Biology subjects

Findeiss, S.

Publications and source records attributed to Findeiss, S..

6 recordsLinked to original sources

bioRxiv2025

Telomerase RNA gene duplications drive telomeric repeatdiversity and evolution in Andrena bees

Most organisms in the animal kingdom require a non-coding telomerease RNA (TR) in conjunction with the telomerase reverse transcriptase (TERT) to add telomere tandem repeats to chromosome ends to genomic instability. Recent studies reported an extensive diversity in the sequence of telomeric repeats in some insect species. Our investigation of TR genes in the Andrena genus provides convincing evidence for the presence of multiple TR gene copies with different template sequences for synthesis of distinct telomeric repeat sequences in several species. In this study we describe the structure, genomic coordinates and abundance of these TR genes, and correlate our findings with the levels of tandem repeats found in DNAseq data. Based on an analysis of the synthenic context of these newly predicted TR genes, we show evidence for the existence of multiple TR paralogs that diverged during the evolution of the genus Andrena. To our knowledge this is the first time such a phenomenon is observed in animals, although recently reported for plants. Interestingly, the comprehensive annotation of all TERT genes found in yet unannotated Andrena species shows no corresponding evolutionary changes in related TERT proteins encoded by a single copy gene. Our study suggests an evolutionary mechanism for diversification of telomeric repeat sequences in certain insect species through telomerase RNA gene duplication.

bioinformatics↗

bioRxiv2022

Tailored machine learning models for functional RNA detection in genome-wide screens

The in silico prediction of non-coding and protein-coding genetic loci is an area of research that has gathered large attention in the field of comparative genomics. In the last decade, much effort has been made to investigate numerous properties of nucleotide sequences that hint at their biological role in the cell. We present here a software framework for the alignment-based training, evaluation and application of machine learning models with user-defined parameters. Instead of focusing on the one-size-fits-all approach of pervasive in silico annotation pipelines, we offer a framework for the structured generation and evaluation of models based on arbitrary features and input data, focusing on stable and explainable results. Furthermore, we showcase the usage of our software package in a full-genome screen of Drosophila melanogaster and evaluate our results against the well-known but much less flexible program RNAz.

bioinformatics↗

bioRxiv2020

Beyond Plug and Pray: Context Sensitivity and in silico Design of Artificial Neomycin Riboswitches

Gene regulation in prokaryotes often depends on RNA elements such as riboswitches or RNA thermometers located in the 5’ untranslated region of mRNA. Rearrangements of the RNA structure in response, e. g., to the binding of small molecules or ions control translational initiation or premature termination of transcription and thus mRNA expression. Such structural responses are amenable to computational modeling, making it possible to rationally design synthetic riboswitches for a given aptamer. Starting from an artificial aptamer, we construct the first synthetic transcriptional riboswitches that respond to the antibiotic neomycin. We show that the switching behavior in vivo critically depends not only on the sequence of the riboswitch itself, but also on its sequence context. We therefore developed in silico methods to predict the impact of the context, making it possible to adapt the design and to rescue non-functional riboswitches. We furthermore analyze the influence of 5’ hairpins with varying stability on neomycin riboswitch activity. Our data highlight the limitations of a simple plug-and-play approach in the design of complex genetic circuits and demonstrate that detailed computational models significantly simplify, improve, and automate the design of transcriptional circuits. Our design software is available under a free license on Github.1Competing Interest StatementThe authors have declared no competing interest.List of Abbreviationsauarbitrary time unitsAURaptamer upstream regionbgaBβ-galactosidasebgaBthe β-galactosidase genedLdecoupling leaderE. coliEscherichia colieGFPthe enhanced green fluorescent proteinegfpthe enhanced green fluorescent protein geneGFPgreen fluorescent proteinLHleader hairpinLMmutated leaderMFEminimum free energymRNAmessenger RNAoLoriginal leaderPospositive controlRBSribosomal binding siteRFUrelative fluorescence unitRNAribonucleic acidRNAPRNA polymeraseRNase ERibonuclease ERppHRNA 5’ pyrophosphohydrolaserRNAribosomal RNASELEXsystematic evolution of ligands by exponential enrichmentSSCsaline sodium citrate bufferTBETris-borate-EDTATDRterminator downstream regionTSStranscription start siteUunstructured regionUTRuntranslated regionView Full Text

synthetic biology↗

bioRxiv2020

Assessing the Quality of Cotranscriptional Folding Simulations

Structural changes in RNAs are an important contributor to controlling gene expression not only at the post-transcriptional stage but also during transcription. A subclass of riboswitches and RNA thermometers located in the 5 region of the primary transcript regulates the downstream functional unit - usually an ORF - through premature termination of transcription. Such elements not only occur naturally but they are also attractive devices in synthetic biology. The possibility to design such riboswitches or RNA thermometers is thus of considerable practical interest. Since these functional RNA elements act already during transcription, it is important to model and understand the dynamics of folding and, in particular, the formation of intermediate structures concurrently with transcription. Cotranscriptional folding simulations are therefore an important step to verify the functionality of design constructs before conducting expensive and labour-intensive wet lab experiments. For RNAs, full-fledged molecular dynamics simulations are far beyond practical reach both because of the size of the molecules and the time scales of interest. Even at the simplified level of secondary structures further approximations are necessary. The BarMap approach is based on representing the secondary structure landscape for each individual transcription step by a coarse-grained representation that only retains a small set of low-energy local minima and the energy barriers between them. The folding dynamics between two transcriptional elongation steps is modeled as a Markov process on this representation. Maps between pairs of consecutive coarse-grained landscapes make it possible to follow the folding process as it changes in response to transcription elongation. In its original implementation, the BarMap software provides a general framework to investigate RNA folding dynamics on temporally changing landscapes. It is, however, difficult to use in particular for specific scenarios such as cotranscriptional folding. To overcome this limitation, we developed the user-friendly BarMap-QA pipeline described in detail in this contribution. It is illustrated here by an elaborate example that emphasizes the careful monitoring of several quality measures. Using an iterative workflow, a reliable and complete kinetics simulation of a synthetic, transcription regulating riboswitch is obtained using minimal computational resources. All programs and scripts used in this contribution are free software and available for download as a source distribution for Linux(R), or as a platform-independent Docker(R) image including support for Apple macOS(R) and Microsoft Windows(R).

bioinformatics↗

bioRxiv2018

Quantitative characterization of translational riboregulators using an in vitro transcription-translation system

Riboregulators are short RNA sequences that, upon binding to a ligand, change their secondary structure and influence the expression rate of a downstream gene. They constitute an attractive alternative to transcription factors for building synthetic gene regulatory networks because they can be engineered de novo and they have a fast turnover and a low metabolic burden. However, riboregulators are generally designed in silico and tested in vivo, which only provides a yes/no evaluation of their performances, thus hindering the improvement of design algorithms. Here we show that a cell-free transcription-translation (TX-TL) system provides valuable quantitative information about the performances of in silico designed riboregulators. In particular, we use the ribosome as an exquisite molecular machine that detects functional riboregulators, precisely measures their concentration and linearly amplifies the signal by generating a fluorescent protein. We apply this method to characterize two types of translational riboregulators composed of a cis-repressed (cr) and a trans-activating (ta) strand. At the DNA level we demonstrate that high concentrations of taDNA poisoned the activator until total shut off. At the RNA level, we show that this approach provides a fast and simple way to measure dissociation constants of functional riboregulators, in contrast to standard mobility-shift assays. Our method opens the route for using cell-free TX-TL systems for the quantitative characterization of functional riboregulators in order to improve their design in silico.

synthetic biology↗

bioRxiv2018

In silico design of ligand triggered RNA switches

This contribution sketches a work flow to design an RNA switch that is able to adapt two structural conformations in a ligand-dependent way. A well characterized RNA aptamer, i. e., knowing its Kd and adaptive structural features, is an essential ingredient of the described design process. We exemplify the principles using the well-known theophylline aptamer throughout this work. The aptamer in its ligand-binding competent structure represents one structural conformation of the switch while an alternative fold that disrupts the binding-competent structure forms the other conformation. To keep it simple we do not incorporate any regulatory mechanism to control transcription or translation. We elucidate a commonly used design process by explicitly dissecting and explaining the necessary steps in detail. We developed a novel objective function which specifies the mechanistics of this simple, ligand-triggered riboswitch and describe an extensive in silico analysis pipeline to evaluate important kinetic properties of the designed sequences. This protocol and the developed software can be easily extended or adapted to fit novel design scenarios and thus can serve as a template for future needs.

bioinformatics↗