Related Experiment Videos
The mRNA poly(A)-binding protein: localization, abundance, and RNA-binding specificity
M Görlach1, C G Burd, G Dreyfuss
1Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia 19104-6148.
Abstract:
The poly(A)-binding protein (PABP) binds to the messenger (mRNA) 3'-poly(A) tail found on most eukaryotic mRNAs and together with the poly(A) tail has been implicated in governing the stability and the translation of mRNA. In order to further understand the role of the PABP in these processes, we have undertaken a detailed analysis of the cellular localization, the abundance, and the RNA-binding properties of the human PABP (hPABP). We raised monoclonal antibodies against the 70-kDa hPABP and confocal immunofluorescence microscopy with these antibodies reveals that it is localized exclusively to the cytoplasm. The hPABP exhibits a very low turnover rate in these cells and quantitative immunoblotting experiments demonstrated that growing HeLa cells contain a surprisingly high number of approximately 8 x 10(6) PABP molecules per cell, which corresponds to an intracellular concentration of about 4 microM. In an in vitro selection/amplification assay from random sequence oligonucleotide pools the hPABP selects oligo(rA)-rich sequences and it binds oligo(rA)25 with an apparent Kd of 7 nM. The hPABP binds to unrelated RNA sequences with an about 100-fold lower affinity (Kd > or = 0.5 microM). The abundance of the hPABP indicates that there is an approximately three-fold excess of the protein over binding sites on cytoplasmic poly(A). This excess and the high concentration of the hPABP, which is three orders of magnitude above its Kd for oligo(rA)25, suggest that the hPABP may bind to additional, lower affinity binding sites in vivo.
Insights
The poly(A)-binding protein (PABP) is abundant in the cytoplasm and binds strongly to poly(A) tails on mRNA. This suggests PABP may have additional roles beyond its known functions in mRNA stability and translation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Poly(A)-binding protein (PABP) interacts with the 3'-poly(A) tail of eukaryotic mRNA.
- PABP and the poly(A) tail are crucial for mRNA stability and translation regulation.
Purpose of the Study:
- To investigate the cellular localization, abundance, and RNA-binding properties of human PABP (hPABP).
- To elucidate the role of hPABP in mRNA processing and regulation.
Main Methods:
- Monoclonal antibodies were generated against hPABP.
- Confocal immunofluorescence microscopy was used for cellular localization.
- Quantitative immunoblotting assessed protein abundance and turnover.
- In vitro selection/amplification assays determined RNA-binding affinities.
Main Results:
- hPABP exclusively localizes to the cytoplasm.
- HeLa cells contain approximately 8 x 10^6 hPABP molecules per cell (approx. 4 microM concentration).
- hPABP exhibits a low turnover rate and binds oligo(rA)25 with high affinity (Kd = 7 nM), with lower affinity for unrelated RNA (Kd > or = 0.5 microM).
Conclusions:
- The high intracellular concentration of hPABP suggests a potential for binding to additional, lower-affinity sites in vivo.
- The abundance of hPABP indicates a three-fold excess over cytoplasmic poly(A) binding sites.
- These findings provide insights into the regulatory mechanisms of mRNA metabolism governed by PABP.