Integrase mediates nuclear localization of Ty3.

Area of Science:

• Molecular Biology
• Yeast Genetics
• Retroviral Research

Background:

• Retroviruses and yeast retrotransposons require nuclear entry for integration.
• The Ty3 integrase (IN) protein's carboxyl-terminal domain contains a bipartite basic motif crucial for transposition.
• Previous studies indicated mutations in this motif block transposition post-3'-end processing.

Purpose of the Study:

• To investigate the role of the bipartite basic motif in Ty3 integrase (IN) nuclear localization and function.
• To determine if nuclear entry is essential for forming active Ty3 virus-like particles (VLPs).
• To elucidate the target sites of Ty3 integration within the yeast genome.

Main Methods:

• Targeting green fluorescent protein to the nucleolus using Ty3 IN.
• Assessing the localization of a heterologous protein fused to the motif-containing region.
• Evaluating the activity of Ty3 virus-like particles (VLPs) defective for nuclear localization in an in vitro integration assay.
• Employing in situ hybridization and PCR to identify Ty3 insertion sites.

Main Results:

• The Ty3 IN protein targets green fluorescent protein to the nucleolus; mutations in the bipartite basic motif abolish this localization.
• The motif-containing region mediates nuclear but not subnuclear localization of a heterologous protein.
• Ty3 elements defective for nuclear localization form inactive VLPs, suggesting nuclear entry is required for VLP maturation or function.
• Ty3 elements insert at transcription initiation sites of tRNA, 5S, and U6 RNA genes, with a notable preference for 5S genes located near ribosomal DNA (rDNA) and the nucleolus.

Conclusions:

• The bipartite basic motif in Ty3 integrase (IN) is essential for nucleolar localization and likely plays a critical role in nuclear import or subsequent steps required for transposition.
• Nuclear entry is a prerequisite for the formation of active Ty3 virus-like particles (VLPs).
• Ty3 retrotransposons exhibit specific integration site preferences, targeting RNA polymerase III-transcribed genes, including 5S RNA genes associated with the nucleolus.