Control of 3' splice site selection by the yeast splicing factor Fyv6.

Pre-mRNA splicing is catalyzed in two steps: 5' splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (1 st and 2 nd step factors). We recently identified Fyv6 (FAM192A in humans) as a 2 nd step factor in S. cerevisiae ; however, we did not determine how widespread Fyv6's impact is on the transcriptome. To answer this question, we have used RNA-seq to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3' SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-EM structure of a yeast product complex spliceosome containing Fyv6 at 2.3 Å. The structure reveals that Fyv6 is the only 2 nd step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the 1 st step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv61 suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of consensus, BP distal 3' SS.

Biochemical and genetic evidence supports Fyv6 as a second-step splicing factor in Saccharomyces cerevisiae.

Precursor mRNA (pre-mRNA) splicing is an essential process for gene expression in eukaryotes catalyzed by the spliceosome in two transesterification steps. The spliceosome is a large, highly dynamic complex composed of five small nuclear RNAs and dozens of proteins, some of which are needed throughout the splicing reaction while others only act during specific stages. The human protein FAM192A was recently proposed to be a splicing factor that functions during the second transesterification step, exon ligation, based on analysis of cryo-electron microscopy (cryo-EM) density. It was also proposed that Fyv6 might be the Saccharomyces cerevisiae functional and structural homolog of FAM192A; however, no biochemical or genetic data has been reported to support this hypothesis. Herein, we show that Fyv6 is a splicing factor and acts during exon ligation. Deletion of FYV6 results in genetic interactions with the essential splicing factors Prp8, Prp16, and Prp22 and decreases splicing in vivo of reporter genes harboring intron substitutions that limit the rate of exon ligation. When splicing is assayed in vitro, whole-cell extracts lacking Fyv6 accumulate first-step products and exhibit a defect in exon ligation. Moreover, loss of Fyv6 causes a change in 3' splice site (SS) selection in both a reporter gene and the endogenous SUS1 transcript in vivo. Together, these data suggest that Fyv6 is a component of the yeast spliceosome that influences 3' SS usage and the potential homolog of human FAM192A.

Biochemical and Genetic Evidence Supports Fyv6 as a Second-Step Splicing Factor in Saccharomyces cerevisiae.

Precursor mRNA (pre-mRNA) splicing is an essential process for gene expression in eukaryotes catalyzed by the spliceosome in two transesterification steps. The spliceosome is a large, highly dynamic complex composed of 5 small nuclear RNAs and dozens of proteins, some of which are needed throughout the splicing reaction while others only act during specific stages. The human protein FAM192A was recently proposed to be a splicing factor that functions during the second transesterification step, exon ligation, based on analysis of cryo-electron microscopy (cryo-EM) density. It was also proposed that Fyv6 might be the functional S. cerevisiae homolog of FAM192A; however, no biochemical or genetic data has been reported to support this hypothesis. Herein, we show that Fyv6 is a splicing factor and acts during exon ligation. Deletion of FYV6 results in genetic interactions with the essential splicing factors Prp8, Prp16, and Prp22; decreases splicing in vivo of reporter genes harboring intron substitutions that limit the rate of exon ligation; and changes 3’ splice site (SS) selection. Together, these data suggest that Fyv6 is a component of the spliceosome and the potential functional and structural homolog of human FAM192A.

Alternative Splicing and Cleavage of GLUT8.

The GLUT (SLC2) family of membrane-associated transporters are described as glucose transporters. However, this family is divided into three classes and, though the regulated transporter activity of class I proteins is becoming better understood, class III protein functions continue to be obscure. We have cataloged the relative expression and splicing of SLC2 mRNA isomers in tumors and normal tissues, with a focus on breast tumors and cell lines. mRNA for the class III protein GLUT8 is the predominant SLC2 species expressed alongside GLUT1 in many tissues, but GLUT8 mRNA exists mostly as an untranslated splice form in tumors. We confirm that GLUT8 is not presented at the cell surface and does not transport glucose directly. However, we reveal a lysosome-dependent reaction that cleaves the GLUT8 protein and releases the carboxy-terminal peptide to a separate vesicle population. Given the localization of GLUT8 at a major metabolic hub (the late endosomal/lysosomal interface) and its regulated cleavage reaction, we evaluated TXNIP-mediated hexosamine homeostasis and speculate that GLUT8 may function as a sensory component of this reaction.

Experimental Evolution of Extreme Resistance to Ionizing Radiation in Escherichia coli after 50 Cycles of Selection.

In previous work (D. R. Harris et al., J Bacteriol 191:5240-5252, 2009, https://doi.org/10.1128/JB.00502-09; B. T. Byrne et al., Elife 3:e01322, 2014, https://doi.org/10.7554/eLife.01322), we demonstrated that Escherichia coli could acquire substantial levels of resistance to ionizing radiation (IR) via directed evolution. Major phenotypic contributions involved adaptation of organic systems for DNA repair. We have now undertaken an extended effort to generate E. coli populations that are as resistant to IR as Deinococcus radiodurans After an initial 50 cycles of selection using high-energy electron beam IR, four replicate populations exhibit major increases in IR resistance but have not yet reached IR resistance equivalent to D. radiodurans Regular deep sequencing reveals complex evolutionary patterns with abundant clonal interference. Prominent IR resistance mechanisms involve novel adaptations to DNA repair systems and alterations in RNA polymerase. Adaptation is highly specialized to resist IR exposure, since isolates from the evolved populations exhibit highly variable patterns of resistance to other forms of DNA damage. Sequenced isolates from the populations possess between 184 and 280 mutations. IR resistance in one isolate, IR9-50-1, is derived largely from four novel mutations affecting DNA and RNA metabolism: RecD A90E, RecN K429Q, and RpoB S72N/RpoC K1172I. Additional mechanisms of IR resistance are evident.IMPORTANCE Some bacterial species exhibit astonishing resistance to ionizing radiation, with Deinococcus radiodurans being the archetype. As natural IR sources rarely exceed mGy levels, the capacity of Deinococcus to survive 5,000 Gy has been attributed to desiccation resistance. To understand the molecular basis of true extreme IR resistance, we are using experimental evolution to generate strains of Escherichia coli with IR resistance levels comparable to Deinococcus Experimental evolution has previously generated moderate radioresistance for multiple bacterial species. However, these efforts could not take advantage of modern genomic sequencing technologies. In this report, we examine four replicate bacterial populations after 50 selection cycles. Genomic sequencing allows us to follow the genesis of mutations in populations throughout selection. Novel mutations affecting genes encoding DNA repair proteins and RNA polymerase enhance radioresistance. However, more contributors are apparent.