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Studies on dissociation and reconstitution of nuclear 30-S ribonucleoprotein particles containing pre-mRNA
Abstract:
Treatment of nuclear 30-S ribonucleoprotein (RNP) particles containing pre-mRNA (precursor of mRNA) with 2 M NaCl leads to dissociation of RNA and protein. The protein component is present either as an aggregate with a sedimentation coefficient close to 30 S (a free informofer) or as a slowly sedimenting material (monomers or oligomers of informatin). Most of the informofers and slowly sedimenting material are in the equilibrium state. Iodination or aging of the 30-S particles stabilizes informofers. Lowering of NaCl concentration in the mixture of RNA with informofers or informatin subunits leads to reconstitution of RNP particles. In both cases, the particles formed have a sedimentation coefficient of about 30 S and a buoyant density equal to 1.4-1.41 g/cm3 but their response to pancreatic RNAase (EC 3.1.27.5) and high salt treatment is very different. Both the particles reconstituted from RNA and informofers and the original particles are very sensitive to pancreatic RNAase and after high salt treatment free informofers are formed. In contrast, the RNA of the particles reconstituted from slowly sedimenting material is much more protected against pancreatic RNAase action. These particles are also rather stable to high salt treatment. Thus, only if a protein in the form of an informofer aggregate is used, faithful reconstitution takes place. The data obtained are discussed in terms of the structure of the nuclear ribonucleoprotein particles containing precursor of messenger RNA.
Insights
Nuclear ribonucleoprotein (RNP) particles can be reconstituted from RNA and protein components. Faithful reconstitution of precursor messenger RNA particles requires the protein component to be in an aggregated informofer state for proper function.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Nuclear 30-S ribonucleoprotein (RNP) particles contain precursor messenger RNA (pre-mRNA).
- These particles dissociate into RNA and protein components upon treatment with high salt concentrations (2 M NaCl).
- The protein component exists as aggregated informofers or as slowly sedimenting informatin monomers/oligomers.
Purpose of the Study:
- To investigate the reconstitution of nuclear RNP particles from their RNA and protein components.
- To determine the structural requirements for faithful reconstitution of pre-mRNA containing RNP particles.
- To compare the properties of reconstituted particles formed from different protein states.
Main Methods:
- Dissociation of 30-S RNP particles using 2 M NaCl.
- Separation of RNA and protein components (informofer aggregates and informatin subunits).
- Reconstitution of RNP particles by lowering NaCl concentration.
- Analysis of reconstituted particles using sedimentation coefficient, buoyant density, pancreatic RNAase sensitivity, and high salt stability.
Main Results:
- Reconstitution of RNP particles from RNA and informofer aggregates resulted in particles sensitive to RNAase and high salt, similar to original particles.
- Reconstitution from RNA and informatin subunits yielded particles with RNA protected from RNAase and stable to high salt.
- Both reconstitution methods produced particles with similar sedimentation coefficients (~30 S) and buoyant densities (1.4-1.41 g/cm³).
Conclusions:
- Faithful reconstitution of nuclear RNP particles requires the protein component to be in an aggregated informofer state.
- The structure of the protein component significantly influences the properties and RNA protection within reconstituted RNP particles.
- These findings provide insights into the structural organization of nuclear RNP particles involved in pre-mRNA processing.