Molecular basis of cellular localization of poly C binding protein 1 in neuronal cells

Abstract

Poly C binding protein 1 (PCBP) is involved in the transcriptional regulation of neuronal mu-opioid receptor gene. In this study, we examined the molecular basis of PCBP cellular/nuclear localization in neuronal cells using EGFP fusion protein. PCBP, containing three KH domains and a variable domain, distributed in cytoplasm and nucleus with a preferential nuclear expression. Domain-deletional analyses suggested the requirement of variable and KH3 domains for strong PCBP nuclear expression. Within the nucleus, a low nucleolar PCBP expression was observed, and PCBP variable domain contributed to this restricted nucleolar expression. Furthermore, the punctate nuclear pattern of PCBP was correlated to its single-stranded (ss) DNA binding ability, with both requiring cooperativity of at least three sequential domains. Collectively, certain PCBP domains thus govern its nuclear distribution and transcriptional regulatory activity in the nucleus of neurons, whereas the low nucleolar expression implicates the disengagement of PCBP in the ribosomal RNA synthesis.

Fig. 1

Sub-cellular localization of the full length or truncated poly C binding protein 1 (PCBP) A, Cellular distribution of poly C binding protein 1 (PCBP) in the neuronal cells. Neuro2A cells were transiently transfected with the pEGFP-PCBP plasmid, in which the full length of PCBP was fused with EGFP. Transfected cells were fixed and viewed using a laser scanning confocal microscope. PCBP: The cellular distribution of the full length PCBP (indicated by the green color). Arrows point to the punctate nuclear pattern. PI: Nuclear region is shown in red color, which was stained using propidium iodide. Merge: The merged image of PCBP and PI, showing co-localization (indicated as the yellow color) of the green fluorescence labeled PCBP and PI nuclear staining. B, Schematic diagram represents the full length and various two-domain deletions of PCBPs, which were fused with the EGFP of the pEGFP-C1 vector. C, Effects of two-domain deletion of PCBPs on the cellular distribution. Neuronal cells were transfected with various two-domain constructs of PCBP plasmids. Cells were fixed and viewed using a confocal microscope. Truncated PCBP: The subcellular distributions (indicated by the green color) of two-domain PCBPs, including K12 (a), Kv3 (b), K2v (c) and K23 (d). PI (e-h), Nuclear region shown in red color was stained using propidium iodide. Merge (i-l), The merged image (indicated as the yellow color) of truncated PCBP and PI, showing co-localization of the green fluorescence labeled truncated PCBP and PI nuclear staining.

Fig. 2

Sub-cellular localization of the one-domain deletion PCBP A, Schematic representation of one-domain deletions of PCBPs, including K2v3, K12v and K123, which were fused with the EGFP of the pEGFP-C1 mammalian expression vector. B, Effect of one-domain deletion of PCBP on the cellular distribution in neuronal cells. Neuronal cells were transiently transfected with one-domain deletion of PCBP plasmids. The transfected cells were fixed and viewed using a confocal microscope. Truncated PCBP: the subcellular distributions (indicated by the green color) of one-domain deletion of PCBPs, including K2v3 (a), K12v (b) and K123 (c). PI (d-f): Nuclear region shown in red color was stained using propidium iodide. Merge (g-i): The merged image of truncated PCBP and PI, showing co-localization (indicated as the yellow color) of the truncated PCBP and PI nuclear staining.

Fig. 3

Effect of domain deletions of PCBP on its nucleolar expression in neuronal cells A, Low nucleolar expression of PCBP in the nucleus of neuronal cells. Neuronal cells were transiently transfected with the pEGFP-PCBP plasmid, fixed, perforated using Triton X-100 and viewed using a confocal microscope. PCBP (a-b): The cellular distribution of the full length PCBP. Arrows showed the low PCBP expression inside the nucleus which is a round or oval shape. Nucleolin (c-d): The oval or round shape of nucleolar region (indicated by arrows) is shown in red color, which was stained using anti-nucleolin antibody conjugated with rhodamine. Merge (e-f): The merged image of PCBP and nucleolus, showing that there was barely a co-localization of the green fluorescence labeled PCBP and nucleolin staining in the nucleolus region. B, Effect of domain deletion of PCBP on its nucleolar distribution in neuronal cells. Neuronal cells were transfected with one-domain deletion of PCBP plasmids, pEGFP-K12v or pEGFP–K2v3, or two-domain deletion of PCBP plasmids, pEGFP-K2v, pEGFP-Kv3 or pEGFP-K23. The sub-nuclear distributions of various truncated PCBPs shown in green color. Arrows indicate the round or oval shape of nucleolar region inside the nucleus of neuron cells.

Fig. 4

Effect of domain deletion of PCBP on its punctate nuclear pattern in neuronal cells A, The punctate nuclear pattern of full-length and one-domain deletion of PCBP in neuronal cells. Neuronal cells were transfected with the full length or one-domain deletion of PCBP plasmids, pEGFP-K12v or pEGFP-K2v3. Transfected cells were fixed and viewed using a confocal microscope. The punctate nuclear pattern of the full length (a-b) or various truncated PCBPs (c-f) are shown. Arrows point to hotspots in the nucleus. PI (g-l), Nuclear region is stained using propidium iodide. B, No obvious punctate nuclear pattern of two-domain deletion of PCBP was observed in neuronal cells. Neuronal cells were transfected with two-domain deletion of PCBP plasmids, pEGFP-K12, pEGFP-K2v, pEGFP-Kv3 or pEGFP-K23. The nuclear distribution patterns of various truncated PCBPs are shown in a-d. PI (e-h), Nuclear region is stained using propidium iodide.