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Translational activity and functional stability of human fibroblast beta 1 and beta 2 interferon mRNAs lacking
Abstract:
Polyadenylylated mRNA was purified from poly(I).poly(C)- and cycloheximide-superinduced human fibroblast (FS-4) cultures. The mRNA was subjected to electrophoresis through an agarose/CH3HgOH gel, and human fibroblast beta 1 and beta 2 interferon mRNAs were isolated. Each mRNA preparation was phosphorolyzed at 0 degrees C for 20 min by using a molar excess of polynucleotide phosphorylase to produce RNAs lacking poly(A) and then incubated at 37 degrees C for varying lengths of time to allow the phosphorylase to further digest the deadenylylated RNA from the 3' end in a processive and synchronous manner. Removal of the poly(A) (less than or equal to 100 residues) and approximately 100 adjacent residues from human fibroblast beta 1 interferon mRNA (native length, 900 residues, including a 3'-noncoding region of 203 residues) did not alter the translational activity or the functional stability of this mRNA in Xenopus oocytes, whereas deletion of the poly(A) and approximately 200 adjacent residues decreased its translational efficiency. On the other hand, removal of the poly(A) (approximately 200 residues) and approximately 200 adjacent residues from human fibroblast beta 2 interferon mRNA (native length, 1300 residues) did not alter the translational activity or the functional stability of this molecule in oocytes. Thus, neither the poly(A) nor large segments of the 3'-noncoding region (which includes the hexanucleotide A-A-U-A-A-A sequence, at least in the case of beta 1 mRNA) are required for the maintenance of the functional stability of human beta 1 and beta 2 interferon mRNAs in Xenopus oocytes.
Insights
The poly(A) tail and adjacent regions of human interferon mRNAs are not essential for their stability in Xenopus oocytes. Deleting these segments did not affect the functional stability of beta 1 and beta 2 interferon mRNA.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
- RNA Biochemistry
Background:
- Polyadenylation of eukaryotic messenger RNA (mRNA) is crucial for its stability and translation.
- The 3'-noncoding region (3'-NCR) of mRNA plays a significant role in post-transcriptional regulation.
- Interferons are key cytokines involved in the immune response to viral infections.
Purpose of the Study:
- To investigate the role of the poly(A) tail and adjacent 3'-NCR sequences in the functional stability and translational activity of human beta 1 and beta 2 interferon mRNAs.
- To determine if these elements are required for maintaining mRNA function in a heterologous system (Xenopus oocytes).
Main Methods:
- Purification of polyadenylylated mRNA from induced human fibroblast cultures.
- Isolation of specific interferon beta 1 and beta 2 mRNA transcripts using gel electrophoresis.
- Enzymatic removal of poly(A) tails and adjacent 3'-NCR sequences using polynucleotide phosphorylase.
- Assessment of mRNA translational activity and functional stability in Xenopus oocytes.
Main Results:
- Removal of the poly(A) tail and up to 100 adjacent residues from beta 1 interferon mRNA did not affect its translational activity or stability.
- Larger deletions (poly(A) + ~200 residues) from beta 1 mRNA reduced translational efficiency.
- Removal of the poly(A) tail (~200 residues) and ~200 adjacent residues from beta 2 interferon mRNA did not alter its translational activity or stability.
- The hexanucleotide A-A-U-A-A-A sequence, present in the 3'-NCR of beta 1 mRNA, was not required for functional stability.
Conclusions:
- The poly(A) tail and extensive portions of the 3'-noncoding region are not essential for the functional stability of human beta 1 and beta 2 interferon mRNAs in Xenopus oocytes.
- These findings suggest alternative mechanisms may contribute to interferon mRNA stability and translation regulation.
- The study highlights the importance of the heterologous oocyte system for dissecting mRNA regulatory elements.