Biology subjects

Brosius, J.

Publications and source records attributed to Brosius, J..

5 recordsLinked to original sources

bioRxiv2025

Thermal cycling transcription boosts RNA production.

Efficient RNA production is essential for therapeutic applications. Currently, mRNA is generated by in vitro transcription with RNA polymerases at a constant temperature. The success of these transcriptions depends on various factors including sequence composition and size. Sometimes, especially for long RNAs, achieving efficient transcript yields are challenging. We developed a novel approach by incorporating a high-temperature denaturation step. Our protocol includes the following two steps of thermal cycling RNA transcription: the initial enzyme-driven RNA transcription step is done at RNA polymerase active temperature (37 {degrees}C); then the nucleic acids melting step is carried out at elevated temperatures (55-70{degrees}C); after the reaction is cooled to the temperature of optimal polymerase activity, the first step is repeated. Denaturation enables the generation of optimized, more stable mRNAs, which alleviates the mRNA instability that often occurs during RNA-based therapeutic development. For example, this new thermal cycling RNA transcription approach can employ high GC content, thus increasing RNA stability. For long and difficult templates, the combination of sequence optimization by LinearDesign algorithm in conjunction with transcription via thermal cycling significantly improved mRNA production. Thermal cycling transcription dramatically increased the efficiency of most RNA products while reducing costs, facilitating RNA-based therapeutic development.

biochemistry↗

bioRxiv2025

circAβ-a RNA encoded Aβ175--the hidden driver of β-amyloid plaque formation and deposition in sporadic Alzheimer's disease.

Mechanisms that trigger A{beta} production in sporadic Alzheimers disease are still obscure. We recently reported the expression of a human circular RNA (circA{beta}-a) encoded A{beta} peptide precursor variant (A{beta}175). Presently, we demonstrated that AAV9 virus-expressed circA{beta}-a gave rise to extensive extracellular A{beta} plaque depositions and microglial activation in mouse brain; this recapitulates critical pathogenic hallmarks within a sporadic AD mouse model. Specifically developed antibodies detected robust endogenous A{beta}175 expression in HEK293 cells and hNSC-derived human neurons, underscoring the potential of A{beta}175 as a salient A{beta} precursor. Furthermore, we detected high levels of A{beta}175 oligomers in young-adult human brains. In intermediate and old-age human brain samples, accumulation of soluble A{beta}175 pentamers was reduced and A{beta}175 oligomers were components of most insoluble A{beta} plaques in older human brain. We propose a causal relationship between human circA{beta}-a RNA expression, dysregulation of A{beta}175 oligomer processing/aggregation and A{beta} plaque accumulation in sporadic AD.

neuroscience↗

bioRxiv2024

Exploring Alu-Driven DNA Transductions in the Primate Genomes

Long terminal repeats (LTRs) and non-LTRs retrotransposons, aka retroelements, collectively occupy a substantial part of the human genome. Certain non-LTR retroelements, such as L1 and SVA, have the potential for DNA transduction, which involves the concurrent mobilization of flanking non-transposon DNA during retrotransposition. These events can be detected by computational approaches. Despite being the most abundant short interspersed sequences (SINEs) that are still active within the genomes of humans and other primates, the transduction rate caused by Alu sequences remains unexplored. Therefore, we conducted an analysis to address this research gap and utilized an in-house program to probe for the presence of Alu-related transductions in the human genome. We analyzed 118,489 full-length AluY subfamilies annotated within the first complete human reference genome, T2T-CHM13. For comparative insights, we extended our exploration to two non-human primate genomes, the chimpanzee and the rhesus monkey. After manual curation, our findings did not confirm any Alu-mediated transductions, whose source genes are, unlike L1 or SVA, transcribed by RNA polymerase III, implying that they are infrequent or possibly absent not only in the human but also in chimpanzee and rhesus monkey genomes. Although we identified loci in which the 3 Target Site Duplication (TSD) was located distantly from the retrotransposed AluYs, a transduction hallmark, our study could not find further support for such events. The observation of these instances can be explained by the incorporation of other nucleotides into the poly(A) tails in conjunction with polymerase slippage.

genetics↗

bioRxiv2019

Pervasive head-to-tail insertions of DNA templates mask desired CRISPR/Cas9-mediated genome editing events

CRISPR/Cas9 mediated homology-directed DNA repair is the method of choice for precise gene editing in a wide range of model organisms, including mouse and human. Broad use by the biomedical community refined the method, making it more efficient and sequence specific. Nevertheless, the rapidly evolving technique still contains pitfalls. During the generation of six different conditional knock-out mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or non-homologous end-joining mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis--in most cases--failed to identify such multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analysis of modified alleles is essential, and offer practical solutions to correctly identify precisely edited chromosomes.

genetics↗

bioRxiv2016

De-novo emergence and template switching of SINE retroposons during the early evolution of passerine birds

Passeriformes (\"perching birds\" or passerines) make up more than half of all extant bird species. Here, we resolve their deep phylogenetic relationships using presence/absence patterns of short interspersed elements (SINEs), a group of retroposons which is abundant in mammalian genomes but considered largely inactive in avian genomes. The resultant retroposon-based phylogeny provides a powerful and independent corroboration of previous indications derived from sequence-based analyses. Notably, SINE activity began in the common ancestor of Eupasseres (passerines excl. the New Zealand wrens Acanthisittidae) and ceased before the rapid diversification of oscine passerines (songbirds). Furthermore, we find evidence for very recent SINE activity within suboscine passerines, following the emergence of a SINE via acquisition of a different tRNA head as we suggest through template switching. We propose that the early evolution of passerines was unusual among birds in that it was accompanied by activity of SINEs. Their genomic and transcriptomic impact warrants further study in the light of the massive diversification of passerines.

evolutionary biology↗