title: Characterization of MRP RNA-protein interactions within the perinucleolar compartment.
abstract: The perinucleolar compartment (PNC) forms in cancer cells and is highly enriched with a subset of polymerase III RNAs and RNA-binding proteins. Here we report that PNC components mitochondrial RNA-processing (MRP) RNA, pyrimidine tract-binding protein (PTB), and CUG-binding protein (CUGBP) interact in vivo, as demonstrated by coimmunoprecipitation and RNA pull-down experiments. Glycerol gradient analyses show that this complex is large and sediments at a different fraction from known MRP RNA-containing complexes, the MRP ribonucleoprotein ribozyme and human telomerase reverse transcriptase. Tethering PNC components to a LacO locus recruits other PNC components, further confirming the in vivo interactions. These interactions are present both in PNC-containing and -lacking cells. High-resolution localization analyses demonstrate that MRP RNA, CUGBP, and PTB colocalize at the PNC as a reticulated network, intertwining with newly synthesized RNA. Furthermore, green fluorescent protein (GFP)-PTB and GFP-CUGBP show a slower rate of fluorescence recovery after photobleaching at the PNC than in the nucleoplasm, illustrating the different molecular interaction of the complexes associated with the PNC. These findings support a working model in which the MRP RNA-protein complex becomes nucleated at the PNC in cancer cells and may play a role in gene expression regulation at the DNA locus that associates with the PNC.
authors.affiliation: Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
authors.forename: Callie
authors.initials: C
authors.lastname: Pollock
authors.forename: Kelly
authors.initials: K
authors.lastname: Daily
authors.forename: Van Trung
authors.initials: VT
authors.lastname: Nguyen
authors.forename: Chen
authors.initials: C
authors.lastname: Wang
authors.forename: Marzena Anna
authors.initials: MA
authors.lastname: Lewandowska
authors.forename: Olivier
authors.initials: O
authors.lastname: Bensaude
authors.forename: Sui
authors.initials: S
authors.lastname: Huang
mesh_terms: D000818:Animals; D002466:Cell Nucleolus / Q000378:metabolism / Q000648:ultrastructure*; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure*; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D000077278:RNA, Mitochondrial; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D024101:Mitochondrial Proteins; D000077278:RNA, Mitochondrial; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D038941:Polypyrimidine Tract-Binding Protein; D049452:Green Fluorescent Proteins; D012313:RNA