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Characterization of messenger ribonucleoprotein and messenger RNA from KB cells
Abstract:
Messenger ribonucleoprotein and mRNA from KB-cells were isolated under conditions designed to minimize nonspecific RNA-protein interaction and to minimize degradation by contaminating ribonucleases. A large fraction, 60-70%, of the messenger ribonucleoprotein from polysomes dissociated in vitro by either EDTA or puromycin sedimented faster than the large ribosome subunit. Messenger ribonucleoprotein particles with sedimentation coefficients up to 200 S were observed. Released mRNA was also large, with maximal molecular weights around 5 x 10(6).
Insights
Messenger ribonucleoprotein (mRNA) complexes were isolated from KB-cells. These complexes, when dissociated, revealed large mRNA molecules and messenger ribonucleoprotein particles that were larger than ribosome subunits.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Messenger ribonucleoprotein (mRNA) particles play crucial roles in gene expression.
- Understanding the composition and structure of these particles is essential for deciphering post-transcriptional regulation.
Purpose of the Study:
- To isolate and characterize messenger ribonucleoprotein (mRNA) complexes from KB-cells.
- To investigate the size and properties of mRNA and associated proteins after dissociation from polysomes.
Main Methods:
- Isolation of messenger ribonucleoprotein (mRNA) and mRNA from KB-cells under conditions minimizing nonspecific interactions and RNase degradation.
- Dissociation of polysomal messenger ribonucleoprotein in vitro using EDTA or puromycin.
- Sedimentation analysis to determine the size of messenger ribonucleoprotein particles and released mRNA.
Main Results:
- A significant fraction (60-70%) of polysomal messenger ribonucleoprotein dissociated into particles larger than the large ribosomal subunit.
- Messenger ribonucleoprotein particles with sedimentation coefficients up to 200 S were detected.
- Released mRNA molecules were found to be large, with maximal molecular weights of approximately 5 x 10^6 Daltons.
Conclusions:
- Mammalian messenger ribonucleoprotein (mRNA) complexes isolated from polysomes contain large mRNA molecules and associated proteins.
- These complexes, upon dissociation, yield messenger ribonucleoprotein particles and mRNA that are substantially larger than previously assumed.