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Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution
Published on: January 16, 2017
RNA-dependent recruitment of the origin recognition complex
Julie Norseen1, Andreas Thomae, Venkatesh Sridharan
1Program in Gene Expression and Regulation, The Wistar Institute, Philadelphia, PA 19104, USA.
The EMBO Journal
|October 24, 2008
Summary
The origin recognition complex (ORC) is recruited to DNA replication origins via RNA-dependent mechanisms involving EBNA1 and HMGA1a proteins. This suggests structural RNAs play a key role in mediating ORC binding at cellular and viral replication sites.
Area of Science:
- Molecular Biology
- Epigenetics
- Virology
Background:
- The origin recognition complex (ORC) is crucial for DNA replication initiation.
- Mechanisms of ORC recruitment and origin determination in higher eukaryotes are not fully understood due to ORC's lack of sequence-specific DNA binding.
- The Epstein-Barr virus origin of plasmid replication (OriP) serves as a model for studying ORC recruitment via the viral EBNA1 protein.
Purpose of the Study:
- To investigate the role of EBNA1's RGG-like motifs (LR1 and LR2) in ORC recruitment to OriP.
- To determine the involvement of RNA in the ORC recruitment process mediated by EBNA1 and HMGA1a.
- To explore the potential role of RNA in stabilizing ORC association with cellular chromatin and replication origins.
Main Methods:
- Investigated the function of EBNA1's LR1 and LR2 motifs in ORC recruitment and DNA replication.
- Assessed the RNA dependency of ORC recruitment by EBNA1 and HMGA1a.
- Analyzed the binding of EBNA1, HMGA1a RGG motifs, and ORC1 peptides to structured G-rich RNA.
- Utilized RNase A treatment to examine the effect of RNA degradation on ORC association with cellular chromatin.
Main Results:
- EBNA1's LR1 and LR2 motifs are essential for ORC recruitment to OriP and subsequent DNA replication.
- ORC recruitment by both EBNA1 and the functionally similar HMGA1a protein is dependent on RNA.
- EBNA1, HMGA1a RGG motifs, and ORC1 peptides demonstrate binding to structured G-rich RNA.
- RNase A treatment released a fraction of ORC from cellular chromatin, indicating RNA stabilizes ORC-chromatin interactions.
Conclusions:
- Structural RNA molecules are critical mediators of ORC recruitment to the Epstein-Barr virus OriP.
- RNA-dependent mechanisms, involving proteins like EBNA1 and HMGA1a, are likely involved in ORC recruitment to cellular DNA replication origins.
- RNA plays a significant role in stabilizing the association of ORC with chromatin, potentially at cellular replication origins.
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