Integrative omics indicate FMRP sequesters mRNA from translation and deadenylation in human neuronal cells.

How fragile X syndrome protein (FMRP) binds mRNAs and regulates mRNA metabolism remains unclear. Our previous work using human neuronal cells focused on mRNAs targeted for nonsense-mediated mRNA decay (NMD), which we showed are generally bound by FMRP and destabilized upon FMRP loss. Here, we identify >400 high-confidence FMRP-bound mRNAs, only ∼35% of which are NMD targets. Integrative transcriptomics together with SILAC-LC-MS/MS reveal that FMRP loss generally results in mRNA destabilization and more protein produced per FMRP target. We use our established RIP-seq technology to show that FMRP footprints are independent of protein-coding potential, target GC-rich and structured sequences, and are densest in 5' UTRs. Regardless of where within an mRNA FMRP binds, we find that FMRP protects mRNAs from deadenylation and directly binds the cytoplasmic poly(A)-binding protein. Our results reveal how FMRP sequesters polyadenylated mRNAs into stabilized and translationally repressed complexes, whose regulation is critical for neurogenesis and synaptic plasticity.

RNA Exosome Component EXOSC4 Amplified in Multiple Cancer Types Is Required for the Cancer Cell Survival.

The RNA exosome is a multi-subunit ribonuclease complex that is evolutionally conserved and the major cellular machinery for the surveillance, processing, degradation, and turnover of diverse RNAs essential for cell viability. Here we performed integrated genomic and clinicopathological analyses of 27 RNA exosome components across 32 tumor types using The Cancer Genome Atlas PanCancer Atlas Studies' datasets. We discovered that the EXOSC4 gene, which encodes a barrel component of the RNA exosome, was amplified across multiple cancer types. We further found that EXOSC4 alteration is associated with a poor prognosis of pancreatic cancer patients. Moreover, we demonstrated that EXOSC4 is required for the survival of pancreatic cancer cells. EXOSC4 also repressed BIK expression and destabilized SESN2 mRNA by promoting its degradation. Furthermore, knockdown of BIK and SESN2 could partially rescue pancreatic cells from the reduction in cell viability caused by EXOSC4 knockdown. Our study provides evidence for EXOSC4-mediated regulation of BIK and SESN2 mRNA in the survival of pancreatic tumor cells.

Repurposing the PDMA-approved drugs in Japan using an insect model of staphylococcal infection.

A total of 1253 compounds approved as therapeutic drugs in Japan (Pharmaceuticals and Medical Devices Agency (PMDA)-approved compounds) were screened for their therapeutic effects against Staphylococcus aureus infection using the silkworm infection model. In the first stage of screening with an index of prolonged survival, 80 compounds were identified as hits. Of these, 64 compounds were clinically used as antimicrobial agents, and the remaining 16 compounds were not. The 16 compounds were examined for their dose-dependent therapeutic effects on the silkworm model as a second screening step, and we obtained five compounds as a result. One of the compounds (capecitabine) had no documented in vitro minimum inhibitory concentration (MIC) value against S. aureus. The MIC value of capecitabine against S. aureus strains ranged from 125 to 250 µg/ml, and capecitabine was therapeutically effective at a dose of 200 mg/kg in a murine model of S. aureus infection. These results suggest that silkworm-based drug repositioning studies are of potential value. Furthermore, the therapeutic effects of capecitabine demonstrated in this study provide an important scientific rationale for clinical observational studies examining the association between staphylococcal infection events and capecitabine administration in cancer chemotherapy patients.

Loss of the fragile X syndrome protein FMRP results in misregulation of nonsense-mediated mRNA decay.

Loss of the fragile X protein FMRP is a leading cause of intellectual disability and autism1,2, but the underlying mechanism remains poorly understood. We report that FMRP deficiency results in hyperactivated nonsense-mediated mRNA decay (NMD)3,4 in human SH-SY5Y neuroblastoma cells and fragile X syndrome (FXS) fibroblast-derived induced pluripotent stem cells (iPSCs). We examined the underlying mechanism and found that the key NMD factor UPF1 binds directly to FMRP, promoting FMRP binding to NMD targets. Our data indicate that FMRP acts as an NMD repressor. In the absence of FMRP, NMD targets are relieved from FMRP-mediated translational repression so that their half-lives are decreased and, for those NMD targets encoding NMD factors, increased translation produces abnormally high factor levels despite their hyperactivated NMD. Transcriptome-wide alterations caused by NMD hyperactivation have a role in the FXS phenotype. Consistent with this, small-molecule-mediated inhibition of hyperactivated NMD, which typifies iPSCs derived from patients with FXS, restores a number of neurodifferentiation markers, including those not deriving from NMD targets. Our mechanistic studies reveal that many molecular abnormalities in FMRP-deficient cells are attributable-either directly or indirectly-to misregulated NMD.

Human U90926 orthologous long non-coding RNA as a novel biomarker for visual prognosis in herpes simplex virus type-1 induced acute retinal necrosis.

Acute retinal necrosis (ARN) is a form of infectious uveitis caused by alpha herpesviruses, including herpes simplex virus type 1 (HSV-1). We previously found that the long non-coding RNA (lncRNA) U90926 is upregulated in murine retinal photoreceptor cells following HSV-1 infection, leading to host cell death. However, to date, an orthologous transcript has not been identified in humans. We investigated U90926 orthologous transcript in humans and examined its utility as a prognostic marker for visual acuity in patients with ARN. We identified two human orthologous transcripts (1955 and 592 bases) of lncRNA U90926. The amount of the longer human U90926 transcript was approximately 30- and 40-fold higher in the vitreous fluid of patients with ARN than in those with sarcoidosis and intraocular lymphoma, respectively. Furthermore, the expression of the longer human U90926 transcript in the vitreous fluid was highly correlated with the final best-corrected logarithm of the minimum angle of resolution visual acuity in patients with ARN (r = 0.7671, p = 0.0079). This suggests higher expression of the longer human U90926 transcript in the vitreous fluid results in worse visual prognosis; therefore, expression of the longer human U90926 transcript is a potential negative prognostic marker for visual acuity in patients with ARN.

Identification of 2H phosphoesterase superfamily proteins with 2'-CPDase activity.

The 2H phosphoesterase superfamily (2H family) proteins are widely conserved among organisms. The 2H family is classified into several subgroups, including YjcG-like proteins whose enzymatic activity has not been reported. In the present study, we found that two YjcG-like proteins (Staphylococcus aureus SA0873 and Bacillus subtilis YjcG) have 2'-CPDase activity that hydrolyzes a 2',3'-cyclic nucleotide, thereby producing a nucleotide with a 3'-phosphate. The SA0873 protein selectively hydrolyzes a 2',3'-cyclic nucleotide with a purine base. Four SA0873 mutant proteins (H34A, T36A, H115A, and T117A), in which alanine was substituted for amino acid residues in the HxT/Sx motifs that are conserved in the 2H family, abolished the 2'-CPDase activity. Comparison of three-dimensional structures between the YjcG-like proteins with 2'-CPDase activity and another 2H family subgroup, LigT/2'-5' RNA ligase-like proteins with 3'-CPDase activity, revealed that the orientation of the substrate binding pocket is reversed between the two groups. Our findings revealed that YjcG-like proteins not only have a substrate-binding pocket different from that of LigT/2'-5' RNA ligase-like proteins, but they also have 2'-CPDase activity.

Isolation of antibiotic-producing Pseudomonas species with low-temperature cultivation of temperate soil.

We performed low-temperature cultivation of soil samples in Tokyo, Japan, and isolated 30 bacterial strains that formed colonies at 4°C. All the culture supernatants of these bacteria exhibited antibacterial activity against Escherichia coli. The 16S rDNA sequences of 29 strains showed similarity to that of the Pseudomonas genus, whereas the 16S rDNA sequence of one strain showed similarity to that of the Janthinobacterium genus. We classified the 29 strains into 10 groups according to the 16S rDNA sequence similarities, and performed two phylogenetic analyses using the 16S rDNA and rpoD gene sequences. Four groups formed a unique branch within Pseudomonas species in both phylogenetic analyses. Four other groups were closely related to the Pseudomonas species, but the most closely related species differed between the two phylogenic tree analyses. These results suggest that low-temperature cultivation of temperate soil is effective for isolating new bacterial sources for producing antibiotics.