Biology subjects

Sze, S.-H.

Publications and source records attributed to Sze, S.-H..

5 recordsLinked to original sources

bioRxiv2024

Higher-order epistasis within Pol II trigger loop haplotypes

RNA polymerase II (Pol II) has a highly conserved domain, the trigger loop (TL), that controls transcription fidelity and speed. We previously probed pairwise genetic interactions between residues within and surrounding the TL for the purpose of understand functional interactions between residues and to understand how individual mutants might alter TL function. We identified widespread incompatibility between TLs of different species when placed in the Saccharomyces cerevisiae Pol II context, indicating species-specific interactions between otherwise highly conserved TLs and its surroundings. These interactions represent epistasis between TL residues and the rest of Pol II. We sought to understand why certain TL sequences are incompatible with S. cerevisiae Pol II and to dissect the nature of genetic interactions within multiply substituted TLs as a window on higher order epistasis in this system. We identified both positive and negative higher-order residue interactions within example TL haplotypes. Intricate higher-order epistasis formed by TL residues was sometimes only apparent from analysis of intermediate genotypes, emphasizing complexity of epistatic interactions. Furthermore, we distinguished TL substitutions with distinct classes of epistatic patterns, suggesting specific TL residues that potentially influence TL evolution. Our examples of complex residue interactions suggest possible pathways for epistasis to facilitate Pol II evolution.

genetics↗

bioRxiv2023

Widespread epistasis shapes RNA Polymerase II active site function and evolution

Multi-subunit RNA Polymerases (msRNAPs) are responsible for transcription in all kingdoms of life. These enzymes rely on dynamic, highly conserved active site domains such as the so-called "trigger loop" (TL) to accomplish steps in the transcription cycle. Mutations in the RNA polymerase II (Pol II) TL confer a spectrum of biochemical and genetic phenotypes that suggest two main classes, which decrease or increase catalysis or other nucleotide addition cycle (NAC) events. The Pol II active site relies on networks of residue interactions to function, and mutations likely perturb these networks in ways that may alter mechanisms. We have undertaken a structural genetics approach to reveal residue interactions within and surrounding the Pol II TL - determining its "interaction landscape" - by deep mutational scanning in Saccharomyces cerevisiae Pol II. This analysis reveals connections between TL residues and surrounding domains, demonstrating that TL function is tightly coupled to its specific enzyme context.

genetics↗

bioRxiv2021

Diverse pathogens activate the host RIDD pathway to subvert BLOS1-directed immune defense

The phagocytosis and destruction of pathogens in lysosomes constitute central elements of innate immune defense. Here, we show that Brucella, the causative agent of brucellosis, the most prevalent bacterial zoonosis globally, subverts this immune defense pathway by activating regulated IRE1-dependent decay (RIDD) of mRNAs encoding BLOS1, a protein that promotes endosome-lysosome fusion. RIDD-deficient cells and mice harboring a RIDD-incompetent variant of IRE1 were resistant to infection. Non-functional Blos1 struggled to assemble the BLOC-1-related complex (BORC), resulting in differential recruitment of BORC-related lysosome trafficking components, perinuclear trafficking of Brucella-containing vacuoles (BCVs), and enhanced susceptibility to infection. The RIDD-resistant Blos1 variant maintains the integrity of BORC and a higher-level association of BORC-related components that promote centrifugal lysosome trafficking, resulting in enhanced BCV peripheral trafficking and lysosomal-destruction, and resistance to infection. These findings demonstrate that host RIDD activity on BLOS1 regulates Brucella intracellular parasitism by disrupting BORC-directed lysosomal trafficking. Notably, coronavirus MHV also subverted the RIDD-BLOS1 axis to promote intracellular replication. Our work therefore establishes BLOS1 as a novel immune defense factor whose activity is hijacked by diverse pathogens.

microbiology↗

bioRxiv2019

Promoter scanning during transcription initiation in Saccharomyces cerevisiae: Pol II in the \"shooting gallery\"

BackgroundThe majority of eukaryotic promoters utilize multiple transcription start sites (TSSs). How multiple TSSs are specified at individual promoters across eukaryotes is not understood for most species. In S. cerevisiae, a preinitiation complex comprised of Pol II and conserved general transcription factors (GTFs) assembles and opens DNA upstream of TSSs. Evidence from model promoters indicates that the preinitiation complex (PIC) scans from upstream to downstream to identify TSSs. Prior results suggest that TSS distributions at promoters where scanning occurs shift in a polar fashion upon alteration in Pol II catalytic activity or GTF function. ResultsTo determine extent of promoter scanning across promoter classes in S. cerevisiae, we perturbed Pol II catalytic activity and GTF function and analyzed their effects on TSS usage genome-wide. We find that alterations to Pol II, TFIIB, or TFIIF function widely alter the initiation landscape consistent with promoter scanning operating at all yeast promoters, regardless of promoter class. Promoter architecture, however, can determine extent of promoter sensitivity to altered Pol II activity in ways that are predicted by a scanning model. ConclusionsOur observations coupled with previous data validate key predictions of the scanning model for Pol II initiation in yeast - which we term the "shooting gallery". In this model, Pol II catalytic activity, and the rate and processivity of Pol II scanning together with promoter sequence determine the distribution of TSSs and their usage.

molecular biology↗

bioRxiv2019

Functional genomics of the stable fly, Stomoxys calcitrans, reveals mechanisms underlying reproduction, host interactions, and novel targets for pest control

BackgroundThe stable fly, Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the United States alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies.\n\nResultsThis study examines stable fly biology by utilizing a combination of high-quality genome sequencing, microbiome analyses, and RNA-seq analyses targeting multiple developmental stages and tissues. In conjunction, manual curation of over 1600 genes was used to examine gene content related to stable fly reproduction, interactions with their host, host-microbe dynamics, and putative routes for control. Most notable was establishment of reproduction-associated genes and identification of expanded vision, chemosensation, immune repertoire, and metabolic detoxification pathway gene families.\n\nConclusionsThe combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and knowledge will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship of Stomoxys to other blood-feeding (Glossina) and non-blood-feeding flies (medflies, Drosophila, house flies) will allow for understanding the evolution of blood feeding among Cyclorrhapha flies.

genomics↗