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Assay of a ribonuclease that preferentially hydrolyses mRNAs containing cytokine-derived UA-rich instability
B Beutler1, P Thompson, J Keyes
1Howard Hughes Medical Institute, University of Texas Health Science Center, Dallas 75235.
Abstract:
mRNA molecules encoding a number of inflammatory cytokines, as well as certain proto-oncogenes, contain a conserved UA-exclusive sequence in the 3' untranslated region that confers message instability in vivo. This sequence may comprise a critical regulatory element, governing the level of these mRNA molecules, and determining the efficiency with which they are translated. Through the use of a double-label RNAse assay, we have determined that lysates prepared from mouse macrophages selectively degrade mRNA molecules containing the 3' untranslated UA sequence found in the mRNA encoding human cachectin/TNF. The degree of instability is dependent upon the number of copies of inserted UA sequence present in the target mRNA molecule (a Xenopus beta-globin mRNA). mRNAs containing randomly generated UA sequences are more labile than unmodified globin mRNA, but less susceptible to degradation than mRNAs containing the authentic cachectin-derived sequence. mRNA molecules containing synthetic UG-exclusive sequences are normally stable or protected in vitro. The destruction of UA-containing mRNA is inhibited by random adenylate/uridilate copolymers, but not by guanylate/uridilate copolymers. Boiling or proteinase K treatment destroys the selective nucleolytic activity of macrophage lysates. We propose that the nuclease measured here may serve to regulate cellular levels of mRNA molecules encoding cachectin, other inflammatory cytokines, and certain proto-oncogene products.
Insights
A specific UA-rich sequence in mRNA
Area of Science:
- Molecular Biology
- Gene Regulation
- Biochemistry
Background:
- Certain messenger RNA (mRNA) molecules, including those for inflammatory cytokines and proto-oncogenes, possess a conserved sequence rich in uracil and adenine (UA) within their 3' untranslated regions.
- This UA-exclusive sequence has been linked to mRNA instability in living organisms and may function as a key regulatory element controlling mRNA levels and translation efficiency.
Purpose of the Study:
- To investigate the role of the 3' untranslated UA-exclusive sequence in mRNA regulation.
- To identify the mechanism by which this sequence confers instability and to characterize the responsible nucleolytic activity.
Main Methods:
- Utilized a double-label RNAse assay to assess the degradation of modified Xenopus beta-globin mRNA molecules in mouse macrophage lysates.
- Investigated the effect of varying numbers of UA sequence copies and the impact of synthetic UA and UG sequences on mRNA stability.
- Examined the influence of adenylate/uridilate and guanylate/uridilate copolymers, as well as heat and proteinase K treatment, on the nucleolytic activity.
Main Results:
- Mouse macrophage lysates selectively degrade mRNA molecules containing the 3' untranslated UA sequence found in human cachectin/TNF mRNA.
- mRNA instability is dose-dependent on the number of UA sequence copies present.
- Synthetic UA sequences increase mRNA lability, while UG sequences confer stability; degradation is inhibited by A/U copolymers but not G/U copolymers.
- The selective nucleolytic activity is abolished by boiling or proteinase K treatment, indicating a protein-based nuclease.
Conclusions:
- A specific nuclease in mouse macrophages selectively degrades mRNA containing a 3' UA-exclusive sequence.
- This nuclease activity likely plays a role in regulating cellular levels of mRNAs encoding inflammatory cytokines and proto-oncogenes, such as cachectin.
- The findings suggest a novel mechanism for post-transcriptional gene regulation mediated by UA-rich sequences and specific nucleases.