Biology subjects

Sawyer, E. M.

Publications and source records attributed to Sawyer, E. M..

3 recordsLinked to original sources

bioRxiv2023

A Flat Protein Complex Shapes Rough ER Membrane Sheets

Rough ER sheets are a fundamental domain of the ER and the gateway into the secretory pathway. While reticulon proteins stabilize high-curvature ER tubules, it is unclear if other proteins scaffold the flat membranes of rough ER sheets. Through a proteomics screen using ER sheet localized RNA-binding proteins as bait, we identify the Sigma-1 receptor (SigmaR1) as an ER sheet shaping factor. High-resolution live cell imaging and electron tomography assign SigmaR1 as an ER sheet-localized factor whose levels determine the amount of rough ER sheets in cells. Structure-guided mutagenesis and in vitro reconstitution on giant unilamellar vesicles further support a mechanism whereby SigmaR1 oligomers use their extended arrays of amphipathic helices to bind and flatten the lumenal leaflet of ER membranes. Our results demonstrate an unexpected way for proteins to sense and propagate flat membrane sheets.

cell biology↗

bioRxiv2023

Diverse viral cas genes antagonize CRISPR immunity

Prokaryotic CRISPR-Cas immunity is subverted via anti-CRISPRs (Acrs), small proteins that inhibit Cas protein activities when expressed during the phage lytic cycle or from resident prophages or plasmids. CRISPR-Cas defenses are classified into 6 types and 33 subtypes, which employ a diverse suite of Cas effectors and differ in their mechanisms of interference. As Acrs often work via binding to a cognate Cas protein, inhibition is almost always limited to a single CRISPR type. Furthermore, while acr genes are frequently organized together in phage-associated gene clusters, how such inhibitors initially evolve has remained unclear. Here we have investigated the Acr content and inhibition specificity of a collection of Listeria isolates, which naturally harbor four diverse CRISPR-Cas systems (types I-B, II-A, II-C, and VI-A). We observed widespread antagonism of CRISPR, which we traced to 12 novel and 4 known Acr gene families encoded on endogenous mobile genetic elements. Among these were two Acrs that possess sequence homology to type I-B Cas proteins and assemble into a defective interference complex. Surprisingly, an additional type I-B Cas homolog did not affect type I immunity, but instead inhibited the RNA-targeting type VI CRISPR system through sequestration of crRNA. By probing the IMGVR database of viral genomes, we detected abundant orphan cas genes located within putative anti-defense gene clusters. We experimentally verified the Acr activity of one viral cas gene, a particularly broad-spectrum cas3 homolog that inhibits type I-B, II-A, and VI-A CRISPR immunity. Our observations provide direct evidence of Acr evolution via cas gene co-option, and new genes with potential for broad-spectrum control of genome editing technologies.

microbiology↗

bioRxiv2018

Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster

Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis. Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogaster. Adh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter does also result in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, we show that transcription from the distal promoter is sufficient to repress proximal transcription in larvae and that the degree of this repression depends on the degree of distal promoter activity. Finally, repression of the endogenous Adh proximal promoter is associated with the enrichment of histone 3 lysine 36 trimethylation (H3K36me3), a chromatin mark necessary for transcription-coupled gene repression in yeast. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.

genetics↗