An APEX2 proximity ligation method for mapping interactions with the nuclear lamina

Area of Science:

• Cell Biology
• Molecular Biology
• Genomics

Background:

• The nuclear lamina (NL) is a structural meshwork beneath the inner nuclear membrane.
• Studying the NL is challenging due to its insolubility, necessitating advanced proximity ligation techniques.
• Previous methods required complex proximity ligation to identify NL-associated molecules.

Purpose of the Study:

• To develop a simplified and temporally controlled method for mapping proteins, RNA, and DNA associated with the nuclear lamina.
• To identify novel NL-interacting RNAs and proteins and characterize their functions.
• To investigate the dynamics of lamina-associated domains (LADs) during the cell cycle.

Main Methods:

• Fusion of APEX2 (an enzyme) to the NL protein lamin-B1 for proximity-dependent labeling.
• APEX2-based proteomics and transcriptomics to identify proximal molecules.
• Genome-wide mapping of lamina-associated domains (LADs) using APEX2.

Main Results:

• Identified NL-interacting RNAs with a bias for long 3' UTRs, correlating with deregulated genes in lamin-null cells.
• Discovered a C-rich motif in 3' UTRs and identified a regulatory protein binding this motif, with altered localization in lamin-null cells.
• Mapped cell cycle-dependent LADs, revealing short, H3K27me3-rich variable domains.

Conclusions:

• APEX2-based tools offer a simplified and effective approach for identifying proteomes, transcriptomes, and genome elements proximal to the nuclear lamina.
• The findings provide new insights into RNA regulation, protein localization, and chromatin organization at the nuclear periphery.
• This study advances the understanding of nuclear lamina functions in gene regulation and cellular architecture.