www.nature.com/scientificreports OPEN A double helical motif in OCIAD2 is essential for its localization, interactions and STAT3 activation Received: 16 November 2017 Saloni Sinha1, Venkata Anudeep Bheemsetty1 & Maneesha S. Inamdar 1,2 Accepted: 20 April 2018 The Ovarian Carcinoma Immunoreactive Antigen domain (OCIAD) - containing proteins OCIAD1/ Published: xx xx xxxx Asrij and OCIAD2, are implicated in several cancers and neurodegenerative diseases. While Asrij has a conserved role in facilitating STAT3 activation for JAK/STAT signaling, the expression and function of OCIAD2 in non-cancerous contexts remains unknown. Here, we report that ociad2 neighbors ociad1/ asrij in most vertebrate genomes, and the two genes likely arose by tandem gene duplication, probably somewhere between the Ordovician and Silurian eras. We show that ociad2 expression is higher in the mouse kidney, liver and brain relative to other tissues. OCIAD2 localizes to early endosomes and mitochondria, and interacts with Asrij and STAT3. Knockdown and overexpression studies showed that OCIAD2 is essential for STAT3 activation and cell migration, which could contribute to its role in tumor metastasis. Structure prediction programs, protein disruption studies, biochemical and functional assays revealed a double helical motif in the OCIA domain that is necessary and sufcient for its localization, interactions and STAT3 activation. Given the importance of JAK/STAT signaling in development and disease, our studies shed light on the evolution and conserved function of the OCIA domain in regulating this pathway and will be critical for understanding this clinically important protein family. OCIAD1 and OCIAD2 are human cancer-related proteins implicated in ovarian1, thyroid2 and lung cancers3, and in various hematological neoplasms4,5 including multiple myeloma6 and neutrophilia7. Teir names derive from the frst report on OCIAD1 (Ovarian Carcinoma Immunoreactive Antigen domain-containing protein 1), which was found in ascites fuid of patients with metastatic ovarian cancer and mapped to chromosome 4p118. Subsequently, a smaller human protein sharing homology with the N-terminal region of OCIAD1 was identifed and designated OCIAD2. While the developmental expression and cellular function of Asrij, the mouse ortholog of human OCIAD1 is reported9, there is limited information on the normal expression and localization of human OCIAD2 or its orthologs. Asrij localizes to endosomes10 and mitochondria11 and has key conserved roles in the maintenance of stem- ness in Drosophila hematopoiesis12, as well as in mouse embryonic stem cell pluripotency9. Moreover, Asrij reg- ulates blood cell homeostasis in Drosophila and its absence causes fy leukemia12. Asrij has a conserved role in regulating the JAK/STAT and Notch signaling pathways9,12. Although OCIAD2 expression varies among diferent cancers, its precise function remains unknown - while high levels of OCIAD2 are reported in ovarian mucinous tumors1 and lung carcinomas3,13; signifcantly reduced OCIAD2 expression is reported in liver and gastric carcinomas14, glioblastomas15 and chronic lymphocytic leu- kemia16. Further, loss of OCIAD2 function promotes cancer progression by increasing activation of the PI3K/ Akt pathway17. Moreover, the human OCIAD proteins are known to localize to lipid-rafs18,19 and have been proposed to be involved in the amyloidogenic processing of proteins associated with proteinopathies such as Alzheimer’s18,19 and Parkinson’s disease20. In this study, we explore the origin, evolution and function of ociad2. We report that the vertebrate OCIAD family members are genomic neighbors that possibly arose by a tandem gene duplication event in the last com- mon ancestor of jawed vertebrates. Further by in silico, in situ and biochemical approaches, we show that the two OCIAD proteins interact via a double helical region in the OCIA domain. In non-cancerous cells, OCIAD2 also interacts with and regulates STAT3 activation and cell migration, which is important in several developmental 1Jawaharlal Nehru Centre for Advanced Scientifc Research, Jakkur, Bangalore, 560064, India. 2Institute for Stem Cell Biology and Regenerative Medicine, GKVK, Bellary Road, Bangalore, 560065, India. Correspondence and requests for materials should be addressed to M.S.I. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:7362 | DOI:10.1038/s41598-018-25667-3 1 www.nature.com/scientificreports/ and immune processes as well as cancer. Our studies will help decipher the role and regulation of the OCIAD family proteins in various normal and pathological contexts. Results ociad2 is located next to ociad1/asrij and encodes an OCIAD family protein. ociad1/asrij is conserved in vertebrates and invertebrates, and has important functions in development and disease. Since the normal function of OCIAD1 in human is not known, we searched for similar proteins that may suggest its pos- sible function. Querying the NCBI genome database by a BLASTp analysis (https://blast.ncbi.nlm.nih.gov/Blast. cgi?PAGE=Proteins) revealed a shorter protein OCIAD2 of 154 amino acids in mouse and human, with 36.36% sequence identity to the OCIA domain and 14.17% sequence identity in the non-domain region of OCIAD1. In silico analysis mapped the corresponding mouse gene to chromosome 5 at 38.54 cM (73322199–73341028 bp), which is next to asrij (38.44 cM, 73292784–73314069 bp) and is transcribed from the antisense strand in the opposite direction (Fig. 1A). Te human protein OCIAD2 also mapped to a gene neighboring ociad1 on chromo- some 4p11. Detailed in silico analysis showed that the genes coding for ociad1 and ociad2 are neighbors, located in the same position and relative orientation (tail-to-tail) in mammals, birds, reptiles, amphibians and fsh [excep- tions: chicken (Gallus gallus), spotted gar (Lepisosteus oculatus, not shown) and whale shark (Rhincodon typus), where ociad2 is absent and red-bellied piranha (Pygocentrus nattereri), where ociad1 and ociad2 have a tail-to- head orientation] (Fig. 1A). Notably, we found that ociad1 and ociad2 are neighbors only in some fsh belonging to the Actinopterygii (Danio rerio, Clupea harengus, Pygocentrus nattereri) and Chondrichthyes (Callorhinchus milii) classes, whereas the others (Ictalurus punctatus, Oreochromis niloticus and Oryzias latipes) had ociad1 and ociad2 on diferent chromosomes (Fig. 1A). Interestingly, apart from ociad1 and ociad2 (referred to as ociad1a and ociad2a in Fig. 1A), some members of the teleost fsh category such as Danio rerio and Clupea harengus also had an additional ociad1-like gene (referred to as ociad1b in Fig. 1A; Gene IDs: 553528, 105903538). Although we could not find ociad2 in whale shark (Rhincodon typus), it is noteworthy that this spe- cies has a non-coding RNA for ociad2, that seems to have been missed by the automatic annotation procedures. In this regard, a tblastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&PAGE_ TYPE=BlastSearch&LINK_LOC=blasthome) search using elephant shark (Callorhinchus milii) OCIAD2 (Accession ID: XP_007890924.1) against the genome of whale shark (Rhincodon typus), revealed the presence of an uncharacterized non-coding RNA (LOC109914671, Accession ID: XR_002258473.1), present in a similar ori- entation, as expected for ociad2. Moreover, alignment of diferent reading frames of the elephant shark OCIAD2 protein and the translated sequence of the LOC109914671 non-coding RNA revealed overlap of two frames with high identity values (57%, 56%) and aided in the identifcation of several conserved exon stretches. Tus, this suggests that the arrangement of the OCIAD family of genes is very similar across all the vertebrate species. Analysis of gene synteny across diferent species helped in identifcation of conserved fanking genes (fryl and cwh43) in the vicinity of ociad1 and ociad2 in mammals, birds and reptiles (Fig. 1A). Although members of the frog and teleost fsh lineages lack cwh43, we found synteny with the gene next to cwh43, namely, dcun1d4, to be conserved (Fig. 1A). Tese results indicate that the gene synteny of ociad1 and ociad2 is conserved and strongly suggests that this particular genomic arrangement is ancestral to all vertebrate genomes analyzed here. Mapping the nucleotide positions of the start and stop sites of ociad1 and ociad2 showed that these genes were non-overlapping in species where they were neighbors (Fig. S1A,B). Further, comparison of protein lengths across species shows that OCIAD2 (154 aa in mouse) is a smaller protein than OCIAD1 (247 aa in mouse) with a shorter C-terminal region. (Fig. 2A). Multiple sequence alignment of the full-length OCIAD1 and OCIAD2 sequences using MUSCLE21 showed maximum conservation towards the N-terminal (Fig. 2B) and a high degree of similarity between OCIAD2 sequences of mouse, rat and human (Fig. 2C). Although signifcant homology exists among the N-terminal regions of OCIAD1 and OCIAD2 sequences across various species, the C-terminal regions are not as well-conserved (Fig. S2A). ociad1 and ociad2 evolved ca 435–500 MYA via tandem gene duplication from an ancestral ociad gene and have comparable rates of amino acid evolution. To explore the evolutionary rela- tionship between OCIAD family members, we collected a total of 106 protein sequences from NCBI (https:// www.ncbi.nlm.nih.gov/) covering 58 unique species, that included invertebrate OCIAD and vertebrate OCIAD1 and OCIAD2 sequences (see Table S1). We used protein sequences to identify genetic events relevant to the evolution