FAM19A5 Expression During Embryogenesis and in the Adult Traumatic Brain of FAM19A5-LacZ Knock-in Mice.

FAM19A5 is a secretory protein that is predominantly expressed in the brain. Although the FAM19A5 gene has been found to be associated with neurological and/or psychiatric diseases, only limited information is available on its function in the brain. Using FAM19A5-LacZ knock-in mice, we determined the expression pattern of FAM19A5 in developing and adult brains and identified cell types that express FAM19A5 in naïve and traumatic brain injury (TBI)-induced brains. According to X-gal staining results, FAM19A5 is expressed in the ventricular zone and ganglionic eminence at a very early stage of brain development, suggesting its functions are related to the generation of neural stem cells and oligodendrocyte precursor cells (OPCs). In the later stages of developing embryos and in adult mice, FAM19A5 expression expanded broadly to particular regions of the brain, including layers 2/3 and 5 of the cortex, cornu amonis (CA) region of the hippocampus, and the corpus callosum. X-gal staining combined with immunostaining for a variety of cell-type markers revealed that FAM19A5 is expressed in many different cell types, including neurons, OPCs, astrocytes, and microglia; however, only some populations of these cell types produce FAM19A5. In a subpopulation of neuronal cells, TBI led to increased X-gal staining that extended to the nucleus, marked by slightly condensed content and increased heterochromatin formation along the nuclear border. Similarly, nuclear extension of X-gal staining occurred in a subpopulation of OPCs in the corpus callosum of the TBI-induced brain. Together, these results suggest that FAM19A5 plays a role in nervous system development from an early stage and increases its expression in response to pathological conditions in subsets of neurons and OPCs of the adult brain.

SERBP1 affects homologous recombination-mediated DNA repair by regulation of CtIP translation during S phase.

DNA double-strand breaks (DSBs) are the most severe type of DNA damage and are primarily repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) in the G1 and S/G2 phase, respectively. Although CtBP-interacting protein (CtIP) is crucial in DNA end resection during HR following DSBs, little is known about how CtIP levels increase in an S phase-specific manner. Here, we show that Serpine mRNA binding protein 1 (SERBP1) regulates CtIP expression at the translational level in S phase. In response to camptothecin-mediated DNA DSBs, CHK1 and RPA2 phosphorylation, which are hallmarks of HR activation, was abrogated in SERBP1-depleted cells. We identified CtIP mRNA as a binding target of SERBP1 using RNA immunoprecipitation-coupled RNA sequencing, and confirmed SERBP1 binding to CtIP mRNA in S phase. SERBP1 depletion resulted in reduction of polysome-associated CtIP mRNA and concomitant loss of CtIP expression in S phase. These effects were reversed by reconstituting cells with wild-type SERBP1, but not by SERBP1 ΔRGG, an RNA binding defective mutant, suggesting regulation of CtIP translation by SERBP1 association with CtIP mRNA. These results indicate that SERBP1 affects HR-mediated DNA repair in response to DNA DSBs by regulation of CtIP translation in S phase.

ß-Catenin recognizes a specific RNA motif in the cyclooxygenase-2 mRNA 3'-UTR and interacts with HuR in colon cancer cells.

RNA-binding proteins regulate multiple steps of RNA metabolism through both dynamic and combined binding. In addition to its crucial roles in cell adhesion and Wnt-activated transcription in cancer cells, ß-catenin regulates RNA alternative splicing and stability possibly by binding to target RNA in cells. An RNA aptamer was selected for specific binding to ß-catenin to address RNA recognition by ß-catenin more specifically. Here, we characterized the structural properties of the RNA aptamer as a model and identified a ß-catenin RNA motif. Similar RNA motif was found in cellular RNA, Cyclooxygenase-2 (COX-2) mRNA 3'-untranslated region (3'-UTR). More significantly, the C-terminal domain of ß-catenin interacted with HuR and the Armadillo repeat domain associated with RNA to form the RNA-ß-catenin-HuR complex in vitro and in cells. Furthermore, the tertiary RNA-protein complex was predominantly found in the cytoplasm of colon cancer cells; thus, it might be related to COX-2 protein level and cancer progression. Taken together, the ß-catenin RNA aptamer was valuable for deducing the cellular RNA aptamer and identifying novel and oncogenic RNA-protein networks in colon cancer cells.

Annexin A2 binds RNA and reduces the frameshifting efficiency of infectious bronchitis virus.

Annexin A2 (ANXA2) is a protein implicated in diverse cellular functions, including exocytosis, DNA synthesis and cell proliferation. It was recently proposed to be involved in RNA metabolism because it was shown to associate with some cellular mRNA. Here, we identified ANXA2 as a RNA binding protein (RBP) that binds IBV (Infectious Bronchitis Virus) pseudoknot RNA. We first confirmed the binding of ANXA2 to IBV pseudoknot RNA by ultraviolet crosslinking and showed its binding to RNA pseudoknot with ANXA2 protein in vitro and in the cells. Since the RNA pseudoknot located in the frameshifting region of IBV was used as bait for cellular RBPs, we tested whether ANXA2 could regulate the frameshfting of IBV pseudoknot RNA by dual luciferase assay. Overexpression of ANXA2 significantly reduced the frameshifting efficiency from IBV pseudoknot RNA and knockdown of the protein strikingly increased the frameshifting efficiency. The results suggest that ANXA2 is a cellular RBP that can modulate the frameshifting efficiency of viral RNA, enabling it to act as an anti-viral cellular protein, and hinting at roles in RNA metabolism for other cellular mRNAs.

Modulation of transcription by the peroxisome proliferator-activated receptor delta--binding RNA aptamer in colon cancer cells.

Peroxisome proliferator-activated receptor delta (PPAR-delta), one of three PPAR subtypes, is a lipid-sensing nuclear receptor that has been implicated in multiple processes, including inflammation and cancer. To directly establish the role of PPAR-delta in colon cancer development and progression, we selected high-affinity RNA aptamers and expressed them in several colon cancer cell lines. Nuclear-expressed aptamers efficiently inhibited PPAR-delta-dependent transcription from a synthetic peroxisome proliferator response element-driven luciferase reporter. PPAR-delta-specific aptamers suppressed transcription from natural promoters of vascular endothelial cell growth factor-A and cyclooxygenase-2. Moreover, vascular endothelial cell growth factor-A and cyclooxygenase-2 mRNA levels were significantly reduced by the PPAR-delta-specific aptamers in colon cancer cells. Most significantly, HCT116 colon cancer cells with high-level expression of PPAR-delta-specific aptamers exhibited a striking loss of tumorigenic potential. Further study on these RNA aptamers could provide an opportunity to modulate PPAR-delta-mediated colon cancer development and progression. Taken together, our results establish an important role for PPAR-delta in transcription of tumor-promoting genes, which can be specifically modulated by high-affinity RNA intramers in colon cancer cells. The RNA intramers may be further developed as specific inhibitors for cancer therapeutic strategies.