Widespread DNA off-targeting confounds RNA chromatin occupancy studies

Area of Science:

• Molecular Biology
• Genomics
• RNA Biology

Background:

• High-throughput sequencing methods like ChIRP-seq, CHART-seq, and RAP-seq are used to map long noncoding RNA (lncRNA) genome-wide chromatin occupancy.
• Observed datasets frequently report thousands of binding sites, which is inconsistent with the low endogenous abundance of most lncRNAs.

Purpose of the Study:

• To investigate the chromatin interactome of the NESPR lncRNA across varying expression levels.
• To conduct a meta-analysis of existing RNA-chromatin interaction datasets in human and mouse cells.
• To identify potential sources of artifact in lncRNA chromatin occupancy studies.

Main Methods:

• Studied the chromatin interactome of NESPR lncRNA in cells with different endogenous expression levels.
• Performed a meta-analysis of dozens of RNA-chromatin interaction datasets from human and mouse.
• Analyzed DNA fragment complementarity to probes as a source of spurious recovery.

Main Results:

• Thousands of reported lncRNA binding sites are likely artifacts arising from spurious DNA element recovery.
• Spurious recovery is linked to partial complementarity between DNA fragment ends and sequencing probes.
• Crucial control experiments were infrequently implemented in prior studies.
• Most reported trans-acting RNA-chromatin interactions in mammals appear to be technical artifacts.

Conclusions:

• The majority of previously identified lncRNA-chromatin binding sites are likely artifacts.
• Re-evaluation of RNA-chromatin interaction data quality is necessary.
• Recommendations are provided for improving and assessing the reliability of RNA-chromatin datasets.