Galectin-3 does not interact with RNA directly.

Area of Science:

• Molecular Biology
• Glycobiology
• RNA Biology

Background:

• Galectin-3, a known glycan-binding protein, was proposed as a dual-function protein with RNA-binding capabilities.
• This putative role suggested a link between glycobiology and RNA biology, particularly in pre-mRNA splicing.
• Previous evidence relied on affinity reagents lacking genetic validation, leaving direct RNA interaction unconfirmed.

Purpose of the Study:

• To investigate the direct RNA-binding capacity of galectin-3.
• To determine if galectin-3 interacts with RNA in cellular processes like pre-mRNA maturation.
• To clarify the molecular basis of previously observed RNA-protein associations involving galectin-3.

Main Methods:

• Utilized antibodies against endogenous human galectin-3 for immunoprecipitation (IP) of RNA-protein crosslinks.
• Performed proteomic analysis of IP isolates.
• Generated LGALS3 knock-out and HNRNPA2B1 knock-out cell lines.
• Introduced an epitope tag to the endogenous C-terminal locus of LGALS3 for targeted isolation.

Main Results:

• Anti-galectin-3 antibodies isolated RNA-protein crosslinks, but this was independent of LGALS3 gene expression.
• Proteomic analysis revealed hnRNPA2B1, an RNA-binding protein, was highly abundant in isolates.
• Genetic ablation of HNRNPA2B1, but not LGALS3, abolished RNA-protein crosslink isolation.
• Isolation of epitope-tagged galectin-3 did not yield RNA-protein crosslinks.

Conclusions:

• Galectin-3 does not appear to directly interact with RNA.
• Observed RNA associations were likely due to cross-reactivity or indirect interactions involving hnRNPA2B1.
• The identification of galectin-3 as an RNA-binding protein may be a misinterpretation, particularly in HeLa cells.
• Recommends using gene deletions and endogenous epitope tags for precise evaluation of protein-RNA interactions.