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A cDNA clone for a polyadenylated RNA-binding protein of Xenopus laevis oocytes hybridizes to four developmentally
Abstract:
Xenopus laevis oocytes contain a unique group of proteins which decrease during oogenesis, bind poly(A) RNA, and possibly play a role in the regulation of translation. A monoclonal antibody generated against one of these proteins was used to screen an expression vector cDNA library. A cDNA clone was isolated and confirmed to code for the binding protein by in vitro translation of hybrid-selected RNA followed by immunoprecipitation. This cDNA, when used in RNA gel blots, hybridized to four transcripts of 2.0, 1.7 (two transcripts of similar size), and 1.2 kilobases. All of the transcripts decreased in amount during oogenesis and were not evident in somatic cells. In addition, the fraction of the transcripts associated with polysomes decreased during oogenesis. Digestion of the cDNA insert with PstI generated two fragments of 220 and 480 base pairs which, when used as probes in an RNA gel blot, hybridized to unique as well as common transcripts. Genomic Southern blots suggested the presence of a single gene, indicating that these transcripts arose by alternative processing.
Insights
Researchers identified a novel poly(A) RNA-binding protein in Xenopus laevis oocytes. Its unique transcripts decrease during oogenesis, suggesting a role in translational regulation.
Area of Science:
- Molecular Biology
- Developmental Biology
- Xenopus laevis research
Background:
- Oocytes contain unique proteins that decrease during oogenesis and bind poly(A) RNA.
- These proteins may regulate translation during development.
Purpose of the Study:
- To isolate and characterize a cDNA clone for a poly(A) RNA-binding protein from Xenopus laevis oocytes.
- To investigate the expression and regulation of the gene encoding this protein.
Main Methods:
- Screening of a cDNA expression library using a monoclonal antibody.
- In vitro translation of hybrid-selected RNA and immunoprecipitation for confirmation.
- RNA gel blot analysis to detect transcripts.
- Genomic Southern blot analysis to assess gene copy number.
Main Results:
- A cDNA clone encoding the poly(A) RNA-binding protein was isolated.
- Four transcripts (2.0, 1.7, 1.7, and 1.2 kb) were identified, decreasing during oogenesis and absent in somatic cells.
- Polysome association of these transcripts decreased during oogenesis.
- Genomic Southern blots indicated a single gene, suggesting alternative processing generates the different transcripts.
Conclusions:
- The isolated cDNA codes for a Xenopus laevis oocyte-specific poly(A) RNA-binding protein.
- Alternative RNA processing likely generates multiple transcripts from a single gene.
- The protein and its transcripts are developmentally regulated and may play a role in translational control during oogenesis.