Visualizing protein interactions involved in the formation of the 42S RNP storage particle of Xenopus oocytes

Abstract

Background information. During early phases of Xenopus oogenesis, 5S rRNA and tRNAs are stored in the cytoplasm of young oocytes in the form of a common RNA-protein complex termed the 42S particle. These storage particles comprise two kinds of proteins with different RNA binding specificities. The tRNA-binding protein 42Sp50 belongs to the EF1A (eukaryotic translation elongation factor 1A) family of translation elongation factors, while 42Sp43 is a diverged form of the transcription factor TFIIIA (transcription factor IIIA) and binds 5S rRNA. Little is known about the mode of protein-protein interactions that stabilize the 42S particle.Results. We have determined the intracellular localization of the protein components of the 42S particle by expressing fluorescent protein-tagged fusions in transparent previtellogenic oocytes. 42Sp50 and its isoforms (EF1A-S and EF1A-O) were excluded from the nuclei and distributed uniformly throughout the cytoplasm with no enrichment in the Balbiani bodies, as described earlier by immunocytochemistry. In contrast, 42Sp43 accumulated in the amplified nucleoli. However, when both proteins were simultaneously expressed, 42Sp43 was no longer present in the nucleoli but was retained, together with 42Sp50, in the cytoplasm, the most likely site of 42S particle assembly. In contrast, the somatic-type EF1A isoforms were unable to redirect 42Sp43 from the nucleolar to the cytoplasmic compartment. We also tested for in vivo interactions using transiently transfected mammalian cells (COS-7 cell line). In this heterologous cell system 42Sp43 remained bound to the nucleoli but, on co-expression, induced the redistribution of 42Sp50 from the cytoplasm to the nucleoli.Conclusions. The microscopic approach described allows visualization of protein-protein interactions involved in the assembly of 42S storage particles. In particular, the transfection assay using COS-7 cells provides a rapid screening test that should facilitate identification of critical residues and structural determinants that enable the proteins of the 42S storage particle to interact with each other and to establish distinct higher-order RNP (ribonucleoprotein) complexes.

A Potential Role for the Gsdf–eEF1α Complex in Inhibiting Germ Cell Proliferation: A Protein-Interaction Analysis in Medaka (Oryzias latipes) From a Proteomics Perspective

“…3b, 11 counts with highest rich factor in Table S6). Thus, ribosome biogenesis was higher in normal and gsdf null ovaries, but lower in normal testes, which is consistent with the quantity of ribosomes being actively transcribed and accumulated during early oogenesis (25,26). In contrast, pathways of microtubule-based phagosomes and gap junctions, as well as cell cycle, represented by pcna (proliferating cell nuclear antigen), were enriched in B but was downregulated in C and A, reflecting that these proteins are higher in normal testes than in normal ovaries or gsdf-deficient ovaries.…”

“…It was reported that loss of dlc1 in the cyst cells caused gonial cell hyperplasia in Drosophila testes (29), resembling the phenotypes of germ cell overproliferation in gsdf-depletion or amhr2-mutation testes (8,10,18). In addition to ribosomal proteins, eukaryotic translation initiation factor 3 (eIF3) subunits, the structural maintenance of chromosomes 5 (SMC5), elongation factor (eEF1 ), and its ovarian isoform (42Sp50), which are expressed in frog and medaka oocytes (25,26), were found among the 36 identified peptides.…”

Gonadal Transcriptome Analysis of Male and Female Olive Flounder (Paralichthys olivaceus)

“…P43 5S RNA-binding protein is combined with 5S rRNA to comprise 42S ribonucleoprotein storage particle. In addition, transcription factor IIIA acts as both a positive transcription factor for 5S RNA genes and a specific RNA-binding protein that complex with 5S RNA in oocytes to form the 7S ribonucleoprotein storage particle [48]. According to Diaz De Cerio et al, 5 S RNA and associate protein could constitute a sensitive and universal marker of oogenesis and oocyte differentiation in fish [49].…”

Characterization of a Novel Association between Two Trypanosome-Specific Proteins and 5S rRNA

“…Under special circumstances, cells transcribe 5S rRNA in excess and accumulate it in the cytoplasm in storage particles for subsequent mobilization to the nucleolus. This event has been studied in detail in Xenopus laevis oocytes [9], where 5S rRNA associates with p43, a 43 kDa protein diverged from TFIIIA.…”