The major novel findings in this study are that elevated miR-124a expression decreases dendritic branching and dFMR1, the fly homologue of the human protein responsible for fragile X syndrome, modulates the biogenesis and function of miRNAs in the Drosophila nervous system. This conclusion is supported by the presence of dFMR1 and miR-124a in the same RNP complex, the genetic interaction between the two genes, and biochemical analysis of steady-state levels of miRNAs in the absence of dFMR1. Moreover, we found that the Dcr-1-Ago1 complex was less abundant in the nervous system of dfmr1 mutants, providing a mechanistic explanation for the observed effect of loss of dFMR1 activity on miRNA biogenesis.
miR-124a is one of the most abundant miRNAs in the brain and whose nucleotide sequence is 100% conserved across species yet it functions in the brain remain incompletely understood (Lagos-Quintana et al., 2002
; Gao, 2008
). Our findings suggest that elevated pre-miR-124a level, such as in pathological conditions, can lead to decreased dendritic branching, which may be detrimental to neuronal function and connectivity. The effect of pre-miR-124a on dendritic branching in vivo seems to require dFMR1. Although dFMR1 is implicated in neuronal development through translational control, the exact mechanism remains unclear. Our findings shed new light on the precise roles of dFMR1 in the developing nervous system. dFMR1 is not an absolute essential factor for the biogenesis of mature miRNAs in vivo, consistent with earlier findings in cultured S2 cells (Caudy et al., 2002
; Ishizuka et al., 2002
). Nonetheless, the finding that dFMR1 modulates the steady-state levels of miR-124a and other miRNAs is of considerable significance, since multiple genes must be expressed coordinately at precise levels both spatially and temporally during brain development. Because miRNAs fine-tune protein synthesis of many genes (Selbach et al., 2008
; Baek et al., 2008
), the developmental and functional defects seen in dfmr1
mutants are likely the consequence of changes in multiple proteins and pathways.
As an RNA-binding protein, dFMR1 is present in multiple distinct RNP complexes that are likely involved in many aspects of RNA metabolism. For instance, dFMR1 forms a complex with PIWI in germ cells and play a role in the biogenesis and function of piRNAs (Megosh et al., 2006
). dFMR1 is also associated with Ago2 and likely plays a role in the RNAi pathway (Caudy et al., 2002
; Ishizuka et al., 2002
). Here we show that endogenous dFMR1 and Ago1 are associated with each other in the developing Drosophila
nervous system, consistent with a recent finding in oocytes (Yang et al., 2007
). More importantly, we found that the Dcr-1-Ago1 complex in the nervous system is less abundant in the absence of dFMR1, providing novel mechanistic insight into the exact role of dFMR1 in the miRNA pathway. Considering the essential roles of the Dcr-1-Ago1 complex in miRNA biogenesis (Okamura et al., 2004
; Forstemann et al., 2007
), impaired association between Dcr-1 and Ago1 due to the absence of dFMR1 seems to be responsible, at least in part, for the observed lower steady-state levels of the nervous system-specific miR-124a and other miRNAs in vivo. Global regulation of miRNA levels by RNA-binding proteins was also reported in the case of Rbm3, a glycine-rich RNA-binding protein that regulates global protein synthesis under cold-stress conditions (Dresios et al., 2005
In a human B-cell line, FMRP was associated with small 20-nt RNAs with unknown identity in a common RNP complex (Jin et al., 2004
). This small RNA is unlikely to be miR-124a, which is nervous system-specific and 23 nt long as many other miRNAs. dFMR1 has three mammalian homologs, FMRP, FXR1, and FXR2. Their exact functions in the miRNA pathway remain largely unknown, although one recent report suggested that FXR1 was involved in some aspects of miRNA function in HeLa cells (Vasudevan et al., 2007
). It is possible that these three mammalian proteins may have redundant functions in the miRNA pathway. Alternatively, different paralogs may have evolved to carry out at least some distinct molecular functions in postmitotic neurons in mammals. It will be interesting to determine the extent to which dFMR1 and FMRP are functionally conserved and whether the miRNA pathway is also misregulated in mouse models of fragile X syndrome.