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Published on: May 17, 2014
TRIB1 Is Regulated Post-Transcriptionally by Proteasomal and Non-Proteasomal Pathways
Sébastien Soubeyrand1, Amy Martinuk1, Paulina Lau1
1Atherogenomics Laboratory, University of Ottawa Heart Institute, Ottawa, Canada.
Abstract:
The TRIB1 gene has been associated with multiple malignancies, plasma triglycerides and coronary artery disease (CAD). Despite the clinical significance of this pseudo-kinase, there is little information on the regulation of TRIB1. Previous studies reported TRIB1 mRNA to be unstable, hinting that TRIB1 might be subject to post-transcriptional regulation. This work explores TRIB1 regulation, focusing on its post-transcriptional aspects. In 3 distinct model systems (HEK293T, HeLa and arterial smooth muscle cells) TRIB1 was undetectable as assessed by western blot. Using recombinant TRIB1 as a proxy, we demonstrate TRIB1 to be highly unstable at the protein and RNA levels. By contrast, recombinant TRIB1 was stable in cellular extracts. Blocking proteasome function led to increased protein steady state levels but failed to rescue protein instability, demonstrating that the 2 processes are uncoupled. Unlike as shown for TRIB2, CUL1 and TRCPβ did not play a role in mediating TRIB1 instability although TRCPβ suppression increased TRIB1 expression. Lastly, we demonstrate that protein instability is independent of TRIB1 subcellular localization. Following the identification of TRIB1 nuclear localization signal, a cytosolic form was engineered. Despite being confined to the cytosol, TRIB1 remained unstable, suggesting that instability occurs at a stage that precedes its nuclear translocation and downstream nuclear function. These results uncover possible avenues of intervention to regulate TRIB1 function by identifying two distinct regulatory axes that control TRIB1 at the post-transcriptional level.
Insights
The TRIB1 gene
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- The TRIB1 gene is linked to various cancers, plasma triglycerides, and coronary artery disease (CAD).
- Limited information exists regarding TRIB1 gene regulation, particularly its post-transcriptional control mechanisms.
- Previous research suggests TRIB1 mRNA instability indicates potential post-transcriptional regulation.
Purpose of the Study:
- To investigate the post-transcriptional regulation of the TRIB1 gene.
- To elucidate the mechanisms underlying TRIB1 protein and RNA instability.
- To identify regulatory pathways controlling TRIB1 expression.
Main Methods:
- Utilized HEK293T, HeLa, and arterial smooth muscle cells as model systems.
- Employed western blotting to assess TRIB1 protein levels.
- Investigated the role of proteasome function, CUL1, and TRCPβ in TRIB1 regulation.
- Engineered a cytosolic TRIB1 variant to assess the impact of subcellular localization on stability.
Main Results:
- TRIB1 protein and RNA were found to be highly unstable in cellular models.
- Proteasome inhibition increased TRIB1 steady-state levels but did not resolve instability, indicating uncoupled processes.
- CUL1 and TRCPβ were not primary mediators of TRIB1 instability, though TRCPβ suppression enhanced TRIB1 expression.
- TRIB1 instability persisted in the cytosol, independent of nuclear translocation.
Conclusions:
- TRIB1 instability is a post-transcriptional phenomenon occurring before nuclear translocation.
- Two distinct regulatory pathways controlling TRIB1 at the post-transcriptional level were identified.
- These findings offer potential strategies for therapeutic intervention targeting TRIB1 function.
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