Heterogeneous nuclear ribonucleoprotein complexes from Xenopus laevis oocytes and somatic cells.
HnRNP proteins have been implicated in most stages of cellular mRNA metabolism, including processing, nucleocytoplasmic transport, stability, and localization. Several hnRNP proteins are also known to participate in key early developmental decisions. In order to facilitate functional studies of these pre-mRNA- and mRNA-binding proteins in a vertebrate organism amenable to developmental studies and experimental manipulation, we identified and purified the major hnRNP proteins and isolated the hnRNP complex from Xenopus laevis oocytes and somatic cells. Using affinity chromatography and immunological methods, we isolated a family of >15 abundant single-stranded nucleic acid-binding proteins, which range in apparent molecular weight from approximately 20 kDa to >150 kDa, and with isoelectric points from <5 to >8. Monoclonal antibodies revealed that a subset of these proteins are major hnRNP proteins in both oocytes and somatic cells in culture, and include proteins related to human hnRNP A2/B1/B2 and hnRNP K. UV crosslinking in living cells demonstrated that these proteins bind poly(A)+ RNA in vivo. Immunopurification using a monoclonal antibodyto X. aevishnRNPA2 resulted in the isolation of RNP complexes that contain a specific subset of single-stranded nucleic acid-binding proteins. The protein composition of complexes isolated from somatic cells and from oocyte germinal vesicles was similar, suggesting that the overall properties and functions of hnRNP proteins in these two cell types are comparable. These findings, together with the novel probes generated here, will also facilitate studies of the function of vertebrate RNA-binding proteins using the well characterized X. laevis oocyte and early embryo as experimental systems.
Marcu
A
A
Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, NY 10029-6574, USA.
Bassit
B
B
Perez
R
R
Piñol-Roma
S
S
D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D005260:Female; D006367:HeLa Cells; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007120:Immunochemistry; D008970:Molecular Weight; D009865:Oocytes / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D014982:Xenopus laevis
D000911:Antibodies, Monoclonal; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012333:RNA, Messenger; D012261:Ribonucleoproteins; C439768:hnRNP A2