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. 2007 Dec;6(12):2206-13.
doi: 10.1128/EC.00176-07. Epub 2007 Oct 5.

Trypanosoma brucei RNA binding proteins p34 and p37 mediate NOPP44/46 cellular localization via the exportin 1 nuclear export pathway

Kristina Hellman  1 , Kimberly Prohaska, Noreen Williams
Affiliations

Trypanosoma brucei RNA binding proteins p34 and p37 mediate NOPP44/46 cellular localization via the exportin 1 nuclear export pathway

Kristina Hellman et al. Eukaryot Cell. 2007 Dec.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Sucrose gradient sedimentation analysis of NOPP44/46. Nuclear extracts were prepared from wild-type procyclic cells (A) and cells expressing p34/p37 dsRNA (B). The amount of nuclear extract indicated was sedimented through a continuous 10 to 30% sucrose gradient. Fractions were collected and TCA precipitated. Western blot analysis was performed to detect the positions of p34 and p37 as well as the NOPP44/46 proteins. Fraction 2 designates the bottom of the gradient. The positions of 5S rRNA, small subunit (18S) rRNA, and large subunit (28S) rRNA markers are indicated.
FIG. 2.
FIG. 2.
Analysis of NOPP44/46 protein levels within nuclear extracts. The indicated amount of nuclear extracts (NE) from either wild-type or p34/p37 RNAi cells was analyzed by Western blotting using antibodies directed against p34 and p37 (upper panel), NOPP44/46 (middle panel), and a β-tubulin loading control (lower panel).
FIG. 3.
FIG. 3.
Analysis of NOPP44/46 mRNA steady-state levels within wild-type and p34/p37 RNAi cells. Total RNA was isolated from an equivalent number (5 × 106) of wild-type and p34/p37 RNAi cells. Northern blot analysis was performed using oligonucleotide probes specific to NOPP44/46 mRNA (upper panel), SSU rRNA (middle panel), and LSU rRNA (lower panel). The left panels include dilutions of total RNA from wild-type cells, and the right panels include dilutions of total RNA from p34/p37 RNAi cells.
FIG. 4.
FIG. 4.
Analysis of NOPP44/46 mRNA stability within wild-type and p34/p37 RNAi cells. Actinomycin D was added to a final concentration of 10 μg/ml. Total RNA was isolated at increasing time points, and 5 μg of each sample was analyzed. Probes specific to NOPP44/46 mRNA (upper panel) and SSU rRNA (lower panel) were utilized in Northern blot analyses.
FIG. 5.
FIG. 5.
Analysis of NOPP44/46 total protein levels. Total cellular protein was prepared from an equivalent number (5 × 106) of wild-type and p34/p37 RNAi cells. In each experiment, half of the cell suspension was applied to two separate SDS-polyacrylamide gels, and samples were processed for Western blot analysis, probing for either p34 and p37 (top panel) or NOPP44/46 (middle panel). A representative blot for the β-tubulin loading control is shown (lower panel).
FIG. 6.
FIG. 6.
NOPP44/46 localization in p34/p37 RNAi cells. Nuclear extracts were prepared from wild-type and p34/p37 RNAi cells. With the amount of extract indicated, samples were separated using SDS-PAGE and processed for Western blot analysis. (A) Results from nuclear extracts; (B) results from cytoplasmic extracts. The presence of p34 and p37 proteins, NOPP44/46, phosphoglycerate kinase (PGK; a cytoplasmic marker), and the RNA polymerase II large subunit (CTD; a nuclear marker) were detected as indicated.
FIG. 7.
FIG. 7.
Motif and sequence analysis of p34 and p37 proteins. (A) Motif maps of the p34 and p37 proteins, including locations of NLS and NES. APK, alanine, proline, lysine-rich domain; RBD, RNA binding domain; KKDX, lysine, lysine, aspartic acid, and “X” repeat motif. (B) Nuclear export signal sequences shown with the consensus sequence. Similar and identical residues between T. brucei p34 and p37 and T. cruzi UBP-1 are underlined. Spaces were introduced to allow for alignment of conserved leucine residues.
FIG. 8.
FIG. 8.
NOPP44/46 associate with exportin 1 through an interaction with p34/p37. Wild-type and RNAi cells were fractionated into cytoplasmic and nuclear extracts. A 500-μg aliquot of nuclear extract was used for subsequent immune capture of exportin 1. Samples were subjected to SDS-PAGE and Western blot analysis to detect p34 and p37 (upper panel). Membranes were stripped and reprobed for detection of NOPP44/46 (middle panel). As a control, membranes were stripped again and reprobed for PABP (lower panel). B, IC beads-alone control; NE, 50 μg nuclear extract; P, IC pellet fraction; S, IC supernatant fraction.
FIG. 9.
FIG. 9.
p34 and/or p37, exportin 1, and NOPP44/46 are involved in a common complex. Procyclic nuclear extracts were used to perform exportin 1 immune capture assays. The first capture was eluted using SDS, DTT, and β-mercaptoethanol. Eluate was renatured and used for subsequent immune capture of p34 and p37. B, IC beads-alone control; NE, 50 μg nuclear extract; P, IC pellet fraction; S, IC supernatant fraction; P2, IC second elution.
FIG. 10.
FIG. 10.
Localization of NOPP44/46 upon treatment of wild-type procyclic cells with leptomycin B. A total of 1.0 × 106 cells/reaction mixture were left untreated or treated with LMB for the indicated time points at a final concentration of 0.001 ng/cell. Cells were fractionated into nuclear and cytoplasmic extracts and subjected to SDS-PAGE. Western blot analyses were performed to detect NOPP44/46 (upper panel) and the large subunit of RNA polymerase II (CTD) (lower panel).
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