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Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

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Efficient Pre-mRNA Cleavage Prevents Replication-Stress-Associated Genome Instability

Federico Teloni¹, Jone Michelena², Aleksandra Lezaja¹

¹Department of Molecular Mechanisms of Disease, University of Zurich, 8057 Zurich, Switzerland; Life Science Zurich Graduate School (LSZGS), 8057 Zurich, Switzerland.

|January 15, 2019

Abstract:

Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability.

Insights

Cancer cells hijack genome integrity mechanisms. Researchers identified key regulators of replication stress resilience, revealing how pre-mRNA cleavage impacts DNA damage and genomic instability.

Area of Science:

Molecular Biology
Cancer Research
Genomics

Background:

Cellular mechanisms maintaining genome integrity are crucial and often dysregulated in cancer.
Identifying genes that protect the genome in cancer is vital for therapeutic strategies.

Purpose of the Study:

To identify novel cancer-related genome caretakers using a multi-screening approach.
To investigate the role of pre-mRNA cleavage and polyadenylation complex in maintaining genome stability under replication stress.

Main Methods:

Convergent multi-screening strategy combined with quantitative image-based cytometry.
Ranking candidate genes based on viability, proliferation, replisome integrity, and DNA damage signaling.
Assessing the impact of pre-mRNA cleavage deregulation on replication fork dynamics and DNA damage.

Main Results:

Identified regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex.
Deregulation of pre-mRNA cleavage impairs replication fork speed and increases origin activity, leading to ATR dependency.
Excessive RNA:DNA hybrid formation correlated with DNA damage; transcription inhibition rescued replication defects.
Uncoupling pre-mRNA cleavage from co-transcriptional processing protected against replication-stress-associated DNA damage.

Conclusions:

Pre-mRNA cleavage plays a critical role in managing replication stress and preventing genomic instability.
Dysregulation of pre-mRNA cleavage contributes to DNA damage in cancer by promoting RNA:DNA hybrid formation and altering replication dynamics.
Targeting pre-mRNA cleavage or its associated processes may offer novel therapeutic avenues for cancer treatment.

Read full analysis

Keywords:

ATR R-loops RNA:DNA hybrids checkpoint activation cleavage gene gating origin firing polyadenylation pre-mRNA processing replication catastrophe replication stress

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