A concert of RNA-binding proteins coordinates mitochondrial function.

Mitochondria are dynamic and plastic organelles, which flexibly adapt morphology, ATP production, and metabolic function to meet extrinsic challenges and demands. Regulation of mitochondrial biogenesis is essential during development and in adult life to survive stress and pathological insults, and is achieved not only by increasing mitochondrial mass, but also by remodeling the organellar proteome, metabolome, and lipidome. In the last decade, the post-transcriptional regulation of the expression of nuclear-encoded mitochondrial proteins has emerged as a fast, flexible, and powerful mechanism to shape mitochondrial function and coordinate it with other cellular processes. At the heart of post-transcriptional responses are a number of RNA-binding proteins that specifically bind mRNAs encoding mitochondrial proteins and define their fate, by influencing transcript maturation, stability, translation, and localization. Thus, RNA-binding proteins provide a uniquely complex regulatory code that orchestrates mitochondrial function during physiological and pathological conditions.

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins.

Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.

RNA-binding protein RBM8A (Y14) and MAGOH localize to centrosome in human A549 cells.

RBM8A (Y14) is carrying RNA-binding motif and forms the tight heterodimer with MAGOH. The heterodimer is known to be a member of exon junction complex on exporting mRNA and is required for mRNA metabolisms such as splicing, mRNA export and nonsense-mediated mRNA decay. Almost all RBM8A-MAGOH complexes localize in nucleoplasm and shuttle between nuclei and cytoplasm for RNA metabolism. Recently, the abnormality of G2/M transition and aberrant centrosome regulation in RBM8A- or MAGOH-deficient cells has been reported. These results prompt us to the reevaluation of the localization of RBM8A-MAGOH in human cells. Interestingly, our immunostaining experiments showed the localization of these proteins in centrosome in addition to nuclei. Furthermore, the transiently expressed eYFP-tagged RBM8A and Flag-tagged MAGOH also co-localized with centrosome signals. In addition, the proximity ligation in situ assay was performed to detect the complex formation in centrosome. Our experiments clearly showed that Myc-tagged RBM8A and Flag-tagged MAGOH formed a complex in centrosome. GFP-tagged PLK1 also co-localized with Myc-RBM8A. Our results show that RBM8A-MAGOH complex is required for M-phase progression via direct localization to centrosome rather than indirect effect.

Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth.

The development of short interfering RNA (siRNA), has provided great hope for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake and bioavailability of siRNA remain a major obstacle to their clinical development and most strategies that propose to improve siRNA delivery remain limited for in vivo applications. In this study, we report a novel peptide-based approach, MPG-8 an improved variant of the amphipathic peptide carrier MPG, that forms nanoparticles with siRNA and promotes their efficient delivery into primary cell lines and in vivo upon intra-tumoral injection. Moreover, we show that functionalization of this carrier with cholesterol significantly improves tissue distribution and stability of siRNA in vivo, thereby enhancing the efficiency of this technology for systemic administration following intravenous injection without triggering any non-specific inflammatory response. We have validated the therapeutic potential of this strategy for cancer treatment by targeting cyclin B1 in mouse tumour models, and demonstrate that tumour growth is compromised. The robustness of the biological response achieved through this approach, infers that MPG 8-based technology holds a strong promise for therapeutic administration of siRNA.

Endothelial-monocyte activating polypeptide II, a novel antitumor cytokine that suppresses primary and metastatic tumor growth and induces apoptosis in growing endothelial cells.

Neovascularization is essential for growth and spread of primary and metastatic tumors. We have identified a novel cytokine, endothelial-monocyte activating polypeptide (EMAP) II, that potently inhibits tumor growth, and appears to have antiangiogenic activity. Mice implanted with Matrigel showed an intense local angiogenic response, which EMAP II blocked by 76% (P < 0.001). Neovascularization of the mouse cornea was similarly prevented by EMAP II (P < 0.003). Intraperitoneally administered EMAP II suppressed the growth of primary Lewis lung carcinomas, with a reduction in tumor volume of 65% versus controls (P < 0.003). Tumors from human breast carcinoma-derived MDA-MB 468 cells were suppressed by >80% in EMAP II-treated animals (P < 0.005). In a lung metastasis model, EMAP II blocked outgrowth of Lewis lung carcinoma macrometastases; total surface metastases were diminished by 65%, and of the 35% metastases present, approximately 80% were inhibited with maximum diameter <2 mm (P < 0.002 vs. controls). In growing capillary endothelial cultures, EMAP II induced apoptosis in a time- and dose-dependent manner, whereas other cell types were unaffected. These data suggest that EMAP II is a tumor-suppressive mediator with antiangiogenic properties allowing it to target growing endothelium and limit establishment of neovasculature.

The dynamic organization of the perinucleolar compartment in the cell nucleus.

The perinucleolar compartment (PNC) is a unique nuclear structure preferentially localized at the periphery of the nucleolus. Several small RNAs transcribed by RNA polymerase III (e.g., the Y RNAs, MRP RNA, and RNase P H1 RNA) and the polypyrimidine tract binding protein (PTB; hnRNP I) have thus far been identified in the PNC (Ghetti, A., S. PinolRoma, W.M. Michael, C. Morandi, and G. Dreyfuss. 1992. Nucleic Acids Res. 20:3671-3678; Matera, A.G., M.R. Frey, K. Margelot, and S.L. Wolin. 1995. J. Cell Biol. 129:1181-1193; Lee, B., A.G. Matera, D.C. Ward, and J. Craft. 1996. Proc. Natl. Acad. Sci. USA. 93: 11471-11476). In this report, we have further characterized this structure in both fixed and living cells. Detection of the PNC in a large number of human cancer and normal cells showed that PNCs are much more prevalent in cancer cells. Analysis through the cell cycle using immunolabeling with a monoclonal antibody, SH54, specifically recognizing PTB, demonstrated that the PNC dissociates at the beginning of mitosis and reforms at late telophase in the daughter nuclei. To visualize the PNC in living cells, a fusion protein between PTB and green fluorescent protein (GFP) was generated. Time lapse studies revealed that the size and shape of the PNC is dynamic over time. In addition, electron microscopic examination in optimally fixed cells revealed that the PNC is composed of multiple strands, each measuring approximately 80-180 nm diam. Some of the strands are in direct contact with the surface of the nucleolus. Furthermore, analysis of the sequence requirement for targeting PTB to the PNC using a series of deletion mutants of the GFP-PTB fusion protein showed that at least three RRMs at either the COOH or NH2 terminus are required for the fusion protein to be targeted to the PNC. This finding suggests that RNA binding may be necessary for PTB to be localized in the PNC.

Drug targeting to the brain using avidin-biotin technology in the mouse; (blood-brain barrier, monoclonal antibody, transferrin receptor, Alzheimer's disease).

A beta1-40 peptide radiopharmaceuticals could be used to image A beta brain amyloid in transgenic mouse models of Alzheimer's disease should the A beta peptide radiopharmaceutical be made transportable through the blood-brain barrier (BBB) in vivo. The present studies used the RI7-217 rat monoclonal antibody to the mouse transferrin receptor as a BBB drug targeting vector for the delivery to brain of A beta1-40 radiolabeled with either 125-Iodine or 111-Indium. The A beta peptide radiopharmaceutical is conjugated to the RI7 MAb using avidin biotin technology, wherein the A beta1-40 peptide radiopharmaceutical is monobiotinylated (bio) and bound to a conjugate of the RI7 MAb and streptavidin (SA). The [125 I]-bio-A beta1-40 or the [111 In]-bio-A beta1-40 either free or bound to the RI7/SA conjugate was injected intravenously into anesthetized adult mice and plasma pharmacokinetics and organ uptake were measured over the next 60 minutes. The A beta1-40 peptide radiopharmaceutical radiolabeled with 111-Indium was the preferred formulation, compared to peptide labeled with 125-Iodine, because there was a greater metabolic stability and reduced artifactual organ uptake of metabolites associated with the use of the 111-Indium nuclide. However, biotinylated A beta1-40 peptide radiopharmaceuticals conjugated to the RI7/SA brain drug targeting system were metabolically unstable in mice in vivo owing to active biotinidase activity. Future work involving brain drug targeting in mice that utilizes avidin biotin technology will need to incorporate biotin analogues that are resistant to biotinidase.

Antitumor activity and pharmacokinetic properties of ARS-interacting multi-functional protein 1 (AIMP1/p43).

Although AIMP1 was identified as a component of the macromolecular aminoacyl tRNA synthetase complex involved in the cellular translation process, it was also found to be secreted as a cytokine having complex physiological functions. Among these, AIMP1's angiostatic and immune stimulating activities suggest its potential use as a novel antitumor therapeutic protein. Here we evaluated its antitumor efficacy in a mouse xenograft model bearing human stomach cancer cells. Intravenous injection of recombinant AIMP1 for 6 days resulted in significant decreases in both tumor volume and weight. Tumor volume decreased 31.1% and 54.0% when treated with AIMP1 at a concentration of 2mg/kg and 10mg/kg, respectively. Tumor weight decreased 29.1% and 52.2% when treated with AIMP1 at a concentration of 2mg/kg and 10mg/kg, respectively. Proliferating cell nuclear antigen (PCNA) staining of tumor tissues from AIMP1-treated mice (at both 2mg/kg and 10mg/kg) showed a 53% reduction of cells exhibiting an active cell cycle progression. Blood levels of tumor-suppressing cytokines such as TNF-alpha and IL-1beta increased in AIMP1-treated mice, whereas IL-12p40 and IFN-gamma levels remained unaltered. Thus, this work suggests that AIMP1 may exert its antitumor activity by inducing tumor-suppressing cytokines. In a pharmacokinetic study in rats after a single intravenous injection, AMP1 exhibited a low clearance showing a one-compartmental disposition. However, due to a low volume of distribution, AIMP1 had a short half-life of 0.1h. In a serum stability test, AIMP1 showed a half life of >60 min in human serum, 52 min in dog serum and 32 min in rat serum.