Recent studies have shown that miRNAs can target the promoter and CDS region. Thus, we predicted miRNA target sites in the 5'-UTR, CDS and 3'-UTR of Homo sapiens, Mus musculus and Drosophila melanogaster using miRanda and TargetScan. Target-site densities normalized with the average region length were higher in the 5'-UTR than 3'-UTR in all three organisms but were lower in the negative data set. Interestingly, the putative target sites were more conserved than non-target regions in both the 5'-UTR and 3'-UTR, implying that target sites in the 5'-UTR are subject to high selective pressure and might be functional. In Drosophila, 48 of 78 (61.5%) miRNAs showed high similarities with predicted siRNAs. Based on the results of previous experimental studies and a large-scale statistical analysis, we conclude that miRNA-mediated regulation is not limited to the 3'-UTR. However, the functionality of target sites in the 5'-UTR and CDS requires thorough investigation.

Insulinoma-associated1a (insm1a) is a zinc-finger transcription factor playing a series of functions in cell formation and differentiation of vertebrate central and peripheral nervous systems and neuroendocrine system. However, its roles on the development of motor neuron have still remained uncovered. Here, we provided evidences that insm1a was a vital regulator of motor neuron development, and provided a mechanistic understanding of how it contributes to this process. Firstly, we showed the localization of insm1a in spinal cord, and primary motor neurons (PMNs) of zebrafish embryos by in situ hybridization, and imaging analysis of transgenic reporter line Tg(insm1a: mCherry)ntu805. Then we demonstrated that the deficiency of insm1a in zebrafish larvae lead to the defects of PMNs development, including the reduction of caudal primary motor neurons (CaP), and middle primary motor neurons (MiP), the excessive branching of motor axons, and the disorganized distance between adjacent CaPs. Additionally, knockout of insm1 impaired motor neuron differentiation in the spinal cord. Locomotion analysis showed that swimming activity was significantly reduced in the insm1a-null zebrafish. Furthermore, we showed that the insm1a loss of function significantly decreased the transcript levels of both olig2 and nkx6.1. Microinjection of olig2 and nkx6.1 mRNA rescued the motor neuron defects in insm1a deficient embryos. Taken together, these data indicated that insm1a regulated the motor neuron development, at least in part, through modulation of the expressions of olig2 and nkx6.1.

Background: Nonsyndromic hearing loss is clinically and genetically heterogeneous. In this study, we characterized the clinical features of 12 Chinese Han deaf families in which mutations in common deafness genes GJB2, SLC26A4, and MT-RNR1 were excluded. Methods: Targeted next-generation sequencing of 147 known deafness genes was performed in probands of 10 families, while whole-exome sequencing was applied in those of the rest two. Results: Pathogenic mutations in a total of 11 rare deafness genes, OTOF, CDH23, PCDH15, PDZD7, ADGRV1, KARS, OTOG, GRXCR2, MYO6, GRHL2, and POU3F4, were identified in all 12 probands, with 16 mutations being novel.Intrafamilial cosegregation of the mutations and the deafness phenotype were confirmed by Sanger sequencing. Conclusion: Our results expanded the mutation spectrum and genotype-phenotype correlation of nonsyndromic hearing loss in Chinese Hans and also emphasized the importance of combining both next-generation sequencing and detailed auditory evaluation to achieve a more accurate diagnosis for nonsyndromic hearing loss. K E Y W O R D Sdeafness, gene mutation, next-generation sequencing (NGS), nonsyndromic, phenotype

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Abstract:The control of cellular N-acetylmannosamine (ManNAc) levels has been postulated to be an effective way to modulate the decoration of cell surfaces with sialic acid. N-acetylglucosamine 2-epimerase catalyzes the interconversion of N-acetylglucosamine (GlcNAc) and ManNAc. Herein, we describe the cloning, expression, purification and biochemical characterization of an unstudied N-acetylglucosamine 2-epimerase from Pedobacter heparinus (PhGn2E). To further characterize the enzyme, several N-acylated glucosamine derivatives were chemically synthesized, and subsequently used to test the substrate specificity of PhGn2E. Furthermore, NMR studies of deuterium/hydrogen exchange at the anomeric hydroxy group and C-2 positions of the substrate in the reaction mixture confirmed for the first time the postulated epimerization reaction via ring-opening/enolate formation. Site-directed mutagenesis of key residues in the active site showed that Arg63 and Glu314 are directly involved in proton abstraction and re-incorporation onto the substrate. As all mechanistically relevant active site residues also occur in all mammalian isoforms, PhGn2E can serve as a model N-acetylglucosamine 2-epimerase for further elucidation of the active site mechanism in these enzymes.