonneuronal lineages, while nPTB protein is largely restricted to neurons. Interestingly, nPTB mRNA was readily detectable in a variety of nonneuronal cell types and down-regulation of PTB induced nPTB protein synthesis by at least two post-transcriptional mechanisms.
It is known that PTB feedback regulates its own expression by binding to intronic silencers around the alternative PTB exon 11 to promote its skipping (Wollerton et al. 2004). As illustrated in Figure 1, PTB binding causes skipping of exon 11, resulting in a reading frame shift and the premature termination of translation. This premature termination induces degradation of the aberrantly spliced PTB transcript by the nonsense-mediated RNA decay (NMD) pathway (Maquat 2005). This mechanism is thought to maintain homeostasis of PTB expression in nonneuronal cells (Wollerton et al. 2004). PTB is now found to operate similarly on the nPTB pre-mRNA through ultraconserved cis-acting elements in the introns surrounding the nPTB alternative exon 10, which is equivalent to exon 11 in PTB. In response to PTB down-regulation by RNAi in N2A or other cells, nPTB splicing was derepressed to include more exon 10. It appears that this same mechanism will affect nPTB after PTB is lost during retinoic acid induction in P19 cells. Furthermore, inhibition of NMD action by the translational inhibitor Cycloheximide or impairment of the NMD machinery by RNAi against Upf1 led to the stabilization of the exon 10 skipped nPTB in a variety of cell types. This demonstrates that the coupling of alternative splicing to NMD is suppressing nPTB expression in nonneuronal cells. Since PTB is controlling both PTB and nPTB alternative splicing, it will be interesting to determine whether nPTB has a similar activity that contributes to the repression of PTB expression, and perhaps the maintenance of nPTB expression in post-mitotic neurons.
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; Hunt and Jackson 1999), and thus, it is possible that PTB may act directly in repressing nPTB translation. Alternatively, PTB may affect the expression of other translational regulators to indirectly modulate nPTB protein synthesis. Consistent with the possibility that these splicing regulators may be regulated in the cytoplasm, recent studies indicate that both PTB and nPTB are subject to translational regulation by microRNAs in muscles and neurons (Lim et al. 2005; Boutz et al. 2007a). It will therefore be interesting in its own right to investigate how PTB and nPTB may control the translation of their own mRNAs in a feedback regulatory loop and the potential involvement of microRNAs in the process. |
Splicing reprogramming induced by the PTB/nPTB switch |
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TopSplicing control by PTBPTB regulation of nPTB...Splicing reprogramming induced...Transcriptional and post...The road ahead: linking...References |
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; Markovtsov et al. 2000). Using the splicing array coupled with single and double RNAi knockdown, the current report demonstrates that the two protein paralogs indeed exhibit partially redundant functions in the regulation of alternative splicing. It is, however, important to emphasize their differences: The alternative exons regulated by PTB differ significantly from those regulated by nPTB, and double knockdown gives rise to a further distinct splicing profile. Together, these results clearly illustrate the different target spectra for PTB and nPTB, and it is the down-regulation of PTB and induction of nPTB that commits cells to a unique and neuronal-specific splicing program. The redundant and distinct functions associated with PTB and nPTB have important biological implications for the splicing program in both neuronal and nonneuronal cells. Alternative splicing regulated by PTB/nPTB may be critical for a variety of cellular functions. Thus, to prevent a complete loss of PTB function, mammalian cells have evolved the mechanism to maintain a level of nPTB transcription to permit a rapid response to potential malfunction of PTB. In developing neurons, on the other hand, the PTB/nPTB switch appears to represent a key post-transcriptional reprogramming event. Instead of dramatically altering the transcriptional program, the PTB/nPTB switch may fine tune the existing program to meet the functional requirements of the nervous system. Further studies on single- and double-knockout animal models will address these important biological questions (see below).
It is interesting to note that, in addition to the PTB/nPTB switch from nonneuronal to neuronal cells, the splicing program in different neuronal cell types may be further fine tuned by the differential expression of other pairs of neuronal-specific splicing regulators. For example, both Nova-1 and Nova-2 are neuronal splicing factors, whose expression in postnatal brain seems largely reciprocal, most obviously in the neocortex where Nova-2 is nearly exclusively expressed (Yang et al. 1998). Furthermore, because Nova-1 is also autoregulated at the splicing level (Dredge et al. 2005), Nova-1 and Nova-2 may undergo both feedback and feed-forward regulation similar to PTB and nPTB. The resulting Nova1/2 switch in different brain regions may relay the PTB/nPTB switch from nonneuronal to neuronal cells to reprogram alternative splicing in different subtypes of neurons.
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Transcriptional and post-transcriptional induction of the neuronal splicing program |
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TopSplicing control by PTBPTB regulation of nPTB...Splicing reprogramming induced...Transcriptional and post...The road ahead: linking...References |
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), although it is unclear how many Nova-dependent splicing events are induced in the P19 system. It may be that a large fraction of neuronal-specific splicing events are controlled by a panel of neuronal-specific splicing regulators that have already been biochemcially and/or genetically characterized, such as Nova (Ule et al. 2005a, b, 2006), Fox (Jin et al. 2003; Auweter et al. 2006), and Hu (Perrone-Bizzozero and Bolognani 2002; Zhu et al. 2006) families of proteins. These neuronal-specific splicing regulators, as well as more general splicing regulators, are likely to act in a combinatorial fashion with large overlapping target sets to establish the alternative splicing program in neurons.
Important questions that remain as to how PTB expression is actively suppressed, and how of the other neuronal-specific splicing regulators are induced in neurons, will hold the key to our understanding of the transcriptome in neurons. Previous studies have established that a large number of neuronal genes are specifically repressed at the transcriptional level in nonneuronal cells by the REST/CoREST complex (Andres et al. 1999; Ballas et al. 2001, 2005; Lunyak et al. 2002). It will be interesting to determine whether any neuronal-specific splicing regulators are coordinately regulated by the RSET/CoREST complex. MicroRNAs may also play a big part in this regulation (Johnston et al. 2005; Boutz et al. 2007a). The PTB/nPTB switch certainly adds an interesting post-transcriptional mechanism for the induction and maintenance of neuronal-specific genes.
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The road ahead: linking regulated splicing to neuronal phenotype |
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TopSplicing control by PTBPTB regulation of nPTB...Splicing reprogramming induced...Transcriptional and post...The road ahead: linking...References |
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Splicing arrays have revealed numerous neuronal-specific mRNA isoforms regulated by Nova (Ule et al. 2005a), and now by PTB and nPTB. Many of those detected isoforms appear directly relevant to neuronal functions as discussed by Ule et al. (2005a) and Boutz et al. (2005b). The link between regulated splicing and neuronal phenotypes can be established by developing specific functional assays to correlate a specific expressed isoform to a defined functional consequence in neurons. Technically, this is challenging, but it has been done in other experimental systems where a specific splicing regulator is either overexpressed or genetically inactivated and a specific alternative splicing event is directly linked to a measurable phenotype (e.g., see Ghigna et al. 2005; Xu et al. 2005; Karni et al. 2007). Antisense technology and isoform-specific inactivation by RNAi also provide powerful tools for functional dissection of alternative splicing induced by the PTB/nPTB switch (Garcia-Blanco et al. 2004; Hua et al. 2007). Future genotype/phenotype studies of regulated splicing during development will clearly benefit from the construction of genetically engineered cell and animal models.
Besides regulated splicing, it is important to keep in mind that PTB is also known to play roles in other aspects of RNA metabolism, including 3' end formation (Castelo-Branco et al. 2004; Le Sommer et al. 2005), and the regulation of RNA stability and translation (Irwin et al. 1997; Hunt and Jackson 1999; Knoch et al. 2004). By extrapolation, nPTB may play both similar and distinct roles in these processes, which may also contribute to important neuronal functions. The current work in conjunction with the published work on Nova and general splicing factors, such as SR proteins, illustrates a general experimental approach to understand regulated splicing in development and disease (Xu and Fu 2005; Möröy and Heyd 2007).
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Footnotes |
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E-MAIL xdfu{at}ucsd.edu; FAX (858) 822-6920. 
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1575607
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