Trypanosoma brucei RNA binding proteins p34 and p37 mediate NOPP44/46 cellular localization via the exportin 1 nuclear export pathway.
We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to interact with a family of nucleolar phosphoproteins, NOPP44/46, in Trypanosoma brucei. These proteins are nearly identical, the major difference being an 18-amino-acid insert in the N terminus of p37. In order to characterize the interaction between p34 and p37 and NOPP44/46, we have utilized an RNA interference (RNAi) cell line that specifically targets p34 and p37. Within these RNAi cells, we detected a disruption of a higher-molecular-weight complex containing NOPP44/46, as well as a dramatic increase in nuclear NOPP44/46 protein levels. We demonstrated that no change occurred in NOPP44/46 mRNA steady-state levels or stability, nor was there a change in cellular protein levels. These results led us to investigate whether p34 and p37 regulate NOPP44/46 cellular localization. Examination of the p34 and p37 amino acid sequences revealed a leucine-rich nuclear export signal, which interacts with the nuclear export factor exportin 1. Immune capture experiments demonstrated that p34, p37, and NOPP44/46 associate with exportin 1. When these experiments were performed with p34/p37 RNAi cells, NOPP44/46 no longer associated with exportin 1. Sequential immune capture experiments demonstrated that p34, p37, NOPP44/46, and exportin 1 exist in a common complex. Inhibiting exportin 1-mediated nuclear export led to an increase in nuclear NOPP44/46 proteins, indicating that they are exported from the nucleus via this pathway. Together, our results demonstrate that p34 and p37 regulate NOPP44/46 cellular localization by facilitating their association with exportin 1.
Hellman
Kristina
K
Department of Microbiology and Immunology and The Witebsky Center for Microbial Pathogenesis and Immunology, 253 Biomedical Research Building, University at Buffalo, Buffalo, New York 14214, USA.
Prohaska
Kimberly
K
Williams
Noreen
N
D021581:Active Transport, Cell Nucleus; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D007107:Immune System; D028884:Karyopherins / Q000378:metabolism*; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000378:metabolism*; D000097606:Exportin 1 Protein
D005231:Fatty Acids, Unsaturated; D028884:Karyopherins; D009687:Nuclear Proteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein; C100391:NOPP44-46 protein, Trypanosoma brucei; C476232:p34 protein, Trypanosoma brucei; C476233:p37 protein, Trypanosoma brucei; C038753:leptomycin B