Related Experiment Videos
Partial purification of a 6-methyladenine mRNA methyltransferase which modifies internal adenine residues
1Department of Chemistry, Ohio University, Athens 45701.
Abstract:
Two forms of a 6-methyladenine mRNA methyltransferase have been partially purified using a T7 transcript coding for mouse dihydrofolate reductase as an RNA substrate. Both enzyme forms modify internal adenine residues within the RNA substrate. The enzymes were purified 357- and 37-fold respectively from nuclear salt extracts prepared from HeLa cells using DEAE-cellulose and phosphocellulose chromatography. The activity of the first form of the enzyme eluted from DEAE-cellulose (major form) was at least 3-fold greater than that of the second (minor form). H.p.l.c. analysis of the hydrolysed, methylated mRNA substrates demonstrated that both forms of the enzyme produced only 6-methyladenine. The two forms of the enzyme differed in their RNA substrate specificity as well as in the dependence for a 5' cap structure. The 6-methyladenine mRNA methyltransferase activity was found to be elevated in HeLa nuclei as compared with nuclear extracts from rat kidney and brain. Enzymic activity could not be detected in nuclei from either normal rat liver or regenerating rat liver. In the case of the HeLa cell, activity could only be detected in nuclear extracts, with a small amount in the ribosomal fraction. Other HeLa subcellular fractions were void of activity.
Insights
Researchers purified two forms of 6-methyladenine mRNA methyltransferase from HeLa cells. Both enzymes modify adenine residues in RNA, with distinct substrate specificities and cap structure dependencies.
Area of Science:
- Molecular Biology
- Enzymology
- RNA Modification
Background:
- Messenger RNA (mRNA) undergoes various post-transcriptional modifications.
- 6-methyladenine is a critical mRNA modification influencing RNA stability and function.
- The enzymes responsible for mRNA methylation are crucial for gene expression regulation.
Purpose of the Study:
- To purify and characterize forms of 6-methyladenine mRNA methyltransferase.
- To investigate the substrate specificity and properties of these enzymes.
- To compare enzyme activity across different cellular and species sources.
Main Methods:
- Partial purification of 6-methyladenine mRNA methyltransferase using DEAE-cellulose and phosphocellulose chromatography.
- Utilizing a T7 transcript coding for mouse dihydrofolate reductase as an RNA substrate.
- High-performance liquid chromatography (HPLC) analysis of methylated mRNA hydrolysates.
Main Results:
- Two forms of 6-methyladenine mRNA methyltransferase were purified from HeLa cell nuclear extracts.
- Both enzyme forms methylated internal adenine residues, producing 6-methyladenine.
- The major form exhibited higher activity than the minor form and differed in substrate specificity and cap dependence.
- Enzyme activity was elevated in HeLa nuclei compared to rat kidney and brain, and absent in rat liver nuclei.
Conclusions:
- HeLa cells possess distinct 6-methyladenine mRNA methyltransferase activities.
- These enzymes play a role in mRNA modification within the nucleus.
- Differential expression and properties suggest specific roles for each enzyme form.