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mRNA structure, in situ, as assessed by microscopic techniques
J H Eberwine1, C Spencer, D Newell
1Department of Pharmacology, University of Pennsylvania, Philadelphia 19104.
Microscopy Research and Technique
|May 1, 1993
Summary
In situ transcription (IST) reveals RNA secondary structure influences gene translation. Pharmacological agents altering POMC translation also changed IST banding patterns, suggesting structural roles in gene regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- RNA secondary and tertiary structures play roles in RNA localization, enzymatic activity, and translation control.
- In situ transcription (IST) is a method to analyze mRNA structure within fixed cells or tissues.
Purpose of the Study:
- To assess mRNA structure in situ using IST.
- To investigate the relationship between RNA structure and POMC translation control.
Main Methods:
- In situ transcription (IST) using fixed cells and tissues.
- Employing an oligonucleotide primer complementary to a putative POMC stem-loop structure.
- Utilizing pharmacological agents to modulate POMC translation and observing changes in IST banding patterns.
Main Results:
- Distinct IST banding patterns were observed for mouse and rat POMC mRNA.
- Specific banding patterns emerged when using a primer targeting a predicted POMC stem-loop structure.
- Pharmacological agents modulating POMC translation affected IST banding patterns, with inhibitors increasing high molecular weight bands and stimulators having the opposite effect.
- Cytosolic protein fractions from AtT20 pituitary cells specifically bound to the IST-predicted RNA structure.
Conclusions:
- mRNA structure can be effectively assessed in situ using IST.
- mRNA secondary and tertiary structures likely influence mRNA translation.
- A stem-loop structure is predicted to exist in the 5'-end of POMC mRNA in situ.