AUTS2 Syndrome: Molecular Mechanisms and Model Systems

Abstract

AUTS2 syndrome is a genetic disorder that causes intellectual disability, microcephaly, and other phenotypes. Syndrome severity is worse when mutations involve 3' regions (exons 9-19) of the AUTS2 gene. Human AUTS2 protein has two major isoforms, full-length (1259 aa) and C-terminal (711 aa), the latter produced from an alternative transcription start site in exon 9. Structurally, AUTS2 contains the putative "AUTS2 domain" (∼200 aa) conserved among AUTS2 and its ohnologs, fibrosin, and fibrosin-like-1. Also, AUTS2 contains extensive low-complexity sequences and intrinsically disordered regions, features typical of RNA-binding proteins. During development, AUTS2 is expressed by specific progenitor cell and neuron types, including pyramidal neurons and Purkinje cells. AUTS2 localizes mainly in cell nuclei, where it regulates transcription and RNA metabolism. Some studies have detected AUTS2 in neurites, where it may regulate cytoskeletal dynamics. Neurodevelopmental functions of AUTS2 have been studied in diverse model systems. In zebrafish, auts2a morphants displayed microcephaly. In mice, excision of different Auts2 exons (7, 8, or 15) caused distinct phenotypes, variously including neonatal breathing abnormalities, cerebellar hypoplasia, dentate gyrus hypoplasia, EEG abnormalities, and behavioral changes. In mouse embryonic stem cells, AUTS2 could promote or delay neuronal differentiation. Cerebral organoids, derived from an AUTS2 syndrome patient containing a pathogenic missense variant in exon 9, exhibited neocortical growth defects. Emerging technologies for analysis of human cerebral organoids will be increasingly useful for understanding mechanisms underlying AUTS2 syndrome. Questions for future research include whether AUTS2 binds RNA directly, how AUTS2 regulates neurogenesis, and how AUTS2 modulates neural circuit formation.

Keywords: AUTS2 syndrome; FBRSL1; RNA-binding protein; cerebellar hypoplasia; cerebral organoids; dentate gyrus hypoplasia; intellectual disability; microcephaly.

FIGURE 1

AUTS2 transcripts and protein isoforms. (A) The full-length AUTS2 transcript has 19 exons. An alternative transcription start site (tss) in exon 9 produces mRNA for the C-terminal protein isoform. (B) The full-length and C-terminal isoforms are indicated with source exons in black (odd) and blue (even). Green boxes enclose splice junctions in which amino acids were encoded across the junctions, comprising exon blocs. Red asterisks indicate source exons that encoded a non-integer number of amino acids. Features mapped from protein sequence: ExB, exon bloc; NLS, nuclear localization sequence; IDR, intrinsically disordered region; PRR, Pro-rich repeat; HRR, His-rich repeat; HX, HX repeat; H8, polyhistidine (8×) repeat; SRR, Ser-rich repeat; PY, PY protein binding motif; CDD, conserved domain database; PD, ProDom predicted domains (domain names in bold also identified in FBRSL1); AFsh, AUTS2-FBRSL1 short homology region. (C) Both major AUTS2 isoforms have a high content of IDRs (zigzag lines) and regions enriched in amino acids Pro, His, or Ser (red lines). The C-terminal isoform includes the AUTS2 domain, but lacks N-terminal features such as the HX repeat.

FIGURE 2

AUTS2 expression in developing mouse and marmoset brain. (A) By in situ hybridization, Auts2 expression is observed in multiple areas of E14.5 mouse brain, including cerebral neocortex (Ncx), hippocampus (Hp), dorsal thalamus (DT), pons, cerebellum (Cb), and medulla (Med). Sagittal section from Genepaint (https://gp3.mpg.de/). (B,C) In P7 mouse cerebellum, AUTS2 protein (red) localizes in the nuclei of calbindin + (green) Purkinje cells, and in scattered Golgi neurons (arrows). Panel (C) magnified 5× from (B). (D) In neonatal marmosets, AUTS2 is expressed in many brain regions, with highest levels in amygdala (Am). (E,F) In 1-year-old (young adult) marmosets, AUTS2 levels remain high in amygdala and dentate gyrus (DG). Data for panels (D–F) from the Marmoset Gene Atlas (https://gene-atlas.brainminds.riken.jp/).

FIGURE 3

AUTS2 molecular functions and interactions. (A) In HEK293 cells, AUTS2 associates with variant forms of polycomb repressive complex 1 (vPRC1), which also contains PCGF3/5, RING1B, and other PRC1 subunits (PMID: 25519132). In turn, AUTS2 recruits P300, a histone acetyltransferase that opens chromatin, and binds NRF1, a transcriptional activator (PMID: 34637754). Also, vPRC1 recruits CK2, a protein kinase that phosphorylates and inactivates RING1B, a ubiquitin ligase and core subunit of PRC1 for chromatin inactivation. The net effect of AUTS2 is to convert PRC1 from a repressor to an activator of transcription. (B) In developing cerebral cortex, AUTS2 associates with multiple RNA-binding proteins (RBPs), including the scaffolds NONO and SFPQ; splicing factors SRSF3 and SRSF7; and RNA helicases DDX5 and DDX7 (PMID: 34013328). Also, AUTS2 co-immunoprecipitated multiple RNA species, suggesting that AUTS2 binds RNA directly or indirectly. (C) In HEK293 cells, AUTS2 associates with a guanine nucleotide exchange factor (GEF) complex containing DOCK1 (also known as DOCK180) and ELMO2 (PMID: 25533347). Through this interaction, AUTS2 is thought to cause activation of Rac1 (a small G protein), remodeling of the cytoskeleton, neurite elongation, and lamellipodia formation. AUTS2 was also found to interact with another GEF, P-REX1; and may regulate multiple small G proteins.

FIGURE 4

Modeling AUTS2 syndrome using cerebral organoids. (A) AUTS2 mutant cerebral organoids have proliferative deficits in neural progenitor cells and impaired neuritogenesis in cortical neurons (left). Utilization of scRNA-seq to determine cell type specific changes in gene expression within diverse progenitor, neuronal, and glial cell populations (right). (B) Generation of assembloids through the fusion of brain region specific organoids (cortical-thalamic assembloid shown, left) investigating neural circuitry using MEA and patch-clamp electrophysiology (right).