Phillips, M. | bioRxiv Science

bioRxiv2019

Regulation of KRAS4A/B splicing in cancer stem cells by the RBM39 splicing complex.

The KRAS oncogene is expressed as major KRAS4B and minor KRAS4A splice isoforms, the distinct functions of which in cancer are unknown. We demonstrate here that KRAS4A is enriched in cancer stem-like cells, and is activated by hypoxia, whereas KRAS4B is more widely expressed and responds to ER stress. Mice completely lacking either isoform are viable but resistant to lung cancer development, as are Kras4A/B double heterozygous mice expressing both isoforms, but in which splice regulation has been uncoupled. Splicing of KRAS4A, but not KRAS4B, in human tumor cells can be inhibited by treatment with the splice inhibitor indisulam, or by CRISPR/Cas inhibition of the RBM39 splicing complex. Our data suggest that control of KRAS4A/B splicing is a targetable vulnerability in KRAS mutant tumors.

cancer biology↗

bioRxiv2019

Comprehensive analysis of the internal structure and firmness in American cranberry (Vaccinium macrocarpon Ait.) fruit

Cranberry (Vaccinium macrocarpon Ait.) fruit quality traits encompass many properties. Among these, fruit firmness has become a quality standard due to the recent demand increase for sweetened and dried cranberries (SDC). Traditionally, this trait has been measured by the cranberry industry using compression tests; however, it is poorly understood how fruit firmness is influenced by other characteristics. In this study, we developed a high-throughput computer-vision method to measure the internal structure of cranberry fruit, which may in turn influence cranberry fruit firmness. We measured the internal structure of 16 cranberry cultivars measured over a 40-day period. Internal structure data paired with fruit firmness values at each evaluation period allowed us to explore the correlations between firmness and internal morphological characteristics.

plant biology↗

bioRxiv2018

Structure and mechanism of TagA, a novel membrane-associated glycosyltransferase that produces wall teichoic acids in pathogenic bacteria

Staphylococcus aureus and other bacterial pathogens affix wall teichoic acids (WTAs) to their surface. These highly abundant anionic glycopolymers have critical functions in bacterial physiology and their susceptibility to {beta}-lactam antibiotics. The membrane-associated TagA glycosyltranserase (GT) catalyzes the first-committed step in WTA biosynthesis and is a founding member of the WecB/TagA/CpsF GT family, more than 6,000 enzymes that synthesize a range of extracellular polysaccharides through a poorly understood mechanism. Crystal structures of TagA from T. italicus in its apo- and UDP-bound states reveal a novel GT fold, and coupled with biochemical and cellular data define the mechanism of catalysis. We propose that enzyme activity is regulated by interactions with the bilayer, which trigger a structural change that facilitates proper active site formation and recognition of the enzymes lipid-linked substrate. These findings inform upon the molecular basis of WecB/TagA/CpsF activity and could guide the development of new anti-microbial drugs.\n\nAUTHOR SUMMARYGram-positive bacteria cause thousands of deaths in the United States each year and are a growing health concern because many bacterial strains have become resistant to commonly used antibiotics. One of the most abundant polymers displayed on the surface of Gram-positive bacteria is wall teichoic acid (WTA), a negatively charged carbohydrate polymer that has critical functions in cell division, morphology, adhesion and pathogenesis. The WTA biosynthetic pathway has drawn significant interest as a drug target because clinically important methicillin-resistant S. aureus (MRSA) strains that lack WTA are defective in host colonization and re-sensitized to {beta}-lactam antibiotics. To understand how bacteria produce WTA, we determined the structure and deduced the enzymatic mechanism of TagA, an important enzyme that is required for WTA synthesis. This research reveals a new method for enzyme regulation, whereby peripheral membrane association enables TagA to adopt its active form as a monomer. As TagA enzymes are highly conserved in bacteria, they can be expected to operate through a similar mechanism. The results of this work provide insight into WTA biosynthesis and could lead to innovative approaches to treat infections caused by pathogenic bacteria.

biochemistry↗