Molecular Psychiatry (2012) 17, 1103–1115 & 2012 Macmillan Publishers Limited All rights reserved 1359-4184/12 www.nature.com/mp ORIGINAL ARTICLE Transcriptome profiling of UPF3B/NMD-deficient lymphoblastoid cells from patients with various forms of intellectual disability LS Nguyen1,2, L Jolly2, C Shoubridge1,2, WK Chan3, L Huang4, F Laumonnier5,6,7, M Raynaud5,7,8, A Hackett9, M Field9, J Rodriguez10, AK Srivastava10, Y Lee11, R Long11, AM Addington11, JL Rapoport11, S Suren12, CN Hahn13, J Gamble14, MF Wilkinson4, MA Corbett2 and J Gecz1,2 1Department of Paediatrics, University of Adelaide, Adelaide, SA, Australia; 2Department of Genetic Medicine, SA Pathology, Adelaide, SA, Australia; 3Department of Bioinformatics and Computational Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 4Department of Reproductive Medicine, University of California, San Diego, CA, USA; 5INSERM, U930, Tours, France; 6CNRS, ERL3106, Tours, France; 7University Francois-Rabelais, UMR ‘Imaging and Brain’, Tours, France; 8CHRU de Tours, Service de Genetique, Tours, France; 9GOLD Service, Hunter Genetics, Newcastle, Australia; 10J.C. Self Research Institute, Greenwood Genetic Centre, Greenwood, SC, USA; 11Child Psychiatry Branch, National Institute of Mental Health, Bethesda, MD, USA; 12Human Developmental Biology Resource, Neural Development Unit, UCL Institute of Child Health, London, UK; 13Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia and 14Centenary Institute of Cancer Medicine & Cell Biology, University of Sydney, NSW, Australia The nonsense-mediated mRNA decay (NMD) pathway was originally discovered by virtue of its ability to rapidly degrade aberrant mRNAs with premature termination codons. More recently, it was shown that NMD also directly regulates subsets of normal transcripts, suggesting that NMD has roles in normal biological processes. Indeed, several NMD factors have been shown to regulate neurological events (for example, neurogenesis and synaptic plasticity) in numerous vertebrate species. In man, mutations in the NMD factor gene UPF3B, which disrupts a branch of the NMD pathway, cause various forms of intellectual disability (ID). Using Epstein Barr virus—immortalized B cells, also known as lymphoblastoid cell lines (LCLs), from ID patients that have loss-of-function mutations in UPF3B, we investigated the genome-wide consequences of compromised NMD and the role of NMD in neuronal development and function. We found that B5% of the human transcriptome is impacted in UPF3B patients. The UPF3B paralog, UPF3A, is stabilized in all UPF3B patients, and partially compensates for the loss of UPF3B function. Interestingly, UPF3A protein, but not mRNA, was stabilised in a quantitative manner that inversely correlated with the severity of patients’ phenotype. This suggested that the ability to stabilize the UPF3A protein is a crucial modifier of the neurological symptoms due to loss of UPF3B. We also identified ARHGAP24, which encodes a GTPase-activating protein, as a canonical target of NMD, and we provide evidence that deregulation of this gene inhibits axon and dendrite outgrowth and branching. Our results demonstrate that the UPF3B-dependent NMD pathway is a major regulator of the transcriptome and that its targets have important roles in neuronal cells. Molecular Psychiatry (2012) 17, 1103–1115; doi:10.1038/mp.2011.163; published online 20 December 2011 Keywords: intellectual disability; Nonsense-mediated mRNA decay; RNA-SEQ; UPF3A; UPF3B Introduction degrades transcripts harboring mutations that intro- duce premature termination codons (PTCs), prevent- Nonsense-mediated mRNA decay (NMD) is a con- ing the truncated proteins with possible dominant served pathway in eukaryotes ranging from Sacchro- negative effects to be made. How NMD carries out its myces cerevisiae to mammals.1 NMD recognizes and function is taxonomically dependent. In metazoan, the conserved UPF1, UPF2 and UPF3 proteins Correspondence: Professor J Gecz, Department of Genetic Medi- constitute the core components of the classical NMD cine, SA Pathology, 72 King William Road, Womens and pathway.1 UPF3 is associated with the exon-junction Childrens Hospital, North Adelaide, South Australia 5006, complex that marks the exon–exon junction during Australia. pre-mRNA splicing.2,3 UPF2 interacts with UPF3 to E-mail: [email protected] Received 1 August 2011; revised 27 September 2011; accepted 24 bridge the exon-junction complex to UPF1 and other October 2011; published online 20 December 2011 NMD factors when the ribosome stalls at the PTC UPF3B/NMD deficiency in intellectual disability LS Nguyen et al 1104 during the pioneer round of translation.1,4 UPF1 is an Materials and methods ATP-ase RNA helicase whose role is to trigger RNA extraction recruitment of downstream NMD factors to degrade RNA was extracted from frozen lymphoblastoid cell transcripts bearing PTC.1,5–7 In addition to this pellet using Trizol (Invitrogen, Grand Island, NY, USA) classical pathway, it has been shown that NMD can and RNeasy Mini Kit (QIAGEN, Hilden, Germany), function in alternative cascades independent of UPF2 according to the manufacturer’s instruction. or UPF3.8,9 The cascade studied in this paper involves UPF3 proteins, UPF3B and its ortholog UPF3A. RNA-SEQ, exon array and SNP (single nucleotide UPF3B and UPF3A share high sequence similarity variants)-chip sample processing and both compete for interaction with UPF2 to m 10,11 Twenty g of RNA (Supplementary Table S2) was sent activate NMD. This is part of a regulatory switch to Geneworks (Adelaide, Australia) for library pre- that maintains proper NMD function in different 10 paration and sequencing on the Illumina (San Diego, tissues where varying level of UPF3B is observed. CA, USA) GAII platform. Seven mg of RNA (Supple- NMD also regulates normal transcript levels. mentary Table S3) was sent to the Australian Genome Microarray studies on NMD-deficient eukaryotic Research Facility (Melbourne, Australia) for labeling models and human cell lines suggested that NMD 9,10,12–16 and hybridizing to Affymetrix Human Exon 1.0 ST regulates 3–10% of the transcriptome. Tran- arrays (Affymetrix, Santa Clara, CA, USA). Three mgof scripts regulated by NMD have important roles in 9,10,12,13 DNA was sent to the Australian Genome Research cell survival and cell function. In fact, NMD is Facility for labeling and hybridizing to the Illumina crucial for higher eukaryotic development as deletion Human Omniexpress SNP chip. All patients de- of Upf1 or Upf2 in the mouse led to embryonic 14,17 scribed in Supplementary Table S1 and two controls lethality. In man, we showed that mutations in were included in the SNP-chip analysis. UPF3B, an X-chromosome linked gene, cause various forms of syndromic and non-syndromic intellectual Mapping of RNA-SEQ reads disability (ID) (Mendelian Inheritance in Man, MIM, RNA-SEQ reads were mapped to the reference 18,19 no. 300676), thus, NMD also has a central role in sequences, which includes the latest build of the regulating normal brain development and function. human genome (HG19), exon-junction database (see To date, mutations in UPF3B have been found in nine below) and all known ribosomal DNA using Efficient 18–20 families, including one unpublished family (LSN Alignment of Nucleotide Database (ELAND, Illumina) & JG, unpublished data). These are frame-shift and allowing up to two mismatches in the first 32 bp seed missense mutations that introduce PTCs and com- of the reads. Reads were mapped at the original pletely abolish UPF3B function (Patient 1–4 and length, 65 bp, and trimmed, 50 bp (first base and the patient 6–9, Supplementary Table S1), or missense last 14 bases excluded) for comparison (Supplemen- change in a conserved amino acid near the UPF2- tary Figures S1a and S1b). Mapped reads, that have binding motif, which could affect UPF3B ability to more than two mismatches along the entire reads or interact with UPF2 (patient 5, Supplementary Table are mapped to ribosomal or mitochondrial DNA, were S1). UPF3B patients present with a highly hetero- removed from downstream analysis (Supplementary geneous phenotype, which include attention-deficit Figure S1b and S1c). hyperactivity disorder, schizophrenia, autism and ID (Supplementary Table S1). There is considerable Generation of exon-junction database intra- and inter-familial variability in clinical pre- A comprehensive list of all known exon junctions, sentations in patients with UPF3B mutations. As including alternatively spliced exons was compiled such we propose to use the term ‘UPF3B spectrum’ to from all genes annotated in the UCSC database using describe this. Having access to patients’ cell lines script included in ELAND. Each exon junction provided us with a unique opportunity to study sequence was comprised of the flanking sequence natural consequences of compromised NMD on the 5 bp shorter than the read length used for mapping on human transcriptome without the need of manipulat- both sides of the exon–exon junction, that is, if read ing UPF3B or NMD in vitro. length was 65 bp or 50 bp, the flanking sequence Here we identify a set of tentative NMD targets would be 60 bp or 45 bp and the total length of the using a combination of approaches. We also show that exon junction sequence would be 120 bp or 90 bp, UPF3A protein is stabilized in UPF3B patients and respectively. This limit ensured that mapped reads functionally compensates for the loss of UPF3B in a matched at least