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Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
Published on: December 4, 2010
Featured Article: Nuclear export of opioid growth factor receptor is CRM1 dependent
Nancy P Kren1, Ian S Zagon1, Patricia J McLaughlin2
1Department of Neural & Behavioral Sciences, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, PA, USA.
Abstract:
Opioid growth factor receptor (OGFr) facilitates growth inhibition in the presence of its specific ligand opioid growth factor (OGF), chemically termed [Met(5)]-enkephalin. The function of the OGF-OGFr axis requires the receptor to translocate to the nucleus. However, the mechanism of nuclear export of OGFr is unknown. In this study, endogenous OGFr, as well as exogenously expressed OGFr-EGFP, demonstrated significant nuclear accumulation in response to leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export, suggesting that OGFr is exported in a CRM1-dependent manner. One consensus sequence for a nuclear export signal (NES) was identified. Mutation of the associated leucines, L217 L220 L223 and L225, to alanine resulted in decreased nuclear accumulation. NES-EGFP responded to LMB, indicating that this sequence is capable of functioning as an export signal in isolation. To determine why the sequence functions differently in isolation than as a full length protein, the localization of subNES was evaluated in the presence and absence of MG132, a potent inhibitor of proteosomal degradation. MG132 had no effect of subNES localization. The role of tandem repeats located at the C-terminus of OGFr was examined for their role in nuclear trafficking. Six of seven tandem repeats were removed to form deltaTR. DeltaTR localized exclusively to the nucleus indicating that the tandem repeats may contribute to the localization of the receptor. Similar to the loss of cellular proliferation activity (i.e. inhibition) recorded with subNES, deltaTR also demonstrated a significant loss of inhibitory activity indicating that the repeats may be integral to receptor function. These experiments reveal that OGFr contains one functional NES, L217 L220 L223 and L225 and can be exported from the nucleus in a CRM1-dependent manner.
Insights
The opioid growth factor receptor (OGFr) is exported from the nucleus via a CRM1-dependent mechanism, involving a specific nuclear export signal (NES) and C-terminal tandem repeats essential for its growth-inhibitory function.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The opioid growth factor receptor (OGFr) mediates growth inhibition through its ligand, opioid growth factor (OGF).
- OGFr function necessitates nuclear translocation, but its nuclear export mechanism remains unclear.
Purpose of the Study:
- To elucidate the mechanism of OGFr nuclear export.
- To identify the specific sequences and factors involved in OGFr nuclear trafficking and function.
Main Methods:
- Utilized leptomycin B (LMB) to inhibit CRM1-dependent nuclear export and observed OGFr localization.
- Identified and mutated a potential nuclear export signal (NES) sequence (L217, L220, L223, L225).
- Investigated the role of C-terminal tandem repeats by creating a truncated mutant (deltaTR) and assessed localization and growth inhibition.
Main Results:
- Leptomycin B induced significant nuclear accumulation of OGFr, indicating CRM1-dependent export.
- Mutation of the identified NES sequence decreased nuclear accumulation.
- Deletion of C-terminal tandem repeats (deltaTR) resulted in exclusive nuclear localization and loss of growth inhibitory activity.
Conclusions:
- OGFr is exported from the nucleus in a CRM1-dependent manner via a functional NES.
- C-terminal tandem repeats are crucial for OGFr's nuclear trafficking and its role in growth inhibition.
