Related Experiment Videos
Adenosine diphosphate ribosyltransferase and protein acceptors associated with cytoplasmic free messenger
Abstract:
ADP-ribosyltransferase activity has been characterized in free messenger ribonucleoprotein particles (mRNP) from mouse plasmacytoma cells. This enzymatic activity appears to be associated with the free mRNP and not due to nuclear contamination. The enzyme activity is not stimulated by added DNA or histone H1 and represents 34 per cent of the total cellular ADP-ribosyltransferase activity while the DNA contamination in free mRNP is less than 4 per cent of the total cellular DNA. Moreover, the ADP-ribosyltransferase specific activity per mg of DNA is about 75-fold higher in free mRNP than in the nuclei. During CsCl gradient centrifugation of the cytoplasmic fraction, the ADP-ribosylated material separates out at a buoyant density similar to that of free mRNP. This ADP-ribosyltransferase activity is inhibited by thymidine, nicotinamide and 3-aminobenzamide, while it is highly stimulated by exogenous pancreatic RNase. The in vitro synthesized acid insoluble material is rendered partly soluble by treatment by a proteolytic enzyme or by snake venom phosphodiesterase resulting in phosphoribosyl-AMP formation: the pancreatic RNase does not solubilize this material. Several ADP-ribosylated proteins are detected by lithium dodecylsulfate gel electrophoresis. Such an ADP-ribosyltransferase activity has also been detected in free mRNP from rat liver. It is suggested that this ADP-ribosylation of specific free mRNP proteins may be associated with free mRNP structure and/or with some chemical covalent type of modification rendering mRNA available for translation.
Insights
Researchers found ADP-ribosyltransferase activity in messenger ribonucleoprotein particles (mRNP). This enzyme modifies mRNP proteins, potentially impacting mRNA translation and availability.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- ADP-ribosyltransferase (ART) activity is crucial in cellular processes.
- Messenger ribonucleoprotein particles (mRNP) are key players in mRNA transport and translation.
- Understanding ART localization and function within mRNPs is essential for deciphering gene expression regulation.
Purpose of the Study:
- To characterize ADP-ribosyltransferase activity within free messenger ribonucleoprotein particles (mRNP).
- To investigate the association of this enzymatic activity with mRNPs and its potential role in mRNA metabolism.
- To determine the biochemical properties and cellular localization of the mRNP-associated ART.
Main Methods:
- Enzymatic assays to measure ADP-ribosyltransferase activity in isolated free mRNPs from mouse plasmacytoma cells and rat liver.
- Biochemical fractionation using CsCl gradient centrifugation to determine the buoyant density of ADP-ribosylated material.
- Inhibition and stimulation studies using various reagents (e.g., thymidine, nicotinamide, 3-aminobenzamide, pancreatic RNase).
- Analysis of ADP-ribosylated proteins using lithium dodecylsulfate gel electrophoresis.
Main Results:
- Significant ADP-ribosyltransferase activity was detected in free mRNPs, distinct from nuclear contamination.
- The specific activity of ART per mg of DNA was substantially higher in free mRNPs compared to nuclei.
- ADP-ribosylated material co-fractionated with free mRNPs during CsCl gradient centrifugation.
- The activity was inhibited by specific compounds and stimulated by pancreatic RNase, suggesting RNA involvement.
- Several ADP-ribosylated proteins were identified within the mRNP fractions.
Conclusions:
- A distinct ADP-ribosyltransferase activity is associated with free mRNPs.
- This mRNP-associated ART likely modifies specific mRNP proteins.
- The observed protein modification may play a role in regulating mRNA structure, availability, or translation efficiency.