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Point mutations in the stem-loop at the 3' end of mouse histone mRNA reduce expression by reducing the efficiency of
N B Pandey1, A S Williams, J H Sun
1Institute of Molecular Biophysics, Florida State University, Tallahassee 32306.
Abstract:
Mammalian histone mRNAs end in a highly conserved stem-loop structure, with a six-base stem and a four-base loop. We have examined the effect of mutating the stem-loop on the expression of the histone mRNA in vivo by introducing the mutated histone genes into CHO cells by stable transfection. Point mutations have been introduced into the loop sequence and into the UA base pair at the top of the stem. Changing either the first or the third base of the conserved UYUN sequence in the loop to a purine greatly reduced expression, while changing both U's to purines abolished expression. A number of alterations in the stem sequence, including reversing the stem sequence, reversing the two base pairs at the base of the stem, or destroying the UA base pair at the top of the stem, also abolished expression. Changing the UA base pair to a CG or a UG base pair also reduced expression. The loss of expression is due to inefficient processing of the pre-mRNA, as judged by the efficiency of processing in vitro. Addition of a polyadenylation site or the wild-type histone processing signal downstream of a mutant stem-loop resulted in rescuing the processing of the mutant pre-histone mRNA. These results suggest that if the histone pre-mRNA is not rapidly processed, then it is degraded.
Insights
Mutations in the conserved stem-loop of histone mRNA significantly impact gene expression. Efficient processing of histone pre-mRNA is crucial for its stability and expression, with defects leading to degradation.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
Background:
- Mammalian histone mRNAs possess a conserved stem-loop structure critical for their function.
- This structure is essential for the proper processing and expression of histone genes.
Purpose of the Study:
- To investigate the impact of mutations within the histone mRNA stem-loop structure on gene expression in vivo.
- To elucidate the role of the stem-loop in pre-mRNA processing and stability.
Main Methods:
- Stable transfection of Chinese Hamster Ovary (CHO) cells with mutated histone genes.
- Introduction of point mutations in the stem-loop sequence, including the loop and stem regions.
- In vitro processing assays to assess pre-mRNA processing efficiency.
Main Results:
- Mutations in the loop sequence, particularly altering the UYUN motif, reduced or abolished expression.
- Alterations in the stem sequence, including the UA base pair at the stem's top, abolished expression.
- Loss of expression was attributed to inefficient pre-mRNA processing, which could be rescued by adding a polyadenylation site or wild-type processing signal.
Conclusions:
- The conserved stem-loop structure is vital for efficient histone pre-mRNA processing.
- Rapid processing of histone pre-mRNA is necessary to prevent its degradation.
- Mutations disrupting the stem-loop impair gene expression by affecting mRNA processing and stability.