CAPRIN2 upregulation by LINC00941 promotes nasopharyngeal carcinoma ferroptosis resistance and metastatic colonization through HMGCR.

Distant metastasis is the main cause of death in nasopharyngeal carcinoma (NPC) patients. There is an urgent need to reveal the underlying mechanism of NPC metastasis and identify novel therapeutic targets. The ferroptosis resistance and survival ability of extracellular matrix (ECM)-detached tumor cells are important factors in determining the success of distant metastasis. In this study, we found that CAPRIN2 contributes to the ferroptosis resistance and survival of ECM-detached NPC cells. Moreover, CAPRIN2 serves as a positive regulator of NPC cell migration and invasion. HMGCR, the key metabolic enzyme of the mevalonate pathway, was identified as the key downstream molecule of CAPRIN2, which mediates its regulation of ferroptosis, survival, migration and invasion of NPC cells. Lung colonization experiments showed that downregulation of the CAPRIN2/HMGCR axis resulted in reduced lung metastasis of NPC cells. Erastin treatment inhibited the ability of NPC cells to colonize the lungs, which was further enhanced by CAPRIN2/HMGCR axis downregulation. Regulated by upstream LINC00941, CAPRIN2 is abnormally activated in NPC, and its high expression is associated with a poor prognosis. In conclusion, CAPRIN2 is a molecular marker of a poor prognosis in NPC, and the LINC00941/CAPRIN2/HMGCR axis provides a new target for the treatment of NPC metastasis and ferroptosis resistance.

Transcriptional and Post-Transcriptional Regulation of Oxytocin and Vasopressin Gene Expression by CREB3L1 and CAPRIN2.

INTRODUCTION: Water homoeostasis is achieved by secretion of the peptide hormones arginine vasopressin (AVP) and oxytocin (OXT) that are synthesized by separate populations of magnocellular neurones (MCNs) in the supraoptic and paraventricular (PVN) nuclei of the hypothalamus. To further understand the molecular mechanisms that facilitate biosynthesis of AVP and OXT by MCNs, we have explored the spatiotemporal dynamic, both mRNA and protein expression, of two genes identified by our group as being important components of the osmotic defence response: Caprin2 and Creb3l1. METHODS: By RNA in situ hybridization and immunohistochemistry, we have characterized the expression of Caprin2 and Creb3l1 in MCNs in the basal state, in response to dehydration, and during rehydration in the rat. RESULTS: We found that Caprin2 and Creb3l1 are expressed in AVP and OXT MCNs and in response to dehydration expression increases in both MCN populations. Protein levels mirror the increase in transcript levels for both CREB3L1 and CAPRIN2. In view of increased CREB3L1 and CAPRIN2 expression in OXT neurones by dehydration, we explored OXT-specific functions for these genes. By luciferase assays, we demonstrate that CREB3L1 may be a transcription factor regulating Oxt gene expression. By RNA immunoprecipitation assays and Northern blot analysis of Oxt mRNA poly(A) tails, we have found that CAPRIN2 binds to Oxt mRNA and regulates its poly(A) tail length. Moreover, in response to dehydration, Caprin2 mRNA is subjected to nuclear retention, possibly to regulate Caprin2 mRNA availability in the cytoplasm. CONCLUSION: The exploration of the spatiotemporal dynamics of Creb3l1- and Caprin2-encoded mRNAs and proteins has provided novel insights beyond the AVP-ergic system, revealing novel OXT-ergic system roles of these genes in the osmotic defence response.

RNA-binding proteins and post-transcriptional regulation in lens biology and cataract: Mediating spatiotemporal expression of key factors that control the cell cycle, transcription, cytoskeleton and transparency.

Development of the ocular lens - a transparent tissue capable of sustaining frequent shape changes for optimal focusing power - pushes the boundaries of what cells can achieve using the molecular toolkit encoded by their genomes. The mammalian lens contains broadly two types of cells, the anteriorly located monolayer of epithelial cells which, at the equatorial region of the lens, initiate differentiation into fiber cells that contribute to the bulk of the tissue. This differentiation program involves massive upregulation of select fiber cell-expressed RNAs and their subsequent translation into high amounts of proteins, such as crystallins. But intriguingly, fiber cells achieve this while also simultaneously undergoing significant morphological changes such as elongation - involving about 1000-fold length-wise increase - and migration, which requires modulation of cytoskeletal and cell adhesion factors. Adding further to the challenges, these molecular and cellular events have to be coordinated as fiber cells progress toward loss of their nuclei and organelles, which irreversibly compromises their potential for harnessing genetically hardwired information. A long-standing question is how processes downstream of signaling and transcription, which may also participate in feedback regulation, contribute toward orchestrating these cellular differentiation events in the lens. It is now becoming clear from findings over the past decade that post-transcriptional gene expression regulatory mechanisms are critical in controlling cellular proteomes and coordinating key processes in lens development and fiber cell differentiation. Indeed, RNA-binding proteins (RBPs) such as Caprin2, Celf1, Rbm24 and Tdrd7 have now been described in mediating post-transcriptional control over key factors (e.g. Actn2, Cdkn1a (p21Cip1), Cdkn1b (p27Kip1), various crystallins, Dnase2b, Hspb1, Pax6, Prox1, Sox2) that are variously involved in cell cycle, transcription, cytoskeleton maintenance and differentiation in the lens. Furthermore, deficiencies of these RBPs have been shown to result in various eye and lens defects and/or cataract. Because fiber cell differentiation in the lens occurs throughout life, the underlying regulatory mechanisms operational in development are expected to also be recruited for the maintenance of transparency in aged lenses. Indeed, in support of this, TDRD7 and CAPRIN2 loci have been linked to age-related cataract in humans. Here, I will review the role of key RBPs in the lens and their importance in understanding the pathology of lens defects. I will discuss advances in RBP-based gene expression control, in general, and the important challenges that need to be addressed in the lens to define the mechanisms that determine the epithelial and fiber cell proteome. Finally, I will also discuss in detail several key future directions including the application of bioinformatics approaches such as iSyTE to study RBP-based post-transcriptional gene expression control in the aging lens and in the context of age-related cataract.

Upregulated lncRNA Cyclin-dependent kinase inhibitor 2B antisense RNA 1 induces the proliferation and migration of colorectal cancer by miR-378b/CAPRIN2 axis.

LncRNA Cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) plays a role in the progression of multiple cancers like cholangiocarcinoma, osteosarcoma and several gastrointestinal tumors. Few studies have linked its function and mechanism to the development of colorectal cancer (CRC). The expression of CDKN2B-AS1, microRNA (miR)-378b, and cytoplasmic activation/proliferation-associated protein 2 (CAPRIN2) was analyzed in CRC patients and cell lines. The proliferation and migration of CRC cells were evaluated after gain and loss-of function mutations. Interactions between CDKN2B-AS1 and miR-378b, miR-378b and CAPRIN2 were validated by luciferase reporter, RNA pull-down and RNA immunoprecipitation assays. The role of CDKN2B-AS1 was further confirmed in a xenograft mouse model. We found that the expression of CDKN2B-AS1 and CAPRIN2 was upregulated in CRC and they were linked to the poor differentiation and distant metastasis in CRC patients. CDKN2B-AS1 knockdown attenuated while CDKN2B-AS1 overexpression promoted CRC cell proliferation and migration. Notably, the results of Starbase 2.0 database analysis and in vitro experiments demonstrated that CDKN2B-AS1 could interact with miR-378b and regulate its expression. Furthermore, CAPRIN2 acted as a downstream target of CDKN2B-AS1/miR-378b that involved in modulating ß-catenin expression in CRC cells. Upregulation of CDKN2B-AS1 contributed to CRC progression via regulating CAPRIN2 expression by binding to miR-378b. Downregulation of CDKN2B-AS1 suppressed tumor growth and Ki-67 staining in vivo that was related to the miR-378b/CAPRIN2 pathway. This study indicated that lncRNA CDKN2B-AS1 promoted the development of CRC through the miR-378b/CAPRIN2/ß-catenin axis. CDKN2B-AS1 might serve as a potential and useful target in CRC diagnosis and treatment.

Implications of RNG140 (caprin2)-mediated translational regulation in eye lens differentiation.

Regulation of gene expression at the translational level is key to determining cell fate and function. An RNA-binding protein, RNG140 (caprin2), plays a role in eye lens differentiation and has been reported to function in translational regulation. However, the mechanism and its role in eyes has remained unclear. Here, we show that RNG140 binds to the translation initiation factor eukaryotic initiation factor 3 (eIF3) and suppresses translation through mechanisms involving suppression of eIF3-dependent translation initiation. Comprehensive ribosome profiling revealed that overexpression of RNG140 in cultured Chinese hamster ovary cells reduces translation of long mRNAs, including those associated with cell proliferation. RNG140-mediated translational regulation also operates in the mouse eye, where RNG140 knockout increased the translation of long mRNAs. mRNAs involved in lens differentiation, such as crystallin mRNAs, are short and can escape translational inhibition by RNG140 and be translated in differentiating lenses. Thus, this study provides insights into the mechanistic basis of lens cell transition from proliferation to differentiation via RNG140-mediated translational regulation.

LINC00941 promotes oral squamous cell carcinoma progression via activating CAPRIN2 and canonical WNT/ß-catenin signaling pathway.

Dysregulation of long non-coding RNAs (lncRNAs) has been implicated in many cancer developments. Previous studies showed that lncRNA LINC00941 was aberrantly expressed in oral squamous cell carcinoma (OSCC). However, its role in OSCC development remains elusive. In this study, we demonstrated that in OSCC cells, EP300 activates LINC00941 transcription through up-regulating its promoter H3K27ac modification. Up-regulated LINC00941 in turn activates CAPRIN2 expression by looping to CAPRIN2 promoter. Functional assays suggest that both LINC00941 and CAPRIN2 play pivotal roles in promoting OSCC cell proliferation and colony formation. In vivo assay further confirmed the role of LINC00941 in promoting OSCC cell tumour formation. Lastly, we showed that the role of LINC00941 and CAPRIN2 in OSCC progression was mediated through activating the canonical WNT/ß-catenin signaling pathway. Thus, LINC00941/CAPRIN2/ WNT/ß-catenin signaling pathway provides new therapeutic targets for OSCC treatment.

Crystal structure of a 123 amino acids dimerization domain of Drosophila Caprin.

Cytoplasmic activation/proliferation-associated protein (Caprin) proteins assume diverse functions in many important biological processes, including synaptic plasticity, stress response, innate immune response, and cellular proliferation. The Caprin family members are characterized by the presence of a highly conserved homologous region (HR1) at the N-terminus and arginine-glycine-rich (RGG) boxes at the C-terminus. We had previously determined the crystal structures of human Caprin-1 and Caprin-2 fragments corresponding to the C-terminal 2/3 of HR1. Both fragments adopt homodimeric structures. Based on sequence conservation, we speculated that all Caprin proteins should have similar homodimeric structures. Here we report the crystal structure of a fragment (residues 187-309) of Drosophila melanogaster Caprin (dCaprin). The dCaprin fragment adopts an all α-helical fold which self-associates to form a homodimer. The overall dCaprin homodimeric structure is similar to the Caprin-1 and Caprin-2 homodimeric structures. Most of the amino acids residues mediating homodimerization in the three structures are conserved among all Caprin family members. These structural and sequence data suggest that homodimerization through a conserved dimerization domain is a common structural feature of the Caprin protein family. The dimeric structures may also be involved in interaction with Caprin partners. Dimer formation creates a V-shape concave surface that may serve as a protein binding groove. The concave surfaces in Caprin-1, Caprin-2, and dCaprin should have different and specific binding partners due to the large difference in electrostatic potentials. We propose the existence of a multi-functional domain in Caprin proteins, which not only mediate homodimerization but also involve in interaction with specific Caprin partners.

Crystal structure of a dimerization domain of human Caprin-2: similar overall dimeric fold but different molecular surface properties to that of human Caprin-1.

Human Caprin-1 and Caprin-2 are prototypic members of the caprin (cytoplasmic activation/proliferation-associated protein) protein family. Vertebrate caprin proteins contain two highly conserved homologous regions (HR1 and HR2) and C-terminal RGG motifs. Drosophila caprin (dCaprin) shares HR1 and RGG motifs but lacks HR2. Caprin-1 and Caprin-2 have important and non-redundant functions. The detailed molecular mechanisms of their actions remain largely unknown. Previously, we determined the crystal structure of a ∼120-residue fragment of Caprin-1 within the HR1 region. The structure has a novel all α-helical fold that self-associates to form a homodimer. In this study, the crystal structure of a corresponding fragment from Caprin-2 is reported. The Caprin-2 fragment has similar protein fold and dimeric structure as that of the Caprin-1 fragment. Structural comparison reveals that the molecular interactions mediating homodimerization of Caprin-1 and Caprin-2 are largely conserved in the two systems. Structural-modelling study of the corresponding dCaprin fragment indicates that dCaprin may also adopt a similar dimeric structure. The presence of a dimerization domain within HR1 may represent an evolutionarily conserved feature of the caprin protein family. Interestingly, while Caprin-1 and Caprin-2 adopt similar overall dimeric structures, the two structures have quite different molecular surface properties. In the Caprin-1 dimeric structure, some of the surface areas are known or suspected to function as binding sites for Carpin-1-interacting proteins. The different surface properties of the caprin dimeric structures may dictate their intermolecular interaction with specific protein partners. Communicated by Ramaswamy H. Sarma.

Unsupervised Network Analysis of the Plastic Supraoptic Nucleus Transcriptome Predicts Caprin2 Regulatory Interactions.

The supraoptic nucleus (SON) is a group of neurons in the hypothalamus responsible for the synthesis and secretion of the peptide hormones vasopressin and oxytocin. Following physiological cues, such as dehydration, salt-loading and lactation, the SON undergoes a function related plasticity that we have previously described in the rat at the transcriptome level. Using the unsupervised graphical lasso (Glasso) algorithm, we reconstructed a putative network from 500 plastic SON genes in which genes are the nodes and the edges are the inferred interactions. The most active nodal gene identified within the network was Caprin2. Caprin2 encodes an RNA-binding protein that we have previously shown to be vital for the functioning of osmoregulatory neuroendocrine neurons in the SON of the rat hypothalamus. To test the validity of the Glasso network, we either overexpressed or knocked down Caprin2 transcripts in differentiated rat pheochromocytoma PC12 cells and showed that these manipulations had significant opposite effects on the levels of putative target mRNAs. These studies suggest that the predicative power of the Glasso algorithm within an in vivo system is accurate, and identifies biological targets that may be important to the functional plasticity of the SON.

Crystal structure of a dimerization domain of human Caprin-1: insights into the assembly of an evolutionarily conserved ribonucleoprotein complex consisting of Caprin-1, FMRP and G3BP1.

Caprin-1 plays roles in many important biological processes, including cellular proliferation, innate immune response, stress response and synaptic plasticity. Caprin-1 has been implicated in several human diseases, including osteosarcoma, breast cancer, viral infection, hearing loss and neurodegenerative disorders. The functions of Caprin-1 depend on its molecular-interaction network. Direct interactions have been established between Caprin-1 and the fragile X mental retardation protein (FMRP), Ras GAP-activating protein-binding protein 1 (G3BP1) and the Japanese encephalitis virus (JEV) core protein. Here, crystal structures of a fragment (residues 132-251) of Caprin-1, which adopts a novel all-α-helical fold and mediates homodimerization through a substantial interface, are reported. Homodimerization creates a large and highly negatively charged concave surface suggestive of a protein-binding groove. The FMRP-interacting sequence motif forms an integral α-helix in the dimeric Caprin-1 structure in such a way that the binding of FMRP would not disrupt the homodimerization of Caprin-1. Based on insights from the structures and existing biochemical data, the existence of an evolutionarily conserved ribonucleoprotein (RNP) complex consisting of Caprin-1, FMRP and G3BP1 is proposed. The JEV core protein may bind Caprin-1 at the negatively charged putative protein-binding groove and an adjacent E-rich sequence to hijack the RNP complex.

Bacterial expression and preliminary crystallographic studies of a 149-residue fragment of human Caprin-1.

Caprin-1 is an RNA-binding protein which plays critical roles in several important biological processes, including cellular proliferation, the interferon-mediated antiviral innate immune response, the maintenance of synaptic plasticity and the formation of RNA stress granules. Caprin-1 has been implicated in the pathogenesis of several human diseases, including osteosarcoma, breast cancer, viral infections, hearing loss and neurodegenerative disorders. Despite the emerging biological and physiopathological significance of Caprin-1, no structural information is available for this protein. Moreover, Caprin-1 does not have sequence similarity to any other protein with a known structure. It is therefore expected that structural studies will play a particularly crucial role in revealing the functional mechanisms of Caprin-1. Here, a protein fragment of human Caprin-1 consisting of residues 112-260 was expressed, purified and crystallized. Native and Se-SAD data sets were collected to resolutions to 2.05 and 2.65 Å, respectively, in different space groups.

Structural insights into the C1q domain of Caprin-2 in canonical Wnt signaling.

Previously, we have identified Caprin-2 as a new regulator in canonical Wnt signaling through a mechanism of facilitating LRP5/6 phosphorylation; moreover, we found that its C-terminal C1q-related domain (Cap2_CRD) is required for this process. Here, we determined the crystal structures of Cap2_CRD from human and zebrafish, which both associate as a homotrimer with calcium located at the symmetric center. Surprisingly, the calcium binding-deficient mutant exists as a more stable trimer than its wild-type counterpart. Further studies showed that this Caprin-2 mutant disabled in binding calcium maintains the activity of promoting LRP5/6 phosphorylation, whereas the mutations disrupting Cap2_CRD homotrimer did impair such activity. Together, our findings suggested that the C-terminal CRD domain of Caprin-2 forms a flexible homotrimer mediated by calcium and that such trimeric assembly is required for Caprin-2 to regulate canonical Wnt signaling.