Alternative role of HuD splicing variants in neuronal differentiation.

HuD is a neuronal RNA-binding protein that plays an important role in neuronal differentiation of the nervous system. HuD has been reported to have three RNA recognition motifs (RRMs) and three splice variants (SVs) that differ in their amino acid sequences between RRM2 and RRM3. This study investigates whether these SVs have specific roles in neuronal differentiation. In primary neural epithelial cells under differentiating conditions, HuD splice variant 1 (HuD-sv1), which is a general form, and HuD-sv2 were expressed at all tested times, whereas HuD-sv4 was transiently expressed at the beginning of differentiation, indicating that HuD-sv4 might play a role compared different from that of HuD-sv1. Indeed, HuD-sv4 did not promote neuronal differentiation in epithelial cells, whereas HuD-sv1 did promote neuronal differentiation. HuD-sv4 overexpression showed less neurite-inducing activity than HuD-sv1 in mouse neuroblastoma N1E-115 cells; however, HuD-sv4 showed stronger growth-arresting activity. HuD-sv1 was localized only in the cytoplasm, whereas HuD-sv4 was localized in both the cytoplasm and the nuclei. The Hu protein has been reported to be involved in translation and alternative splicing in the cytoplasm and nuclei, respectively. Consistent with this observation, HuD-sv1 showed translational activity on p21, which plays a role in growth arrest and neuronal differentiation, whereas HuD-sv4 did not. By contrast, HuD-sv4 showed stronger pre-mRNA splicing activity than did HuD-sv1 on Clasp2, which participates in cell division. Therefore, HuD SVs might play a role in controlling the timing of proliferation/differentiation switching by controlling the translation and alternative splicing of target genes.

Proposing a new RNA quadruplex structure: j-motif, with possible links to neural development.

An RNA-binding protein, hnRNP K, has been studied extensively because of its involvement in neural development through the post-transcriptional regulation of its downstream target genes; however, its binding mode remains unclear. According to structural features of the binding sites, we have presumed the existence of possible unique structures 'j-motifs' that are similar to known i-motifs, the difference being that the initial cluster comprises successive U nucleic acids instead of C. It was suspected that the motifs could be recognized by hnRNP K to regulate the translation levels of target proteins, however, there were virtually no methods to verify their existence except computational methods: regular expression searches and theoretical molecular orbital (MO) calculations. Here, we first show a list of 16 genes having j-motif-like sequences we discovered under refined search conditions. The list was highly related to neural development from both subjective and objective aspects. Additionally, MO calculations revealed the similarity of non-canonical base pairs found in i- and j-motifs qualitatively, leading to a proposal of the possible existence of the j-motifs. When taken into consideration, it was indicated that the j-motifs could be formed and play some role in the neural development.

The miR-17/106-p38 axis is a key regulator of the neurogenic-to-gliogenic transition in developing neural stem/progenitor cells.

Neural stem/progenitor cell (NSPC) multipotency is highly regulated so that specific neural networks form during development. NSPCs cannot respond to gliogenic signals without acquiring gliogenic competence and decreasing their neurogenic competence as development proceeds. Coup-tfI and Coup-tfII are triggers of these temporal NSPC competence changes. However, the downstream effectors of Coup-tfs that mediate the neurogenic-to-gliogenic competence transition remain unknown. Here, we identified the microRNA-17/106 (miR-17/106)-p38 axis as a critical regulator of this transition. Overexpression of miR-17 inhibited the acquisition of gliogenic competence and forced stage-progressed NSPCs to regain neurogenic competence without altering the methylation status of a glial gene promoter. We also identified Mapk14 (also known as p38) as a target of miR-17/106 and found that Mapk14 inhibition restored neurogenic competence after the neurogenic phase. These results demonstrate that the miR-17/106-p38 axis is a key regulator of the neurogenic-to-gliogenic NSPC competence transition and that manipulation of this axis permits bidirectional control of NSPC multipotency.

Neural RNA-binding protein Musashi1 controls midline crossing of precerebellar neurons through posttranscriptional regulation of Robo3/Rig-1 expression.

Precisely regulated spatiotemporal gene expression is essential for the establishment of neural circuits. In contrast to the increasing evidence for transcriptional regulation of axon guidance cues and receptors, the role of posttranscriptional regulation in axon guidance, especially in vivo, remains poorly characterized. Here, we demonstrate that the expression of Slit receptor Robo3/Rig-1, which plays crucial roles in axonal midline crossing, is regulated by a neural RNA-binding protein Musashi1 (Msi1). Msi1 binds to Robo3 mRNA through RNA recognition motifs and increases the protein level of Robo3 without affecting its mRNA level. In Msi1-deficient precerebellar neurons, Robo3 protein, but not its mRNA, is dramatically reduced. Moreover, similar to defects in Robo3-deficient mice, axonal midline crossing and neuronal migration of precerebellar neurons are severely impaired in Msi1-deficient mice. Together, these findings indicate that Msi1-mediated posttranscriptional regulation of Robo3 controls midline crossing of precerebellar neurons.