Biology subjects

Djuranovic, S.

Publications and source records attributed to Djuranovic, S..

11 recordsLinked to original sources

bioRxiv2026

RAPPL-Seq Enables Rapid, High-Sensitivity Ribosome Profiling of Scarce Samples

Ribosome profiling is a powerful method for transcriptome-wide monitoring of translation activity in vivo, but its use is limited by lengthy protocols, reliance on specialized equipment and inefficient recovery of ribosome-protected fragments (RPFs). To overcome these challenges, we developed RAPPL-Seq, a magnetic bead-based affinity capture method that enables fast and efficient RPF isolation. RAPPL-Seq achieves high recovery efficiency, short processing time, and robust performance with limited input material. It is broadly applicable across multiple sample types, including cell lines, primary tissues, and human biospecimens. RAPPL-Seq can also be paired with translation inhibitors to characterize inhibitor- and tissue-specific mRNA translation. RAPPL-Seq efficiently captured translatomes of glioblastoma (GBM) and adjacent non-tumor brain tissues from patient biopsies. Together, these findings establish RAPPL-Seq as a straightforward, easily accessible, and highly sensitive approach that extends translatome studies to scarce experimental and clinical samples, opening new avenues for mechanistic insight and therapeutic discovery.

molecular biology↗

bioRxiv2026

Protein expression is amplified by stabilizing mRNA using gene-specific antisense oligonucleotides

Effective and safe strategies to restore gene expression in haploinsufficiency disorders or to augment protective protein signaling have long been attractive but challenging therapeutic goals. Here, we demonstrate the potential of antisense oligonucleotides (ASOs) to upregulate gene targets through mRNA stabilization. The 3 untranslated regions (UTRs) of mRNAs regulate stability via activating and repressive elements. One prominent destabilizing cis-element found in ~30% of transcripts is the Pumilio RNA-binding protein recognition element (PRE), which can be targeted to enable gene-specific protein upregulation. A scan for PREs highlights hundreds of disease-relevant transcripts that may be targetable via ASO-mediated gene-specific blocking of Pumilio binding. We show that masking specific PREs with ASOs produces sustained protein upregulation through mRNA stabilization using multiple reporter and endogenous gene targets. Using PRE-masking ASOs, we neutralized PRE-mediated destabilization of TANK-binding kinase-1 (TBK1) mRNA and restored TBK1 protein expression in participant-derived fibroblasts with TBK1 haploinsufficiency, causal for amyotrophic lateral sclerosis. Additionally, by targeting PRE sites in the myoprotective gene follistatin (FST), we achieved effective FST mRNA upregulation in tested mouse tissues. These findings suggest that ASO strategies that neutralize destabilizing 3UTR sequences like PREs may provide a powerful approach to upregulate haploinsufficiency-related genes or enhance protective protein levels for therapeutic benefit.

molecular biology↗

bioRxiv2024

Reduction in ETFDH expression optimizes cancer cell bioenergetics

Mitochondrial electron transport flavoprotein (ETF) insufficiency causes metabolic diseases known as a multiple acyl-CoA dehydrogenase deficiency (MADD). In contrast to muscle, ETFDH is a non-essential gene in acute lymphoblastic leukemia NALM-6 cells, and its expression is reduced across human cancers. ETF insufficiency caused by decreased ETFDH expression limits flexibility of OXPHOS fuel utilization but paradoxically increases cancer cell bioenergetics and accelerates neoplastic growth by activation of the mTORC1/BCL-6/4E-BP1 axis. Collectively, these findings reveal that while ETF insufficiency is rare and has detrimental effects in non-malignant tissues, it is common in neoplasia, where ETFDH downregulation leads to bioenergetic and signaling reprogramming that accelerate neoplastic growth.

cancer biology↗

bioRxiv2024

A rapid, facile, and economical method for the isolation of ribosomes and translational machinery for structural and functional studies

Short abstractRibosomes are essential RNA-protein complexes involved in protein synthesis and quality control. Traditional methods for ribosome isolation are labor intensive, expensive, and require substantial biological material. In contrast, our new method, RAPPL (RNA Affinity Purification using Poly-Lysine), offers a rapid, simple, and cost-effective alternative. This method enriches ribosomes and associated factors from various species and sample types, including cell lysates, whole cells, organs, and whole organisms, and is compatible with traditional isolation techniques. Here, we use RAPPL to facilitate the rapid isolation, functional screening, and structural analysis of ribosomes with associated factors. We demonstrate ribosome-associated resistance mechanisms from patient uropathogenic Escherichia coli samples and generate a 2.7[A] cryoEM structure of ribosomes from Cryptococcus neoformans. By significantly reducing the amount of the starting biological material and the time required for isolation, the RAPPL approach improves the study of ribosomal function, interactions, and antibiotic resistance, providing a versatile platform for academic, clinical, and industrial research on ribosomes. Long abstractRibosomes are macromolecular RNA-protein complexes that constitute the central machinery responsible for protein synthesis and quality control in the cell. Ribosomes also serve as a hub for multiple non-ribosomal proteins and RNAs that control protein synthesis. However, the purification of ribosomes and associated factors for functional and structural studies requires a large amount of starting biological material and a tedious workflow. Current methods are challenging as they combine ultracentrifugation, the use of sucrose cushions or gradients, expensive equipment, and multiple hours to days of work. Here, we present a rapid, facile, and cost-effective method to isolate ribosomes from in vivo or in vitro samples for functional and structural studies using single-step enrichment on magnetic beads - RAPPL (RNA Affinity Purification using Poly-Lysine). Using mass spectrometry and western blot analyses, we show that poly-lysine coated beads incubated with E. coli and HEK-293 cell lysates enrich specifically for ribosomes and ribosome-associated factors. We demonstrate the ability of RAPPL to isolate ribosomes and translation-associated factors from limited material quantities, as well as a wide variety of biological samples: cell lysates, cells, organs, and whole organisms. Using RAPPL, we characterized and visualized the different effects of various drugs and translation inhibitors on protein synthesis. Our method is compatible with traditional ribosome isolation. It can be used to purify specific complexes from fractions of sucrose gradients or in tandem affinity purifications for ribosome-associated factors. Ribosomes isolated using RAPPL are functionally active and can be used for rapid screening and in vitro characterization of ribosome antibiotic resistance. Lastly, we demonstrate the structural applications of RAPPL by purifying and solving the 2.7[A] cryo-EM structure of ribosomes from the Cryptococcus neoformans, an encapsulated yeast causing cryptococcosis. Ribosomes and translational machinery purified with this method are suitable for subsequent functional or structural analyses and provide a solid foundation for researchers to carry out further applications - academic, clinical, or industrial - on ribosomes.

biochemistry↗

bioRxiv2024

Role of U11/U12 minor spliceosome gene ZCRB1 in Ciliogenesis and WNT Signaling

Despite the fact that 0.5% of human introns are processed by the U11/U12 minor spliceosome, the latter influences gene expression across multiple cellular processes. The ZCRB1 protein is a recently described core component of the U12 mono-snRNP minor spliceosome, but its functional significance to minor splicing, gene regulation, and biological signaling cascades is poorly understood. Using CRISPR-Cas9 and siRNA targeted knockout and knockdown strategies, we show that human cell lines with a partial reduction in ZCRB1 expression exhibit significant dysregulation of the splicing and expression of U12-type genes, primarily due to dysregulation of U12 mono-snRNA. RNA-Seq and targeted analyses of minor intron-containing genes indicate a downregulation in the expression of genes involved in ciliogenesis, and consequentially an upregulation in WNT signaling. Additionally, zcrb1 CRISPR-Cas12a knockdown in zebrafish embryos led to gross developmental and body axis abnormalities, disrupted ciliogenesis, and upregulated WNT signaling, complementing our human cell studies. This work highlights a conserved and essential biological role of the minor spliceosome in general, and the ZCRB1 protein specifically in cellular and developmental processes across species, shedding light on the multifaceted relationship between splicing regulation, ciliogenesis, and WNT signaling.

cell biology↗

bioRxiv2024

U-rich elements drive pervasive cryptic splicing in 3' UTR massively parallel reporter assays

Non-coding RNA sequences play essential roles in orchestrating gene expression. However, the sequence codes and mechanisms underpinning post-transcriptional regulation remain incompletely understood. Here, we revisit the finding from a prior massively parallel reporter assay (MPRA) that AU-rich (U-rich) elements in 3 untranslated regions (3 UTRs) can drive upregulation or downregulation of mRNA expression depending on 3 UTR context. We unexpectedly discover that this variable regulation arises from widespread cryptic splicing, predominately from an unannotated splice donor in the coding sequence of GFP to diverse acceptor sites in reporter 3 UTRs. Splicing is activated by U-rich sequences, which function as potent position-dependent regulators of 5 and 3 splice site choice and overall splicing efficiency. Splicing has diverse impacts on reporter expression, causing both increases and decreases in reporter expression via multiple mechanisms. We further provide evidence that cryptic splicing impacts between 10 to 50% of measurements made by other published 3 UTR MPRAs. Overall, our work emphasizes U-rich sequences as principal drivers of splicing and provides strategies to minimize cryptic splicing artifacts in reporter assays.

biochemistry↗

bioRxiv2023

Poly-basic peptides and polymers as new drug candidate against Plasmodium falciparum

Plasmodium falciparum, the malaria-causing parasite, is a leading cause of infection-induced deaths worldwide. The preferred treatment approach is artemisinin-combination therapy, which couples fast-acting artemisinin derivatives with longer-acting drugs like lumefantrine, mefloquine, and amodiaquine. However, the urgency for new treatments has risen due to the parasites growing resistance to existing therapies. Our study shows that a common characteristic of the P. falciparum proteome - stretches of poly-lysine residues such as those found in proteins related to adhesion and pathogenicity - can serve as an effective peptide treatment for infected erythrocytes. A single dose of these poly-basic peptides can successfully diminish parasitemia in human erythrocytes in vitro with minimal toxicity. The effectiveness of the treatment correlates with the length of the poly-lysine peptide, with 30 lysine peptides supporting the eradication of erythrocytic parasites within 72 hours. PEG-ylation of the poly-lysine peptides or utilizing poly-lysine dendrimers and polymers further increases parasite clearance efficiency and bolsters the stability of these potential new therapeutics. Lastly, our affinity pull-downs and mass-spectrometry identify P. falciparums outer membrane proteins as likely targets for polybasic peptide medications. Since poly-lysine dendrimers are already FDA-approved for drug delivery, their adaptation as antimalarial drugs presents a promising new therapeutic strategy. One-Sentence SummaryOur study demonstrates that poly-lysine peptides, particularly those modified through PEG-ylation or in the form of poly-lysine dendrimers, can effectively reduce Plasmodium falciparum, the causative agent of malaria, in human erythrocytes in vitro, with potential for use as a promising new antimalarial therapy.

microbiology↗

bioRxiv2021

Association of RACK1 protein with ribosomes in Plasmodium falciparum

The receptor for activated C-kinase 1 (RACK1), a highly conserved eukaryotic protein, is known to have many, varying biological roles and functions. Previous work has established RACK1 as a ribosomal protein, with defined regions important for binding ribosomes in both human and yeast cells. In Plasmodium falciparum, RACK1 has been shown to be required for parasite growth, however conflicting evidence has been presented about the RACK1 ribosome binding and its role in mRNA translation. Given the importance of RACK1 as a regulatory component of mRNA translation and ribosome quality control, the case could be made in parasites for either of the binary options: bound or unbound to the ribosome. Here we used bioinformatics and transcription analyses to describe P. falciparum RACK1 protein. Based on homology modeling and structural analyses, we generated a model of P. falciparum RACK1 protein. We created and explored mutant and chimeric human and P. falciparum RACK1 proteins binding properties to the human and P. falciparum ribosome. Wild type, chimeric and mutant RACK1 proteins suggest different binding characteristics for P. falciparum and human RACK1 proteins and different regions being involved in their ribosome association. The ribosomal binding of RACK1 variants in human and parasite cells shown here demonstrates that although RACK1 proteins have highly conserved sequences and structures across species, ribosomal binding is affected by species specific alterations to the protein. In conclusion, we show that in the case of P. falciparum, contrary to the structural data, RACK1 is found to be bound to ribosomes and in actively translating polysomes of P. falciparum cells.

molecular biology↗

bioRxiv2021

Gene dosage effects of polyA track engineered hypomorphs

The manipulation of gene activity through the creation of hypomorphic mutants has been a long-standing tool in examining gene function. Our previous studies have indicated that hypomorphic mutants could be created by inserting cis-regulatory sequences composed of consecutive adenosine nucleotides called polyA tracks. Here we use polyA tracks to create hypomorphic mutants and functional characterization of membrane, secretory and endogenous proteins. Insertion of polyA tracks into the sequences of interleukin-2 and membrane protein CD20 results in a programmable reduction of mRNA stability and attenuation of protein expression regardless of the presence of signaling sequence. Likewise, CRISPR/Cas9 targeted insertion of polyA tracks in the coding sequence of endogenous human genes AUF1 and TP53 results in a programmable reduction of targeted protein and mRNA levels. Functional analyses of AUF1 engineered hypomorphs indicate a direct correlation between AUF1 gene levels and the stability of AUF1-regulated mRNAs. Hypomorphs of TP53 affect the expression of the target genes differentially depending upon the severity of the hypomorphic mutation. Finally, decreases in TP53 protein affect the same cellular pathways in polyA track engineered cells as in cancer cells, indicating these variants biological relevance. These results highlight this technologys power to create predictable, stable hypomorphs in recombinant or endogenous genes in combination with CRISPR/Cas9 engineering tools.

genetics↗

bioRxiv2019

Short translational ramp determines efficiency of protein synthesis

It is generally assumed that translation efficiency is governed by translation initiation. However, the efficiency of protein synthesis is regulated by multiple factors including tRNA abundance, codon composition, mRNA motifs and amino-acid sequence1-4. These factors influence the rate of protein synthesis beyond the initiation phase of translation, typically by modulating the rate of peptide-bond formation and to a lesser extent that of translocation. The slowdown in translation during the early elongation phase, known as the 5 translational ramp, likely contributes to the efficiency of protein synthesis 5-9. Multiple mechanisms, which could explain the molecular basis for this translational ramp, have been proposed that include tRNA abundance bias6,9, the rate of translation initiation10-15, mRNA and ribosome structure 11,12,14,16-18, or retention of initiation factors during early elongation events 19. Here, we show that the amount of synthesized protein (translation efficiency) depends on a short translational ramp that comprises the first 5 codons in mRNA. Using a library of more than 250,000 reporter sequences combined with in vitro and in vivo protein expression assays, we show that differences in the short ramp can lead to 3 to 4 orders of magnitude changes in protein abundance. The observed difference is not dependent on tRNA abundance, efficiency of translation initiation, or overall mRNA structure. Instead, we show that translation is regulated by amino-acid-sequence composition and local mRNA sequence. Single-molecule measurements of translation kinetics indicate substantial pausing of ribosome and abortion of protein synthesis on the 4th or 5th codon for distinct amino acid or nucleotide compositions. Introduction of preferred sequence motifs, only at the exact positions within the mRNA, improves protein synthesis for recombinant proteins, indicating an evolutionarily conserved mechanism for controlling translational efficiency.

molecular biology↗

bioRxiv2018

PolyA tracks and poly-lysine repeats are the Achilles heel of Plasmodium falciparum

Plasmodium falciparum, the causative agent of human malaria, is an apicomplexan parasite with a complex, multi-host life cycle. Sixty percent of transcripts from its extreme AT-rich (81%) genome possess coding polyadenosine (polyA) runs, distinguishing the parasite from its hosts and other sequenced organisms. Recent studies indicate that transcripts with polyA runs encoding poly-lysine are hot spots for ribosome stalling and frameshifting, eliciting mRNA surveillance pathways and attenuating protein synthesis in the majority of prokaryotic and eukaryotic organisms. Here, we show that the P. falciparum translational machinery is paradigm-breaking. Using bioinformatic and biochemical approaches, we demonstrate that both endogenous genes and reporter sequences containing long polyA runs are efficiently and accurately transcribed and translated in P. falciparum cells. Translation of polyA tracks in the parasite does not elicit any response from mRNA surveillance pathways usually seen in host human cells or organisms with similar AT content. The translation efficiency and accuracy of the parasite protein synthesis machinery reveals a unique role of ribosomes in the evolution and adaptation of P. falciparum to an AU-rich transcriptome and polybasic amino sequences. Finally, we show that the ability of P. falciparum to synthesize long poly-lysine repeats has given this parasite a unique protein exportome and an advantage in infectivity that can be suppressed by addition of exogenous poly-basic polymers.

microbiology↗