NS-specific alternative pre-mRNA splicing plays an important role in the establishment of neuronal identity (Black, 2003
; Licatalosi and Darnell, 2006
; Lipscombe, 2005
; Ule and Darnell, 2006
). However, the regulatory network that underlies the transition from nonneuronal to NS-specific alternative splicing is poorly understood. Similarly, several brain-specific miRNAs have been identified, but relatively little is known about their functions (Kosik, 2006
). Here we show that miR-124 is a critical regulator of NS-specific splicing, and that it functions by controlling a switch in the ratio of the splicing repressors PTBP1 and PTBP2. This, in turn, leads to the initiation of a global NS-specific splicing program and promotes neuronal differentiation ().
We show that PTBP1 protein is present at high levels in neural progenitor cells and outside of the NS where little or no miR-124 is expressed. In nonneuronal cells PTBP1 represses the inclusion of alternative exon 10 in PTBP2 mRNA and destabilizes PTBP2 mRNA via the NMD pathway. In differentiating and mature neurons, expression of miR-124 reduces PTBP1 levels, triggering NS-specific splicing events and increasing the abundance of PTBP2 protein by allowing the inclusion of exon 10 in PTBP2 mRNA.
Similar to previously studied examples of miRNA/mRNA interactions, the 3′UTR of PTBP1 mRNA contains several miR-124 target sites that render the expression of a luciferase reporter sensitive to miR-124. Rescue of the PTBP1 3′UTR reporter activity required the inactivation of the strongest 8-mer MTS and four additional 6- and 7-mer MTSs. We therefore conclude that the regulation of PTBP1 by miR-124 requires several closely positioned MTSs in the 3′ UTR of PTBP1, which may function synergistically to achieve optimal sensitivity to miR-124 levels (Doench and Sharp, 2004
). Considering that one consequence of the upregulation of miR-124 is a dramatic increase in the level of PTBP2 protein, it was surprising to find that the 3′UTR of PTBP2 is also targeted by miR-124, albeit weakly. This miR-124-mediated repression of PTBP2 expression is due primarily to a single 9-mer MTS in the PTBP2 3′UTR. At present, the functional significance of this miR-124 targeting of PTBP2 is not understood.
Data presented in this study and in a number of earlier reports (Black, 2003
; Spellman and Smith, 2006
; Wagner and Garcia-Blanco, 2001
) implicate PTBP1 as a global repressor of NS-specific alternative splicing. Our microarray data indicate that at least 16 exons are upregulated by miR-124 in CAD cells. It is likely that even more exons are affected, because the microarray we used was not designed to specifically detect alternative splicing events. In all the cases we examined closely (Cdc42, Gabbr1, Mtap4, Rufy3, Pdlim7, Ptbp2, Rtn4/Nogo, and Tpm3), siRNA knockdown of PTBP1 was sufficient to induce NS-specific alternative splicing confirming that they are regulated by PTBP1.
PTBP2 was identified as an NS-enriched homolog of PTBP1 that interacts with the global splicing regulator Nova, and this interaction was shown to interfere with the inclusion of a Nova-induced exon in the glycine receptor α2 mRNA (Polydorides et al., 2000
). In addition, PTBP2 weakly represses Src N1 cassette exon splicing in vitro, whereas PTBP1 acts as a stronger repressor (Markovtsov et al., 2000
). Here we provide in vivo evidence that PTBP2 acts as a weak repressor of NS-specific exons in several pre-mRNAs (). Understanding the role of PTBP2 in NS-specific alternative splicing is complicated by the recent finding that Nova can function as either an activator or repressor of alternative splicing depending on the position of its binding sites relative to the regulated exons (Ule et al., 2006
). Thus, the increase in PTBP2 protein in response to miR-124 could, in principle, lead to either the repression or activation of exon inclusion via the inhibition of Nova by PTBP2.
Recently, the muscle-specific miRNA miR-133 was shown to repress both PTBP1 and PTBP2 protein synthesis in differentiating myoblasts (Boutz et al., 2007
). In this system, PTBP2 appears to be the primary target, opposite to the miRNA regulation in the NS, where miR-124 efficiently downregulates PTBP1 expression but only weakly represses PTBP2 mRNA. Thus, individual 3′UTRs may contain arrays of MTSs for distinct tissue-specific miR-NAs, which could fine-tune gene expression levels in corresponding tissues.
To determine whether the miRNA processing machinery, and thus microRNAs in general, are required for the downregulation of PTBP1 in the mouse brain, we made use of a conditional knockout mouse in which the Dicer
gene is inactivated in the embryonic telencephalon. Examination of these mice revealed that the forebrain was substantially smaller than in the wild-type mice, likely the consequence of a high-frequency apoptosis, consistent with previously described conditional Dicer
mutant phenotypes (Cobb et al., 2005
; Harfe et al., 2005
; Harris et al., 2006
; Muljo et al., 2005
). However, many of the surviving cells expressed high levels of PTBP1 mRNA and protein, which correlated with a decrease in the levels of PTBP2; a low abundance of the NS-specific splice forms of Mtap4, Rufy3, and Cdc42; and normal or high abundance of the corresponding general splice forms (, Figure S18
, and data not shown). These data indicate that the Dicer/miRNA pathway controls the level of PTBP1 expression and is essential for normal brain development. However, further experiments will be required for a more complete understanding of the role of miRNAs in the developing NS.
Evidence that miR-124 plays an important role in brain development was provided by the observation that expression of miR-124 at physiological levels in either CAD or N2a cells induces neurite outgrowth under conditions in which spontaneous differentiation is not normally observed ( and Figures S2–S4
). Notably, miR-124 reduces the levels of PTBP1 mRNA >2-fold and complementation of PTBP1 is sufficient to inhibit miR-124-induced neurite outgrowth in CAD cells ( and Figure S5A
). In addition, miR-124 may directly target mRNAs encoding proteins required for cell proliferation (Figures S5E and S5F
; Wang, 2006
). Consistent with the latter observation, miR-124 induces cell-cycle arrest in neuroblastoma cells (Figures S4B and S4C
). Thus, miR-124 likely contributes to neuronal differentiation by repressing a range of mRNAs.
We nóte, however, that the role of miR-124 in neuronal differentiation is controversial. An earlier study concluded that miR-124 does not play an essential role in neuronal differentiation in the chick spinal cord (Cao et al., 2007
). However, a more recent study in the same system showed that miR-124 stimulates neurogenesis and that anti-miR-124 2′-OMe-RNA oligonucleotides decreased the expression of neuronal markers and increased the number of proliferating cells (Visvanathan et al., 2007
). We have shown that miR-124 enhances the neurodifferentiation of P19 cells stimulated by RA but does not promote detectable differentiation in the absence of RA. We therefore conclude that miR-124 is necessary, but not sufficient, for neuronal differentiation. Consistent with this view, neurons undergoing differentiation express dramatically higher levels of miR-124 compared to neuronal progenitor cells ( and Figure S16
) (Cao et al., 2007
; Deo et al., 2006
; Visvanathan et al., 2007
We have shown that downregulation of PTBP1 protein levels is required for optimal miR-124-induced differentiation of both CAD and P19 cells. Two observations are consistent with the possibility that corresponding changes in alternative splicing are required for neuronal differentiation. First, most of the alternatively spliced forms induced by miR-124 are enriched in the NS (). Second, several of the corresponding genes encode proteins essential for neuronal morphogenesis and function (Table S4
). However, PTBP1 is known to function in processes other than alternative splicing, including regulation of mRNA translation and stability (Bushell et al., 2006
; Cho et al., 2005
; Kim et al., 2000
; Knoch et al., 2004
; Pautz et al., 2006
; Pilipenko et al., 2001
). Therefore, further studies will be required to determine whether any of these additional activities contribute to the PTBP1-mediated inhibition of neuronal differentiation.
Our conclusion that miR-124 regulates the expression of a global repressor of NS-specific alternative splicing in nonneuronal cells has an interesting parallel at the level of transcription. As previously mentioned, miR-124 was recently shown to target SCP1 mRNA during neuronal differentiation (Visvanathan et al., 2007
). SCP1 has been implicated in the function of the NRSF/REST complex, a global repressor of NS-specific transcription in nonneuronal cells (Yeo et al., 2005
). Thus, miR-124, and possibly other tissue-specific miR-NAs, can contribute to establishing correct cell identity by controlling the levels of global repressors of gene expression.