A B23-interacting sequence as a tool to visualize protein interactions in a cellular context.

International audienceWe report the characterization of a nucleolar localization sequence (NoLS) that targets the green fluorescent protein (GFP) into the granular component (GC) of nucleoli. This NoLS interacts in vitro specifically and directly with the major nucleolar protein B23 and more precisely with the region of B23 including the two acidic stretches. The affinity of NoLS for B23 is stronger than that of the HIV-1 Rev protein in vitro. Moreover, B23-NoLS interaction also occurs in vivo. Indeed, (1) NoLS confers on the GFP the behavior of B23 throughout the cell cycle, (2) the GFP-NoLS fusion and B23 remain colocalized after drug treatments, (3) a selective delocalization of B23 from nucleoli to nucleoplasm induces a concomitent delocalization of the GFP-NoLS fusion, and (4) the fusion of NoLS to fibrillarin makes it possible to colocalize fibrillarin and B23. Interestingly, by fusing NoLS to fibrillarin, both fibrillarin and the fibrillarin partner Nop56 are mislocalized in the GC of nucleoli. Similarly, by fusing the NoLS to MafG, part of the nuclear transcription factor NF-E2 composed of both MafG and p45 NF-E2, NF-E2 is redirected from the nucleoplasm to the nucleoli. Thus, we propose that the NoLS may be used as a tool to visualize and prove protein interactions in a cellular context

Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

BACKGROUND: The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv) has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. RESULTS: Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. CONCLUSION: The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv

The unusually large Plasmodium telomerase reverse-transcriptase localizes in a discrete compartment associated with the nucleolus

Telomerase replicates chromosome ends, a function necessary for maintaining genome integrity. We have identified the gene that encodes the catalytic reverse transcriptase (RT) component of this enzyme in the malaria parasite Plasmodium falciparum (PfTERT) as well as the orthologous genes from two rodent and one simian malaria species. PfTERT is predicted to encode a basic protein that contains the major sequence motifs previously identified in known telomerase RTs (TERTs). At ∼2500 amino acids, PfTERT is three times larger than other characterized TERTs. We observed remarkable sequence diversity between TERT proteins of different Plasmodial species, with conserved domains alternating with hypervariable regions. Immunofluorescence analysis revealed that PfTERT is expressed in asexual blood stage parasites that have begun DNA synthesis. Surprisingly, rather than at telomere clusters, PfTERT typically localizes into a discrete nuclear compartment. We further demonstrate that this compartment is associated with the nucleolus, hereby defined for the first time in P.falciparum

Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery

Copyright @ 2014 Booth et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.When the nucleolus disassembles during open mitosis, many nucleolar proteins and RNAs associate with chromosomes, establishing a perichromosomal compartment coating the chromosome periphery. At present nothing is known about the function of this poorly characterised compartment. In this study, we report that the nucleolar protein Ki-67 is required for the assembly of the perichromosomal compartment in human cells. Ki-67 is a cell-cycle regulated protein phosphatase 1-binding protein that is involved in phospho-regulation of the nucleolar protein B23/nucleophosmin. Following siRNA depletion of Ki-67, NIFK, B23, nucleolin, and four novel chromosome periphery proteins all fail to associate with the periphery of human chromosomes. Correlative light and electron microscopy (CLEM) images suggest a near-complete loss of the entire perichromosomal compartment. Mitotic chromosome condensation and intrinsic structure appear normal in the absence of the perichromosomal compartment but significant differences in nucleolar reassembly and nuclear organisation are observed in post-mitotic cells

Depletion of nucleophosmin leads to distortion of nucleolar and nuclear structures in HeLa cells

NPM (nucleophosmin; also known as B23) is an abundantly and ubiquitously expressed multifunctional nucleolar phosphoprotein, which is involved in numerous cellular processes, including ribosome biogenesis, protein chaperoning and centrosome duplication; however, the role of NPM in the cell cycle still remains unknown. In the present study, we show dynamic localization of NPM throughout the cell cycle of HeLa cells. Using a combination of RNAi (RNA interference) and three-dimensional microscopy we show that NPM is localized at the chromosome periphery during mitosis. We also demonstrate that depletion of NPM causes distortion of nucleolar structure as expected and leads to unexpected dramatic changes in nuclear morphology with multiple micronuclei formation. The defect in nuclear shape of NPM-depleted cells, which is clearly observed by live-cell imaging, is due to the distortion of cytoskeletal (α-tubulin and β-actin) structure, resulting from the defects in centrosomal microtubule nucleation. These results indicate that NPM is an essential protein not only for the formation of normal nucleolar structure, but also for the maintenance of regular nuclear shape in HeLa cells

Expression and Functional Studies on the Noncoding RNA, PRINS.

PRINS, a noncoding RNA identified earlier by our research group, contributes to psoriasis susceptibility and cellular stress response. We have now studied the cellular and histological distribution of PRINS by using in situ hybridization and demonstrated variable expressions in different human tissues and a consistent staining pattern in epidermal keratinocytes and in vitro cultured keratinocytes. To identify the cellular function(s) of PRINS, we searched for a direct interacting partner(s) of this stress-induced molecule. In HaCaT and NHEK cell lysates, the protein proved to be nucleophosmin (NPM) protein as a potential physical interactor with PRINS. Immunohistochemical experiments revealed an elevated expression of NPM in the dividing cells of the basal layers of psoriatic involved skin samples as compared with healthy and psoriatic uninvolved samples. Others have previously shown that NPM is a ubiquitously expressed nucleolar phosphoprotein which shuttles to the nucleoplasm after UV-B irradiation in fibroblasts and cancer cells. We detected a similar translocation of NPM in UV-B-irradiated cultured keratinocytes. The gene-specific silencing of PRINS resulted in the retention of NPM in the nucleolus of UV-B-irradiated keratinocytes; suggesting that PRINS may play a role in the NPM-mediated cellular stress response in the skin