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3' RNA processing efficiency plays a primary role in generating termination-competent RNA polymerase II elongation
G Edwalds-Gilbert1, J Prescott, E Falck-Pedersen
1Department of Microbiology, W. R. Hearst Research Foundation, Cornell University Medical College, New York, New York 10021.
Molecular and Cellular Biology
|June 1, 1993
Summary
Efficient RNA processing at the polyadenylation (poly(A)) site is crucial for proper transcription termination in mammals. This study shows that the efficiency of 3' end processing directly impacts the termination of RNA polymerase II transcription.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
- RNA Processing
Background:
- Transcription termination in eukaryotes is linked to 3' RNA processing.
- The efficiency of polyadenylation (poly(A)) site processing varies.
- The mouse beta maj-globin transcription unit requires an intact poly(A) site for efficient termination.
Purpose of the Study:
- To investigate the relationship between poly(A) site processing efficiency and poly(A) site-dependent transcription termination.
- To determine if varying processing efficiencies of pre-mRNAs affect termination events.
- To elucidate the role of 3' processing complex formation in RNA polymerase II termination.
Main Methods:
- In vitro assays to measure pre-mRNA cleavage efficiency.
- In vivo transient transfection assays to assess mRNA and gene product expression.
- Nuclear run-on assays using recombinant adenovirus termination vectors.
- Analysis of chimeric poly(A) sites with modified downstream elements.
Main Results:
- Efficient processing of beta maj-globin pre-mRNA correlated with high termination efficiency.
- Replacing the beta maj-globin poly(A) site with a poorly processed L1 site decreased termination.
- Replacing with a highly processed L3 site maintained high termination efficiency.
- Variations in downstream GU-rich elements affected both processing and termination efficiency.
Conclusions:
- Transcription termination efficiency is directly dependent on the yield of the 3' RNA processing event.
- The efficiency of 3' processing complex formation correlates with RNA polymerase II termination at the 3' end of mammalian transcription units.