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CIRP2, a major cytoplasmic RNA-binding protein in Xenopus oocytes
K Matsumoto1, K Aoki, N Dohmae
1RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan. matsumok@postman.riken.go.jp
Abstract:
In an attempt to isolate mRNA-binding proteins we fractionated Xenopus oocyte lysate by oligo(dT)-cellulose chromatography. A 20 kDa protein was the major component of the eluate. cDNA cloning revealed that this protein is a Xenopus homolog of the cold-inducible RNA-binding protein (CIRP) which was originally identified in mammalian cells as a protein that is overexpressed upon a temperature downshift. This Xenopus protein, termed here xCIRP2, is highly expressed in ovary, testis and brain in adult Xenopus tissues. In oocytes it is predominantly localized in the cytoplasm. By biochemical fractionation we provide evidence that xCIRP2 is associated with ribosomes, suggesting that it participates in translational regulation in oocytes. Microinjection of labeled mRNA into oocytes followed by UV cross-linking of the oocyte lysate led to identification of two major RNA-binding activities. Immunoprecipitation of the RNA-binding proteins demonstrated that one is xCIRP2 and that the other contains FRGY2. FRGY2, which is one of the principal constituents of mRNA storage particles involved in translational masking of maternal mRNA, has an RNA-binding domain conserved to those of bacterial cold shock proteins. Possible implications of the highly abundant expression in oocytes of cold shock RNA-binding proteins of both eukaryotic and prokaryotic types are discussed.
Insights
Researchers identified xCIRP2, a Xenopus homolog of cold-inducible RNA-binding protein (CIRP), in oocytes. This protein associates with ribosomes, suggesting a role in translational regulation during early development.
Area of Science:
- Molecular Biology
- Developmental Biology
- Xenopus laevis research
Background:
- mRNA-binding proteins play crucial roles in post-transcriptional gene regulation.
- Cold-inducible RNA-binding proteins (CIRPs) are involved in cellular responses to temperature changes.
- Understanding protein interactions in oocytes is key to deciphering early developmental processes.
Purpose of the Study:
- To identify and characterize novel mRNA-binding proteins in Xenopus oocytes.
- To investigate the function and localization of a specific 20 kDa protein identified in oocyte lysate.
- To explore the role of cold shock RNA-binding proteins in Xenopus oogenesis.
Main Methods:
- Oligo(dT)-cellulose chromatography for protein fractionation.
- cDNA cloning for protein identification.
- Biochemical fractionation and UV cross-linking assays for RNA-binding analysis.
- Immunoprecipitation to identify interacting proteins.
Main Results:
- A 20 kDa protein, identified as Xenopus cold-inducible RNA-binding protein 2 (xCIRP2), was isolated.
- xCIRP2 is highly expressed in Xenopus ovary, testis, and brain, and localized to the cytoplasm of oocytes.
- Evidence suggests xCIRP2 associates with ribosomes, indicating a role in translational regulation.
- Two major RNA-binding activities were identified: xCIRP2 and FRGY2, a known component of translational masking particles.
Conclusions:
- xCIRP2 is a significant mRNA-binding protein in Xenopus oocytes, likely involved in translational control.
- The co-occurrence of eukaryotic (xCIRP2) and prokaryotic-like (FRGY2) cold shock RNA-binding proteins in oocytes suggests conserved regulatory mechanisms.
- These findings contribute to understanding the complex regulation of maternal mRNA in early development.