Galectin-3 does not interact with RNA directly.

Galectin-3, well characterized as a glycan binding protein, has been identified as a putative RNA binding protein, possibly through participation in pre-mRNA maturation through interactions with splicosomes. Given recent developments with cell surface RNA biology, the putative dual-function nature of galectin-3 evokes a possible non-classical connection between glycobiology and RNA biology. However, with limited functional evidence of a direct RNA interaction, many molecular-level observations rely on affinity reagents and lack appropriate genetic controls. Thus, evidence of a direct interaction remains elusive. We demonstrate that antibodies raised to endogenous human galectin-3 can isolate RNA-protein crosslinks, but this activity remains insensitive to LGALS3 knock-out. Proteomic characterization of anti-galectin-3 IPs revealed enrichment of galectin-3, but high abundance of hnRNPA2B1, an abundant, well-characterized RNA-binding protein with weak homology to the N-terminal domain of galectin-3, in the isolate. Genetic ablation of HNRNPA2B1, but not LGALS3, eliminates the ability of the anti-galectin-3 antibodies to isolate RNA-protein crosslinks, implying either an indirect interaction or cross-reactivity. To address this, we introduced an epitope tag to the endogenous C-terminal locus of LGALS3. Isolation of the tagged galectin-3 failed to reveal any RNA-protein crosslinks. This result suggests that the galectin-3 does not directly interact with RNA and may be misidentified as an RNA-binding protein, at least in HeLa where the putative RNA associations were first identified. We encourage further investigation of this phenomenon employ gene deletions and, when possible, endogenous epitope tags to achieve the specificity required to evaluate potential interactions.

The INO80 chromatin remodeler sustains metabolic stability by promoting TOR signaling and regulating histone acetylation.

Chromatin remodeling complexes are essential for gene expression programs that coordinate cell function with metabolic status. However, how these remodelers are integrated in metabolic stability pathways is not well known. Here, we report an expansive genetic screen with chromatin remodelers and metabolic regulators in Saccharomyces cerevisiae. We found that, unlike the SWR1 remodeler, the INO80 chromatin remodeling complex is composed of multiple distinct functional subunit modules. We identified a strikingly divergent genetic signature for the Ies6 subunit module that links the INO80 complex to metabolic homeostasis. In particular, mitochondrial maintenance is disrupted in ies6 mutants. INO80 is also needed to communicate TORC1-mediated signaling to chromatin, as ino80 mutants exhibit defective transcriptional profiles and altered histone acetylation of TORC1-responsive genes. Furthermore, comparative analysis reveals subunits of INO80 and mTORC1 have high co-occurrence of alterations in human cancers. Collectively, these results demonstrate that the INO80 complex is a central component of metabolic homeostasis that influences histone acetylation and may contribute to disease when disrupted.

A Genome-Wide CRISPR Screen Identifies Sortilin as the Receptor Responsible for Galectin-1 Lysosomal Trafficking.

Galectins are a family of mammalian glycan-binding proteins that have been implicated as regulators of myriad cellular processes including cell migration, apoptosis, and immune modulation. Several members of this family, such as galectin-1, exhibit both cell-surface and intracellular functions. Interestingly, galectin-1 can be found in the endomembrane system, nucleus, or cytosol, as well as on the cell surface. The mechanisms by which galectin-1 traffics between cellular compartments, including its unconventional secretion and internalization processes, are poorly understood. Here, we determined the pathways by which exogenous galectin-1 enters cells and explored its capacity as a delivery vehicle for protein and siRNA therapeutics. We used a galectin-1-toxin conjugate, modelled on antibody-drug conjugates, as a selection tool in a genome-wide CRISPR screen. We discovered that galectin-1 interacts with the endosome-lysosome trafficking receptor sortilin in a glycan-dependent manner, which regulates galectin-1 trafficking to the lysosome. Further, we show that this pathway can be exploited for delivery of a functional siRNA. This study sheds light on the mechanisms by which galectin-1 is internalized by cells and suggests a new strategy for intracellular drug delivery via galectin-1 conjugation.

The microenvironment dictates glycocalyx construction and immune surveillance.

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium.