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Identification of an mRNA-binding protein and the specific elements that may mediate the pH-responsive induction of
O F Laterza1, W R Hansen, L Taylor
1Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA.
Abstract:
Various segments of the 3'-nontranslated region of the renal glutaminase (GA) mRNA were tested for their ability to enhance turnover and pH responsiveness. The combined effects were retained in the 340-base R-2 segment. However, the combined R-1 and R-3 fragments also imparted a partial destabilization and pH responsiveness to a chimeric beta-globin mRNA. RNA electrophoretic mobility shift assays indicated that cytosolic extracts of rat renal cortex contain a protein that binds to the R-2 and R-3 RNAs. The binding observed with the R-2 RNA was mapped to a direct repeat of an 8-base AU sequence. This binding was effectively competed with an excess of the same RNA, but not by adjacent or unrelated RNAs. UV cross-linking experiments identified a 48-kDa protein that binds to the AU repeats of the R-2 RNA. The apparent binding of this protein was greatly reduced in renal cytosolic extracts prepared from acutely acidotic rats. Two related RNA sequences in the R-3 segment also exhibited specific binding. However, the latter binding was more effectively competed by R-2 RNA than by itself, indicating that the homologous sites may be weaker binding sites for the same 48-kDa protein. Thus, a single protein may bind specifically to multiple instability elements within the 3'-nontranslated region of the GA mRNA and mediate its pH-responsive stabilization.
Insights
A specific protein binds to key regions of renal glutaminase (GA) mRNA, controlling its stability and response to pH changes. This discovery offers insights into kidney function regulation.
Area of Science:
- Molecular Biology
- Biochemistry
- Renal Physiology
Background:
- The 3'-nontranslated region of messenger RNA (mRNA) plays a crucial role in regulating gene expression, including mRNA stability and translation.
- Renal glutaminase (GA) is an enzyme critical for kidney function, and its activity is known to be influenced by pH.
- Understanding the post-transcriptional regulation of GA mRNA is essential for elucidating kidney physiology and pathophysiology.
Purpose of the Study:
- To investigate the functional significance of specific segments within the 3'-nontranslated region of renal glutaminase (GA) mRNA.
- To identify proteins that bind to these regulatory RNA segments and determine their role in mRNA turnover and pH responsiveness.
- To elucidate the mechanism by which GA mRNA stability and pH responsiveness are mediated.
Main Methods:
- RNA manipulation techniques were used to analyze various segments of the GA mRNA 3'-untranslated region.
- RNA electrophoretic mobility shift assays (EMSA) were employed to detect protein-RNA interactions using rat renal cytosolic extracts.
- UV cross-linking experiments were performed to identify and characterize the binding protein.
- Competition assays were utilized to assess the specificity of RNA-protein binding.
Main Results:
- The R-2 segment of the GA mRNA 3'-untranslated region retained the ability to enhance turnover and pH responsiveness.
- A 48-kDa protein was identified that specifically binds to AU-rich repeats within the R-2 segment of GA mRNA.
- Binding of this protein was significantly reduced in renal cytosolic extracts from acidotic rats, suggesting a role in pH-mediated regulation.
- The protein also binds to related sequences in the R-3 segment, indicating it may interact with multiple regulatory elements.
Conclusions:
- A single 48-kDa protein binds to multiple instability elements in the 3'-untranslated region of GA mRNA.
- This protein-mediated binding is responsible for the pH-responsive stabilization of GA mRNA.
- The findings provide a molecular mechanism for the pH-dependent regulation of renal glutaminase expression.