Ruan X*, Hu K*, Yang Y*, Yang R, Tseng E, Kang B, Kauffman A, Zhong R, and Zhang X.

J. Neurosciene. https://doi.org/10.1523/JNEUROSCI.0822-24.2024

How master splicing regulators crosstalk with each other and to what extent transcription regulators are differentially spliced remain unclear in the developing brain. Here, cell-type-specific RNA-Seq analyses of the developing neocortex uncover variable expression of the Rbfox1/2/3 genes and enriched splicing events in transcription regulators, altering protein isoforms or inducing nonsense-mediated mRNA decay. Transient expression of Rbfox proteins in radial glial progenitors induces neuronal splicing events in transcription regulators such as Meis2 and Tead1. Surprisingly, Rbfox proteins promote the inclusion of a mammal-specific alternative exon and a previously undescribed poison exon in Ptbp1. Simultaneous ablation of Rbfox1/2/3 in the neocortex downregulates neuronal isoforms and disrupts radial neuronal migration. These observations indicate functions of Rbfox1/2/3 and their regulation of transcription regulators in the developing neocortex.

Alternative splicing of transcription regulators in cortical development. (2024)

Zhang X.

Front. Mol. Neurosci. 2024;17 (Invited review, as a part of the post-transcriptional regulation collection)

Pediatric neurological disorders are frequently devastating and present unmet needs for effective medicine. The successful treatment of spinal muscular atrophy with splice-switching antisense oligonucleotides (SSO) indicates a feasible path to targeting neurological disorders. SSOs have been actively explored to restore gene functions by redirecting natural mRNA isoforms, correcting translational reading frames, and suppressing variant-specific splice sites. This review discusses recent advances that utilize SSOs to treat pediatric neurological disorders, with an emphasis on genetic models.

SSOs targeting specific variants, exons, and genes. (2024)

Yang Y*, Yang R*, Kang B, Qian S, He X†, Zhang X.†

Cell Reports. 2023;42(11): 113335.

Dysregulation of alternative splicing has been repeatedly associated with neurodevelopmental disorders, but the extent of cell-type-specific splicing in human neural development remains largely uncharted. Here, single-cell long-read sequencing in induced pluripotent stem cell (iPSC)-derived cerebral organoids identifies thousands of uncatalogued isoforms and cell-type-specific splicing events, such as coordinated exons and retained introns. The splicing program in autistic brains is closer to the progenitor state than neurons, and cell-type-specific exons harbor more de novo mutations in autism probands than siblings. These results highlight the role of cell-type-specific splicing in autism and neuronal gene regulation.

Single-cell long-read RNA-Seq in human cerebral organoids. (2023)

Kang B, Yang Y, Hu K, Ruan X, Liu Y, Lee P, Lee J, Wang J† and Zhang X.†

Genome Research. 2023;33:1–14.

Differential polyadenylation sites (PAs) critically regulate gene expression, but their cell type–specific usage and spatial distribution in the brain have not been systematically characterized. Here, we present Infernape to infer and quantify PA usage from single-cell and spatial transcriptomic data. Infernape uncovers alternative intronic PAs and 3′-UTR lengthening during cortical neurogenesis. In the adult mouse brain, we uncover cell type–specific PAs and visualize such events using spatial transcriptomic data. Over two dozen neurodevelopmental disorder–associated genes such as Csnk2a1 and Mecp2 show differential PAs during brain development. This study presents Infernape to identify PAs from scRNA-seq and spatial data, and highlights the role of alternative PAs in neuronal gene regulation.

Infernape uncovers cell type–specific and spatially resolved alternative polyadenylation. (2023)

Ruan X, Hu K, Zhang X.

Nat Commun. 2023;14(1):3275.

Tracking protein-RNA interaction across cell types is challenging. Here in PIE-Seq, we fuse RNA-binding proteins with dual deaminases to mark target RNAs with both C-to-U and A-to-I base editing. We benchmark PIE-Seq and demonstrate its sensitivity in single cells, its application in the developing brain, and its scalability with 25 human RBPs. PIE-Seq provides an orthogonal approach and resource to uncover RBP targets in mice and human cells.

PIE-seq: identifying RNA-binding protein targets by dual RNA-deaminase editing. (2023)

Yang R*, Feng X*, Arias-Cavieres A, Mitchell RM, Polo A, Hu K, Zhong R, Qi C, Zhang RS, Westneat N, Portillo CA, Nobrega MA, Hansel C, Garcia Iii AJ, Zhang X.

Neuron. 2023;111(10):1637-50. Pdf. Cover and Featured. News by BSD and Spectrum.

De novo SYNGAP1 mutations cause autism and intellectual disability. This study shows that PTBP1/2 proteins promote SYNGAP1 mRNA decay through alternative splicing. Redirecting splicing upregulates Syngap1 expression and alleviates neurological deficits in disease models. These results suggest that redirecting splicing can potentially rescue SYNGAP1 haploinsufficiency.

Upregulation of SYNGAP1 expression in models of human diseases. (2023)

Qi C, Feng I, Costa AR, Pinto-Costa R, Neil JE, Caluseriu O, Li D, Ganetzky RD, Brasch-Andersen C, Fagerberg C, Hansen LK, Bupp C, Muraresku CC, Ruan X, Kang B, Hu K, Zhong R, Brites P, Bhoj EJ, Hill RS, Falk MJ, Hakonarson H, Kahle KT, Sousa, M.M.†, Walsh, C.A.†, Zhang, X.†

Genet Med. 2022;24(2):319-31.

ADD1 loss-of-function mutations are associated with intellectual disability and structural brain defects, and Add1 knockout mice recapitulate the corpus callosum dysgenesis and ventriculomegaly phenotypes.

ADD1 variants are associated with structural brain malformations. (2022)

Ruan X*, Kang B*, Qi C, Lin W, Wang J†, Zhang X.†

Proc Natl Acad Sci U S A. 2021;118(10).

How projection neuron types are temporally and sequentially generated in the mammalian neocortex remains unclear. We performed single-cell RNA sequencing analysis of embryonic day (E) 10.5 through E18.5 mouse neocortical cells and uncovered molecular signatures for neuroepithelial cells and temporal gene expression in radial glial progenitors. Importantly, Eomes-positive cells display temporal expression of previously characterized neuronal identity genes. These results suggest transcriptional priming of neocortical progenitor cells for neuron type specification.

Temporal gene expression in cortical progenitor cells. (2021)

Zhang, X.†, Chen, M.H., Wu, X., Kodani, A., Fan, J., Doan, R., Ozawa, M., Ma, J., Yoshida, N., Reiter, J.F., Black, D.L. Kharchenko, P.V. Sharp, P.A. Walsh, C.A.†

Cell. 2016; 166, 1147-1162 e1115.

Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains—especially in cytoskeletal proteins—and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development.

Cell-type-specific RNA splicing regualtes neocortex development. (2016)

Zhang, X.*, Ling, J.*, Barcia, G.*, Jing, L., Wu, J., Barry, B.J., Mochida, G.H., Hill, R.S., Weimer, J.M., Stein, Q., Poduri, A., Partlow, J.N., Ville, D., Dulac, O., Yu, T.W., Lam, A.T., Servattalab, S., Rodriguez, J., Boddaert, N., Munnich, A., Colleaux, L., Zon, L.I., Soll, D., Walsh, C.A.†, and Nabbout, R†.

Am J Hum Genet. 2014; 94, 547-558. (*Co-first authors, †corresponding authors)

We report causal QARS variants in two unrelated families affected by progressive microcephaly, severe seizures in infancy, atrophy of the cerebral cortex and cerebellum. Variants p.Gly45Val and p.Tyr57His were located in the N-terminal domain , and recombinant QARS proteins bearing either substitution showed an over 10-fold reduction in aminoacylation activity. Conversely, variants p.Arg403Trp and p.Arg515Trp, each occurring in a different family, were located in the catalytic core and completely disrupted QARS aminoacylation activity in vitro. In zebrafish, homozygous qars loss of function caused decreased brain and eye size and extensive cell death in the brain. Our results highlight the importance of QARS during brain development.

Mutations in the glutaminyl-tRNA synthetase cause brain atrophy. (2014)

Zhang, X., Zabinsky, R., Teng, Y., Cui, M., and Han, M.

Proc Natl Acad Sci USA. 2011; 108, 17997-18002.

Numerous animal species across multiple phyla enter developmental arrest for long-term survival in unfavorable environments and resume development upon stress removal. Here we show that compromising overall microRNA (miRNA) functions or mutating certain individual miRNAs impairs the long-term survival of nematodes during starvation-induced L1 diapause. We provide evidence that miRNA miR-71 is not required for the animals’ entry into L1 diapause, but plays a critical role in long-term survival by repressing the expression of insulin receptor/PI3K pathway genes . Furthermore, miR-71 plays a prominent role in developmental recovery from L1 diapause partly through repressing the expression of certain heterochronic genes. The presented results indicate that miRNAs especially miR-71 regulate the response to starvation-induced L1 diapause.

miR-71 is required for the timing of cell division under food deprivation. (2011)

Zhang, X., Lei, K., Yuan, X., Wu, X., Zhuang, Y., Xu, T., Xu, R., and Han, M.

Neuron. 2009; 64, 173-187. (Accompanied by a Mini-review by Hiroyuki Koizumi and Joe Gleeson, highlighted by Nature China and Faculty of 1000)

A gap in the radial neuronal migration field was how the nucleus is connected to the centrosome and microtubules during long-distance nucleus translocation. We found both Syne-1;Syne-2 and Sun1;Sun2 double knockout causes perinatal lethality and significantly reduced brain size. Our further studies indicated that SUN-Syne complexes connect centrosomes to the nucleus during interkinetic nuclear migration and radial neuronal migration in the developing mouse cerebral cortex. This study established the critical functions of nuclear envelope proteins in positioning nuclei during brain development.

SUN-KASH links centrosome to the nucleus for radial neuronal migration (2009, accompanied by a mini-review by Hiroyuki Koizumi and Joe Gleeson).

Zhang, X., Xu, R., Zhu, B., Yang, X., Ding, X., Duan, S., Xu, T., Zhuang, Y., and Han, M.

Development. 2007; 134, 901-908. (Highlighted by Faculty of 1000)

Each of our skeletal muscle cell contains dozens to hundreds of nuclei that are stably positioned during drastic muscle contractions. How these nuclei are anchored was an open question. We investigated two classes of nuclear envelope (NE) proteins, outer NE proteins Syne-1 (Nesprin-1) and Syne-2 (Nesprin-2) as well as inner NE proteins Sun1 and Sun2, using knockout and transgenic mouse models. We found that Syne-1 and Sun1/2 are required for myonuclear anchorage and motor neuron innervation.

The anchorage of synaptic nuclei is abolished in Syne-1 -/- mice. (2007)