Biology subjects

Marri, S.

Publications and source records attributed to Marri, S..

4 recordsLinked to original sources

bioRxiv2025

Gene library deep sequencing for protein super-family profiling

Unravelling protein function at scale remains challenging. Gain-of-function (GOF) screens harnessing large open reading frame (ORF) libraries enable systematic functional annotations of cell behavior drivers and drug targets. Currently, few sequencing methods are established to analyze existing ORF libraries and facilitate future library generation. Here, we developed a barcode-free deep sequencing method termed pooled plasmid library sequencing (PPLseq). PPLseq achieved high efficiency and single nucleotide accuracy, including the detection of previously undocumented variants, on a prototypical super-family ORF library (>300 human G-protein-coupled receptors (GPCRs)). We next combined PPLseq with high-throughput gene engineering to generate a new library of 246 GPCR-fluorescent protein (FP) fusions. We quantified expression of all members of this library and identified robustly expressed yet understudied receptors. Collectively, we demonstrate accessible, scalable, and sensitive ORF library sequencing towards a deeper understanding of proteome function.

synthetic biology↗

bioRxiv2023

Versatile toolkit for highly-efficient and scarless overexpression of circular RNAs

Circular RNAs (circRNAs) are a class of single-stranded, covalently closed RNA that contain a unique back-splice junction (bsj) sequence created by the ligation of their 5 and 3 ends via spliceosome-catalyzed back-splicing. A key step in illuminating the cellular roles of specific circRNAs is via increasing their expression. This is frequently done by transfecting cells with plasmid DNA containing cloned exons from which the circRNA is transcribed, flanked by sequences that promote back-splicing. We observed that commonly used plasmids lead to the production of circRNAs with molecular scars at the circRNA bsj. Stepwise redesign of the cloning vector corrected this problem, ensuring bona fide circRNAs are produced with their natural bsj at high efficiency. The fidelity of circRNAs produced from this new construct was validated by RNA sequencing and also functionally validated. To increase the utility of this modified resource for expressing circRNA, we developed an expanded set of vectors incorporating this design that (i) enables selection with a variety of antibiotics and fluorescent proteins, (ii) employs a range of promoters varying in promoter strength and (iii) generated a complementary set of lentiviral plasmids for difficult-to-transfect cells. These resources provide a novel and versatile toolkit for high-efficiency and scarless overexpression of circular RNAs that fulfill a critical need for the investigation of circRNA function.

molecular biology↗

bioRxiv2019

A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development

Carotenoids are core plastid components, yet a regulatory function during plastid biogenesis remains enigmatic. A unique carotenoid biosynthesis mutant, carotenoid chloroplast regulation 2 (ccr2), that has no prolamellar body (PLB) and normal PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR) levels, was used to demonstrate a regulatory function for carotenoids under varied dark-light regimes. A forward genetics approach revealed how an epistatic interaction between a (-carotene isomerase mutant (ziso-155) and ccr2 blocked the biosynthesis of specific cis-carotenes and restored PLB formation in etioplasts. We attributed this to a novel apocarotenoid signal, as chemical inhibition of carotenoid cleavage dioxygenase activity restored PLB formation in ccr2 etioplasts during skotomorphogenesis. The apocarotenoid acted in parallel to the transcriptional repressor of photomorphogenesis, DEETIOLATED1 (DET1), to post-transcriptionally regulate PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR), PHYTOCHROME INTERACTING FACTOR3 (PIF3) and ELONGATED HYPOCOTYL5 (HY5) protein levels. The apocarotenoid signal and det1 complemented each other to restore POR levels and PLB formation, thereby controlling plastid development. One-sentence summaryCarotenoids are not just required as core components for plastid biogenesis, they can be cleaved into an apocarotenoid signal that regulates etioplast and chloroplast development during extended periods of darkness.

plant biology↗

bioRxiv2018

Resistance to pentamidine is mediated by AdeAB, regulated by AdeRS, and influenced by growth conditions in Acinetobacter baumannii ATCC 17978

In recent years, effective treatment of infections caused by Acinetobacter baumannii has become challenging due to the ability of the bacterium to acquire or up-regulate antimicrobial resistance determinants. Two component signal transduction systems are known to regulate expression of virulence factors including multidrug efflux pumps. Here, we investigated the role of the AdeRS two component signal transduction system in regulating the AdeAB efflux system, determined whether AdeA and/or AdeB can individually confer antimicrobial resistance, and explored the interplay between pentamidine resistance and growth conditions in A. baumannii ATCC 17978. Results identified that deletion of adeRS affected resistance towards chlorhexidine and 4,6-diamidino-2-phenylindole dihydrochloride, two previously defined AdeABC substrates, and also identified an 8-fold decrease in resistance to pentamidine. Examination of {Delta}adeA, {Delta}adeB and {Delta}adeAB cells augmented results seen for {Delta}adeRS and identified a set of dicationic AdeAB substrates. RNA-sequencing of {Delta}adeRS revealed transcription of 290 genes were [≥]2-fold altered compared to the wildtype. Pentamidine shock significantly increased adeA expression in the wildtype, but decreased it in {Delta}adeRS, implying that AdeRS activates adeAB transcription in ATCC 17978. Investigation under multiple growth conditions, including the use of Biolog phenotypic microarrays, revealed resistance to pentamidine in ATCC 17978 and mutants could be altered by bioavailability of iron or utilization of different carbon sources. In conclusion, the results of this study provide evidence that AdeAB in ATCC 17978 can confer intrinsic resistance to a subset of dicationic compounds and in particular, resistance to pentamidine can be significantly altered depending on the growth conditions.

microbiology↗