Biology subjects

Backes, S.

Publications and source records attributed to Backes, S..

5 recordsLinked to original sources

bioRxiv2025

Visualizing the transcription and replication of influenza A viral RNAs in cells by multiple direct RNA padlock probing and in-situ sequencing (mudRapp-seq)

Influenza A viruses (IAV) contain eight negative-sense single-stranded viral RNA (vRNA) molecules, which are transcribed into mRNA and replicated via complementary RNA (cRNA). These processes are tightly regulated, but the precise molecular mechanisms governing the switch from transcription to replication remain elusive. Here, we introduce multiple direct-RNA assisted padlock probing in combination with in situ sequencing (mudRapp-seq) to visualize the transcription and replication of all eight IAV vRNA and mRNA molecules at the single-cell level. We demonstrate that direct RNA padlock probing is three times more efficient than conventional probes that target cDNA. Individual probes showed variations in efficiency, partly due to the RNA structure of the target, which was mitigated by employing multiple padlock probes per target. Applying mudRapp-seq to an infection time course, we observed early mRNA expression, followed by vRNA accumulation approximately 3 hours later. Individual viral segments exhibited differential expression, particularly in the mRNA population. Both bulk and single-cell analyses revealed a correlation between the expression of M mRNA and the onset of the transcription-to-replication switch. Our findings demonstrate that mudRapp-seq offers significant potential for elucidating viral replication mechanisms and may be applicable to studying other RNA viruses and cellular RNA processes.

microbiology↗

bioRxiv2024

The Visual Experience Evaluation Tool: A Myopia Research Instrument for Quantifying Visual Experience

Current myopia research has demonstrated the role of extended visual experience in healthy ocular development. Optical cues and the spectrum, intensity, and temporal characteristics of light landing on the retina are all known factors affecting the development of the eye. However, there is still limited understanding as to which of these extrinsic factors are most important or how they interplay with intrinsic physical and neural differences between individuals. Part of the problem is inadequate tooling. Our team at Reality Labs Research created the Visual Environment Evaluation Tool (VEET), a non-commercial research instrument, to accelerate myopia research. In this paper, we describe the VEETs physical design, sensor suite and capabilities, and the associated software which makes it well-suited for research of quantified visual experience.

neuroscience↗

bioRxiv2023

Coronaviruses use ACE2 monomers as entry receptors

The angiotensin-converting enzyme 2 (ACE2) has been identified as entry receptor on cells enabling binding and infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via trimeric spike (S) proteins protruding from the viral surface1,2. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of preferably multimeric ACE2 receptors to achieve a higher binding and infection efficiency1,3. However, our current knowledge about the influence of ACE2 expression and organization in the plasma membrane on SARS-CoV-2 infection efficiency remains elusive. Here we used direct stochastic optical reconstruction microscopy (dSTORM) in combination with different labeling approaches to visualize the distribution and quantify the expression of ACE2 on different cells. Our results reveal that endogenous ACE2 receptors are present as monomers in the plasma membrane with densities of only 1-2 receptors m-2. In addition, binding of trimeric S proteins does not induce clustering of ACE2 receptors in the plasma membrane. Supported by infection studies using vesicular stomatitis virus (VSV) particles bearing S proteins our data demonstrate that a single S protein interaction per virus particle with a monomeric ACE2 receptor is sufficient for infection which attests SARS-CoV-2 a high infectivity.

molecular biology↗

bioRxiv2021

HSV-1 and influenza infection induce linear and circular splicing of the long NEAT1 isoform

The herpes simplex virus 1 (HSV-1) virion host shut-off (vhs) protein cleaves both cellular and viral mRNAs by a translation-initiation-dependent mechanism. Here, we show that vhs-mediated degradation of mRNAs leads to an accumulation of circular RNAs (circRNAs) relative to linear mRNAs during HSV-1 infection. Strikingly, we found that circular splicing of the long isoform (NEAT1_2) of the nuclear paraspeckle assembly transcript 1 (NEAT1) was massively induced during HSV-1 infection. In contrast to other circRNAs, induction of the NEAT1_2 circRNA was independent of vhs and occurred while NEAT1_2 was still bound to the chromatin. This was associated with induction of linear splicing of NEAT1_2 both within and downstream of the circRNA. NEAT1_2 splicing was absent in uninfected cells but can be induced by ectopic co-expression of the HSV-1 immediate-early proteins ICP22 and ICP27. Interestingly, NEAT1_2 circular and linear splicing was also up-regulated in influenza A virus (IAV) infection but absent in stress conditions, which disrupt transcription termination similar to but not by the same mechanisms as HSV-1 and IAV infection. Large-scale analysis of published RNA-seq data uncovered induction of NEAT1_2 splicing in cancer cells upon inhibition or knockdown of cyclin-dependent kinase 7 (CDK7) or the MED1 subunit of the Mediator complex phosphorylated by CDK7. Finally, CDK7 inhibition also disrupted transcription termination, highlighting a possible link between disruption of transcription termination and NEAT1_2 splicing.

bioinformatics↗

bioRxiv2020

The mitochondrial surface receptor Tom70 protects the cytosol against mitoprotein-induced stress

Most mitochondrial proteins are synthesized as precursors in the cytosol and post-translationally transported into mitochondria. The mitochondrial surface protein Tom70 acts at the interface of the cytosol and mitochondria. In vitro import experiments identified Tom70 as targeting receptor, particularly for hydrophobic carriers. Using in vivo methods and high content screens, we revisited the question of Tom70 function and considerably expanded the set of Tom70-dependent mitochondrial proteins. We demonstrate that the crucial activity of Tom70 is its ability to recruit cytosolic chaperones to the outer membrane. Indeed, tethering an unrelated chaperone-binding domain onto the mitochondrial surface complements most of the defects caused by Tom70 deletion. Tom70-mediated chaperone recruitment reduces the proteotoxicity of mitochondrial precursor proteins, in particular of hydrophobic inner membrane proteins. Thus, our work suggests that the predominant function of Tom70 is to tether cytosolic chaperones to the outer mitochondrial membrane, rather than to serve as a mitochondria-specifying targeting receptor.

cell biology↗