ChangHui Pak

MicroRNAs (miRNAs) are small non-coding RNAs that inhibit expression of specific target genes at the post-transcriptional level. Sequence variations in miRNA genes, including pri-miRNAs, pre-miRNAs and mature miRNAs, could potentially influence the processing and/or target selection of miRNAs. In this study, we have systematically identified single nucleotide polymorphisms (SNPs) associated with 227 known human miRNAs. Among 323 total SNPs that we identify, 12 are located within the miRNA precursor and one is at the eighth nucleotide (+8) of the mature miR-125a, which has been proposed to play a critical role in recognition of mRNA targets by miRNAs. Through a series of in vivo analyses, we unexpectedly find that this miR-125a SNP significantly blocks the processing of pri-miRNA to pre-miRNA, in addition to reducing miRNA-mediated translational suppression. Thus, our study reveals an additional structural requirement for pri-miRNA processing and emphasizes the importance of identifying new miRNA SNPs and their contributions to miRNA biogenesis and human genetic disease.

Direct conversion of nonneural cells to functional neurons holds great promise for neurological disease modeling and regenerative medicine. We previously reported rapid reprogramming of mouse embryonic fibroblasts (MEFs) into mature induced neuronal (iN) cells by forced expression of three transcription factors: ASCL1, MYT1L, and BRN2. Here, we show that ASCL1 alone is sufficient to generate functional iN cells from mouse and human fibroblasts and embryonic stem cells, indicating that ASCL1 is the key driver of iN cell reprogramming in different cell contexts and that the role of MYT1L and BRN2 is primarily to enhance the neuronal maturation process. ASCL1-induced single-factor neurons (1F-iN) expressed mature neuronal markers, exhibited typical passive and active intrinsic membrane properties, and formed functional pre- and postsynaptic structures. Surprisingly, ASCL1-induced iN cells were predominantly excitatory, demonstrating that ASCL1 is permissive but alone not deterministic for the inhibitory neuronal lineage.

Heterozygous SHANK3 mutations are associated with idiopathic autism and Phelan-McDermid syndrome. SHANK3 is a ubiquitously expressed scaffolding protein that is enriched in postsynaptic excitatory synapses. Here, we used engineered conditional mutations in human neurons and found that heterozygous and homozygous SHANK3 mutations severely and specifically impaired hyperpolarization-activated cation (Ih) channels. SHANK3 mutations caused alterations in neuronal morphology and synaptic connectivity; chronic pharmacological blockage of Ih channels reproduced these phenotypes, suggesting that they may be secondary to Ih-channel impairment. Moreover, mouse Shank3-deficient neurons also exhibited severe decreases in Ih currents. SHANK3 protein interacted with hyperpolarization-activated cyclic nucleotide-gated channel proteins (HCN proteins) that form Ih channels, indicating that SHANK3 functions to organize HCN channels. Our data suggest that SHANK3 mutations predispose to autism, at least partially, by inducing an Ih channelopathy that may be amenable to pharmacological intervention.