The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli.

Small proteins (<50 amino acids) are emerging as ubiquitous and important regulators in organisms ranging from bacteria to humans, where they commonly bind to and regulate larger proteins during stress responses. However, fundamental aspects of small proteins, such as their molecular mechanism of action, downregulation after they are no longer needed, and their evolutionary provenance, are poorly understood. Here, we show that the MntS small protein involved in manganese (Mn) homeostasis binds and inhibits the MntP Mn transporter. Mn is crucial for bacterial survival in stressful environments but is toxic in excess. Thus, Mn transport is tightly controlled at multiple levels to maintain optimal Mn levels. The small protein MntS adds a new level of regulation for Mn transporters, beyond the known transcriptional and post-transcriptional control. We also found that MntS binds to itself in the presence of Mn, providing a possible mechanism of downregulating MntS activity to terminate its inhibition of MntP Mn export. MntS is homologous to the signal peptide of SitA, the periplasmic metal-binding subunit of a Mn importer. Remarkably, the homologous signal peptide regions can substitute for MntS, demonstrating a functional relationship between MntS and these signal peptides. Conserved gene neighborhoods support that MntS evolved from the signal peptide of an ancestral SitA protein, acquiring a life of its own with a distinct function in Mn homeostasis.

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.

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.

Structure-function analysis of manganese exporter proteins across bacteria.

Manganese (Mn) is an essential trace nutrient for organisms because of its role in cofactoring enzymes and providing protection against reactive oxygen species (ROS). Many bacteria require manganese to form pathogenic or symbiotic interactions with eukaryotic host cells. However, excess manganese is toxic, requiring cells to have manganese export mechanisms. Bacteria are currently known to possess two widely distributed classes of manganese export proteins, MntP and MntE, but other types of transporters likely exist. Moreover, the structure and function of MntP is not well understood. Here, we characterized the role of three structurally related proteins known or predicted to be involved in manganese transport in bacteria from the MntP, UPF0016, and TerC families. These studies used computational analysis to analyze phylogeny and structure, physiological assays to test sensitivity to high levels of manganese and ROS, and inductively coupled plasma-mass spectrometry (ICP-MS) to measure metal levels. We found that MntP alters cellular resistance to ROS. Moreover, we used extensive computational analyses and phenotypic assays to identify amino acids required for MntP activity. These negatively charged residues likely serve to directly bind manganese and transport it from the cytoplasm through the membrane. We further characterized two other potential manganese transporters associated with a Mn-sensing riboswitch and found that the UPF0016 family of proteins has manganese export activity. We provide here the first phenotypic and biochemical evidence for the role of Alx, a member of the TerC family, in manganese homeostasis. It does not appear to export manganese, but rather it intriguingly facilitates an increase in intracellular manganese concentration. These findings expand the available knowledge about the identity and mechanisms of manganese homeostasis proteins across bacteria and show that proximity to a Mn-responsive riboswitch can be used to identify new components of the manganese homeostasis machinery.

Mechanisms and regulation of spliceosome-mediated pre-mRNA splicing in Saccharomyces cerevisiae.

Pre-mRNA splicing, the removal of introns and ligation of flanking exons, is a crucial step in eukaryotic gene expression. The spliceosome, a macromolecular complex made up of five small nuclear RNAs (snRNAs) and dozens of proteins, assembles on introns via a complex pathway before catalyzing the two transesterification reactions necessary for splicing. All of these steps have the potential to be highly regulated to ensure correct mRNA isoform production for proper cellular function. While Saccharomyces cerevisiae (yeast) has a limited set of intron-containing genes, many of these genes are highly expressed, resulting in a large number of transcripts in a cell being spliced. As a result, splicing regulation is of critical importance for yeast. Just as in humans, yeast splicing can be influenced by protein components of the splicing machinery, structures and properties of the pre-mRNA itself, or by the action of trans-acting factors. It is likely that further analysis of the mechanisms and pathways of splicing regulation in yeast can reveal general principles applicable to other eukaryotes. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing.

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 (1st and 2nd step factors). We recently identified Fyv6 (FAM192A in humans) as a 2nd 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 2nd step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the 1st step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv6Δ 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 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.

Age-related differences in the social associative learning of trust information.

Trust is a key component of social interaction. Older adults, however, often exhibit excessive trust relative to younger adults. One explanation is that older adults may learn to trust differently than younger adults. Here, we examine how younger (N = 33) and older adults (N = 30) learn to trust over time. Participants completed a classic iterative trust game with 3 partners. Younger and older adults shared similar amounts but differed in how they shared money. Compared to younger adults, older adults invested more with untrustworthy partners and less with trustworthy partners. As a group, older adults displayed less learning than younger adults. However, computational modeling suggests that this is not because older adults learn differently from positive and negative feedback than younger adults. Model-based fMRI analyses revealed several age- and learning-related differences in neural processing. Specifically, we found that older learners (N = 19), relative to older non-learners (N = 11), had greater reputation-related activity in metalizing/memory areas while making their decisions. Collectively, these findings suggest that older adult learners use social cues differently from non-learners.

The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli.

Small proteins (< 50 amino acids) are emerging as ubiquitous and important regulators in organisms ranging from bacteria to humans, where they commonly bind to and regulate larger proteins during stress responses. However, fundamental aspects of small proteins, such as their molecular mechanism of action, downregulation after they are no longer needed, and their evolutionary provenance are poorly understood. Here we show that the MntS small protein involved in manganese (Mn) homeostasis binds and inhibits the MntP Mn transporter. Mn is crucial for bacterial survival in stressful environments, but is toxic in excess. Thus, Mn transport is tightly controlled at multiple levels to maintain optimal Mn levels. The small protein MntS adds a new level of regulation for Mn transporters, beyond the known transcriptional and post-transcriptional control. We also found that MntS binds to itself in the presence of Mn, providing a possible mechanism of downregulating MntS activity to terminate its inhibition of MntP Mn export. MntS is homologous to the signal peptide of SitA, the periplasmic metal-binding subunit of a Mn importer. Remarkably, the homologous signal peptide regions can substitute for MntS, demonstrating a functional relationship between MntS and these signal peptides. Conserved gene-neighborhoods support that MntS evolved from an ancestral SitA, acquiring a life of its own with a distinct function in Mn homeostasis. Significance: This study demonstrates that the MntS small protein binds and inhibits the MntP Mn exporter, adding another layer to the complex regulation of Mn homeostasis. MntS also interacts with itself in cells with Mn, which could prevent it from regulating MntP. We propose that MntS and other small proteins might sense environmental signals and shut off their own regulation via binding to ligands (e.g., metals) or other proteins. We also provide evidence that MntS evolved from the signal peptide region of the Mn importer, SitA. Homologous SitA signal peptides can recapitulate MntS activities, showing that they have a second function beyond protein secretion. Overall, we establish that small proteins can emerge and develop novel functionalities from gene remnants.

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.