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Control of cleavage site selection during mRNA 3' end formation by a yeast hnRNP
L Minvielle-Sebastia1, K Beyer, A M Krecic
1Department of Cell Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland. minvielle@ubaclu.unibas.ch
Abstract:
Endonucleolytic cleavage of pre-mRNAs is the first step during eukaryotic mRNA 3' end formation. It has been proposed that cleavage factors CF IA, CF IB and CF II are required for pre-mRNA 3' end cleavage in yeast. CF IB is composed of a single polypeptide, Nab4p/Hrp1p, which is related to the A/B group of metazoan heterogeneous nuclear ribonucleoproteins (hnRNPs) that function as antagonistic regulators of 5' splice site selection. Here, we provide evidence that Nab4p/Hrp1p is not required for pre-mRNA 3' end endonucleolytic cleavage. We show that CF IA and CF II devoid of Nab4p/Hrp1p are sufficient to cleave a variety of RNA substrates but that cleavage occurs at multiple sites. Addition of Nab4p/Hrp1p prevents these alternative cleavages in a concentration-dependent manner, suggesting an essential and conserved role for some hnRNPs in pre-mRNA cleavage site selection.
Insights
Yeast cleavage factors CF IA and CF II can cleave pre-mRNA, but Nab4p/Hrp1p is not required for this process. Nab4p/Hrp1p regulates cleavage site selection, preventing alternative cleavage events.
Area of Science:
- Molecular Biology
- RNA Processing
- Eukaryotic Gene Expression
Background:
- Pre-mRNA 3' end formation involves endonucleolytic cleavage, a critical step in eukaryotic gene expression.
- Cleavage factors CF IA, CF IB, and CF II were previously thought essential for yeast pre-mRNA 3' end cleavage.
- CF IB, comprising Nab4p/Hrp1p, is related to metazoan hnRNPs involved in splice site selection.
Purpose of the Study:
- To investigate the role of Nab4p/Hrp1p in pre-mRNA 3' end endonucleolytic cleavage in yeast.
- To determine if Nab4p/Hrp1p is essential for the cleavage activity of CF IA and CF II.
- To elucidate the function of Nab4p/Hrp1p in regulating cleavage site specificity.
Main Methods:
- In vitro cleavage assays using various RNA substrates.
- Analysis of pre-mRNA cleavage by purified cleavage factors (CF IA, CF II) with and without Nab4p/Hrp1p.
- Concentration-dependent studies of Nab4p/Hrp1p addition to assess its effect on cleavage patterns.
Main Results:
- CF IA and CF II, when devoid of Nab4p/Hrp1p, were capable of cleaving RNA substrates.
- Cleavage by CF IA and CF II alone occurred at multiple, non-specific sites.
- The addition of Nab4p/Hrp1p inhibited alternative cleavage events in a concentration-dependent manner, promoting specific cleavage.
Conclusions:
- Nab4p/Hrp1p is not essential for the endonucleolytic cleavage activity of CF IA and CF II.
- Nab4p/Hrp1p plays a crucial role in ensuring accurate pre-mRNA 3' end cleavage site selection.
- This suggests a conserved function for hnRNPs in regulating RNA processing events across eukaryotes.