A novel RNA-binding protein associated with cell plate formation

Building a cell plate during cytokinesis in plant cells requires the participation of a number of proteins in a multistep process. We previously identified phragmoplastin as a cell plate-specific protein involved in creating a tubulovesicular network at the cell plate. We report here the identification and characterization of a phragmoplastin-interacting protein, PHIP1, in Arabidopsis (Arabidopsis thaliana). It contains multiple functional motifs, including a lysine-rich domain, two RNA recognition motifs, and three CCHC-type zinc fingers. Polypeptides with similar motif structures were found only in plant protein databases, but not in the sequenced prokaryotic, fungal, and animal genomes, suggesting that PHIP1 represents a plant-specific RNA-binding protein. In addition to phragmoplastin, two Arabidopsis small GTP-binding proteins, Rop1 and Ran2, are also found to interact with PHIP1. The zinc fingers of PHIP1 were not required for its interaction with Rop1 and phragmoplastin, but they may participate in its binding with the Ran2 mRNA. Immunofluorescence, in situ RNA hybridization, and green fluorescent protein tagging experiments showed the association of PHIP1 with the forming cell plate during cytokinesis. Taken together, our data suggest that PHIP1 is a novel RNA-binding protein and may play a unique role in the polarized mRNA transport to the vicinity of the cell plate.     

RNA-binding protein insulin-like growth factor mRNA-binding protein 3 (IMP-3) promotes cell survival via insulin-like growth factor II signaling after ionizing radiation

Ionizing radiation (IR) induces proapoptotic gene expression programs that inhibit cell survival. These programs often involve RNA-binding proteins that associate with their mRNA targets to elicit changes in mRNA stability and/or translation. The RNA-binding protein IMP-3 is an oncofetal protein overexpressed in many human malignancies. IMP-3 abundance correlates with tumor aggressiveness and poor prognosis. As such, IMP-3 is proving to be a highly significant biomarker in surgical pathology. Among its many mRNA targets, IMP-3 binds to and promotes translation of insulin-like growth factor II (IGFII) mRNA. Our earlier studies showed that reducing IMP-3 abundance with siRNAs reduced proliferation of human K562 chronic myeloid leukemia cells because of reduced IGF-II biosynthesis. However, the role of IMP-3 in apoptosis is unknown. Here, we have used IR-induced apoptosis of K562 cells as a model to explore a role for IMP-3 in cell survival. Knockdown of IMP-3 with siRNA increased susceptibility of cells to IR-induced apoptosis and led to reduced IGF-II production. Gene reporter assays revealed that IMP-3 acts through the 5' UTR of IGFII mRNA during apoptosis to promote translation. Finally, culture of IR-treated cells with recombinant IGF-II partially reversed the effects of IMP-3 knockdown on IR-induced apoptosis. Together, these results indicate that IMP-3 acts in part through the IGF-II pathway to promote cell survival in response to IR. Thus, IMP-3 might serve as a new drug target to increase sensitivity of CML cells or other cancers to IR therapy.     

The splicing factor crooked neck associates with the RNA-binding protein HOW to control glial cell maturation in Drosophila

In both vertebrates and invertebrates, glial cells wrap axonal processes to ensure electrical conductance. Here we report that Crooked neck (Crn), the Drosophila homolog of the yeast Clf1p splicing factor, is directing peripheral glial cell maturation. We show that crooked neck is expressed and required in glial cells to control migration and axonal wrapping. Within the cytoplasm, Crn interacts with the RNA-binding protein HOW and then translocates to the nucleus where the Crn/HOW complex controls glial differentiation by facilitating splicing of specific target genes. By using a GFP-exon trap approach, we identified some of the in vivo target genes that encode proteins localized in autocellular septate junctions. In conclusion, here we show that glial cell differentiation is controlled by a cytoplasmic assembly of splicing components, which upon translocation to the nucleus promote the splicing of genes involved in the assembly of cellular junctions.     

Expression of TAR RNA-binding protein in baculovirus and co-immunoprecipitation with insect cell protein kinase

TAR RNA-binding protein TRBP was originally isolated by its binding affinity for radiolabeled HIV-1 leader RNA. Subsequent studies have suggested that this protein is one member of a family of double-stranded RNA-binding proteins. Recent findings indicate that TRBP might function to antagonize the translational inhibitory effect that can be mediated through cellular protein kinase, PKR. Here, we report on the over-expression of a cDNA coding for TRBP in eukaryotic SF9 cells using baculovirus. We characterized the nuclear localization of TRBP in insect cells, and we demonstrate that TRBP co-immunoprecipitates with a protein in these cells antigenically related to human PKR.     

MicroRNA-24 targeting RNA-binding protein DND1 in tongue squamous cell carcinoma

Deregulations of microRNA have been frequently observed in tongue squamous cell carcinoma (TSCC), but their roles in tumorigenesis are not entirely clear. Here, we reported the up-regulation of miR-24 in TSCC. MiR-24 up-regulation reduced the expression of RNA-binding protein dead end 1 (DND1). Knockdown of miR-24 led to enhanced expression of DND1. The direct targeting of miR-24 to the DND1 mRNA was predicted bioinformatically and confirmed by luciferase reporter gene assays. Furthermore, the miR-24-mediated change in DND1 expression suppressed the expression of cyclin-dependent kinase inhibitor 1B (CDKN1B), and also led to enhanced proliferation and reduced apoptosis in TSCC cells.     

ERα is an RNA-binding protein sustaining tumor cell survival and drug resistance

1. Download : Download high-res image (163KB)
2. Download : Download full-size image

     

RNA-binding protein HuD controls insulin translation

Although expression of the mammalian RNA-binding protein HuD was considered to be restricted to neurons, we report that HuD is present in pancreatic β cells, where its levels are controlled by the insulin receptor pathway. We found that HuD associated with a 22-nucleotide segment of the 5' untranslated region (UTR) of preproinsulin (Ins2) mRNA. Modulating HuD abundance did not alter Ins2 mRNA levels, but HuD overexpression decreased Ins2 mRNA translation and insulin production, and conversely, HuD silencing enhanced Ins2 mRNA translation and insulin production. Following treatment with glucose, HuD rapidly dissociated from Ins2 mRNA and enabled insulin biosynthesis. Importantly, HuD-knockout mice displayed higher insulin levels in pancreatic islets, while HuD-overexpressing mice exhibited lower insulin levels in islets and in plasma. In sum, our results identify HuD as a pivotal regulator of insulin translation in pancreatic β cells.