CED-4 is an mRNA-binding protein that delivers ced-3 mRNA to ribosomes.

Cell death abnormal (ced)-3 and ced-4 genes regulate apoptosis to maintain tissue homeostasis in Caenorhabditis elegans. Apoptosome formation and CED-4 translocation drive CED-3 activation. However, the precise role of CED-4 translocation is not yet fully understood. In this study, using a combination of immunoprecipitation and reverse transcription-polymerase chain reaction methods in cells and a glutathione-S-transferase pull down assay in a cell-free system, we show that CED-4 binds ced-3 mRNA. In the presence of ced-3 mRNA, CED-4 protein is enriched in the microsomal fraction and interacts with ribosomal protein L10a in mammalian cells, increasing the levels of CED-3. These results suggest that CED-4 forms a complex with ced-3 mRNA and delivers it to ribosomes for translation.

Effect of the interaction between ribosomal protein L10a and insulin receptor on carbohydrate metabolism.

The number of patients with insulin-resistant diabetes has significantly increased. Thus, alternative insulin mimetics are required for such patients. Some evidences indicate that ribosomal protein L10a (RpL10a) is involved in the insulin pathway. In addition, we previously demonstrated that recombinant RpL10a from Fenneropenaeus merguiensis (Fm-RpL10a) could stimulate cell proliferation and trehalose metabolism in RpL10a-over-expressing flies by inducing insulin receptor (InR) expression and some insulin signaling mediators phosphorylation. In this study, we investigated the in silico binding between Fm-RpL10a and InR. The results indicated that Fm-RpL10a bound to InR at residues 635-640 and 697-702 of the FnIII2 domain. This binding was confirmed using a pull-down and immunofluorescence assay. Further analysis indicated that Fm-RpL10a could stimulate glucose utilisation by insulin-resistant cells (IRCs) and healthy cells. Additionally, Fm-RpL10a at a low concentration (1 µg/ml) altered some glucose metabolism-related genes expression in Fm-RpL10a treated IRCs. The qRT-PCR result revealed the up-regulation of Hk1, which encode key enzymes in glycolysis. Conversely, the expression of G6pc3, which participates in gluconeogenesis, was down-regulated. Overall, the results suggest that Fm-RpL10a can alleviate insulin resistance by stimulating insulin signaling via the FnIII2 domain of InR and activate glycolysis. Therefore, Fm-RpL10a may be a candidate insulin mimetic for the treatment of diabetes.

Ribosomal protein L10A and signaling pathway.

Ribosome: machinery in control of messenger RNA's (mRNAs) and several ribosomal proteins are in the small and large subunit of the ribosome. Various aspects of ribosomal proteins have related to cell growth, cell cycle, and diseases. Ribosomal protein L10A (RpL10A) in shrimp and fruit fly has been demonstrated to play a role in oogenesis. Interestingly, deletion RpL10A gene (RpL10Ab-/-) germ line clone of the fruit fly showed a loss of follicle cells surrounding the egg chamber, but nurse cells appeared normal. This phenotype is reminiscent of insulin receptor mutants (InR-/-). In contrast, over-expression of RpL10A in the eyes of the fruit flies resulted in abnormal ommatidia with a loss of red pigment in the center of the eyes. In this study, the abnormal rearrangements of nuclei and lack of cell membranes in those eyes were demonstrated. Furthermore, the expression of InR gene and the InR protein were extensively increased as determined by real-time PCR and immunohistochemistry, respectively. In addition, some insulin signaling mediators were also detected. The Akt and FOXO proteins were highly phosphorylated in the RpL10A over-expressed mutant. The results revealed that RpL10A induced over-expression of the insulin receptor and consequently activated in insulin signaling pathway which affects cell proliferation and we suggest RpL10A stimulates cell proliferation via the insulin signaling pathway.

A retrograde adeno-associated virus for collecting ribosome-bound mRNA from anatomically defined projection neurons.

The brain contains a large variety of projection neurons with different functional properties. The functional properties of projection neurons arise from their connectivity with other neurons and their molecular composition. We describe a novel tool for obtaining the gene expression profiles of projection neurons that are anatomically defined by the location of their soma and axon terminals. Our tool utilizes adeno-associated virus serotype 9 (AAV9), which we found to retrogradely transduce projection neurons after injection at the site of the axon terminals. We used AAV9 to express Enhanced Green Fluorescent Protein (EGFP)-tagged ribosomal protein L10a (EGFP-L10a), which enables the immunoprecipitation of EGFP-tagged ribosomes and associated mRNA with a method known as Translating Ribosome Affinity Purification (TRAP). To achieve high expression of the EGFP-L10a protein in projection neurons, we placed its expression under control of a 1.3 kb alpha-calcium/calmodulin-dependent protein kinase II (Camk2a) promoter. We injected the AAV9-Camk2a-TRAP virus in either the hippocampus or the bed nucleus of the stria terminalis (BNST) of the mouse brain. In both brain regions the 1.3 kb Camk2a promoter did not confer complete cell-type specificity around the site of injection, as EGFP-L10a expression was observed in Camk2a-expressing neurons as well as in neuronal and non-neuronal cells that did not express Camk2a. In contrast, cell-type specific expression was observed in Camk2a-positive projection neurons that were retrogradely transduced by AAV9-Camk2a-TRAP. Injection of AAV9-Camk2a-TRAP into the BNST enabled the use of TRAP to collect ribosome-bound mRNA from basal amygdala projection neurons that innervate the BNST. AAV9-Camk2a-TRAP provides a single-virus system that can be used for the molecular profiling of anatomically defined projection neurons in mice and other mammalian model organisms. In addition, AAV9-Camk2a-TRAP may enable the discovery of protein synthesis events that support information storage in projection neurons.