Isolation and characterization of rat liver free and membrane-bound polysomal messenger ribonucleoprotein particles
Abstract:
Rat liver mRNA-labeled free and membrane-bound polysomes uncontaminated with nuclear or cytoplasmic ribonucleoprotein particles were dissociated with EDTA and the released messenger ribonucleoprotein particles isolated using an oligo(dT)-cellulose column. Seventy percent of the labeled mRNA applied bound to the column. Binding of mRNP was dependent on the presence of a poly(A) segment in the RNA. The nonbound material contained most of the ribosomal subunits and also messenger-sized poly(A-)RNA molecules associated with protein. The free mRNP fraction bound to the column, washed with 250 mM NaCl, and subsequently eluted contained seven proteins ranging in molecular weight from 52 000 to 138 000 only one of which was found in the fraction not bound to the column. Furthermore, these proteins were shown to have a higher affinity for poly(A+)RNA as compared with rRNA. The membrane-bound mRNP contained five proteins, four of which were identical with those associated with free mRNP. Membrane-bound mRNP were disrupted at a lower salt concentration than the free. The proteins found associated with free and membrane-bound polysomal mRNP appeared to be clustered in the poly(A) region of the molecule. The implications of these findings are discussed.
Insights
Researchers isolated messenger ribonucleoprotein particles (mRNPs) from rat liver polysomes. These mRNPs contain specific proteins that bind to the poly(A) tail of messenger RNA (mRNA).
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Messenger ribonucleoprotein particles (mRNPs) are complexes of messenger RNA (mRNA) and proteins.
- These complexes play crucial roles in mRNA metabolism, including translation, transport, and stability.
- Understanding the protein composition of mRNPs is essential for elucidating their functions.
Purpose of the Study:
- To characterize the proteins associated with free and membrane-bound polysomal mRNPs in rat liver.
- To investigate the role of the poly(A) tail in the binding of these proteins to mRNA.
- To compare the protein composition and salt sensitivity of free and membrane-bound mRNPs.
Main Methods:
- Isolation of free and membrane-bound polysomes from rat liver.
- Dissociation of polysomes and isolation of messenger ribonucleoprotein particles (mRNPs) using oligo(dT)-cellulose chromatography.
- Analysis of protein composition by SDS-PAGE and molecular weight determination.
- Assessment of protein-RNA binding affinity and salt sensitivity.
Main Results:
- Seventy percent of labeled mRNA bound to oligo(dT)-cellulose, indicating poly(A) tail-dependent binding.
- Free mRNPs contained seven unique proteins (52-138 kDa), with one protein also found in unbound fractions.
- Membrane-bound mRNPs contained five proteins, four of which were identical to those in free mRNPs.
- Proteins associated with both free and membrane-bound mRNPs showed higher affinity for poly(A+)RNA than rRNA.
- Membrane-bound mRNPs dissociated at lower salt concentrations than free mRNPs.
Conclusions:
- Specific proteins associate with the poly(A) tail of mRNA in both free and membrane-bound polysomal mRNPs.
- These proteins exhibit differential salt sensitivity, suggesting distinct roles in mRNP organization and function.
- The findings provide insights into the structural organization and regulatory mechanisms of polysomal mRNPs.
