Biology subjects

Scheibe, M.

Publications and source records attributed to Scheibe, M..

4 recordsLinked to original sources

bioRxiv2026

Identification and binding-site mapping of RNA-binding proteins interacting with microRNA precursors in Arabidopsis

BackgroundMicroRNAs (miRNAs) play key roles in modulating gene expression. Upon transcription, primary miRNA transcripts (pri-miRNAs or MIRNAs) fold into stem-loop structures. Endonucleolytic cleavage releases a miRNA/miRNA* duplex from the stem, which is subsequently matured. In higher plants, the pri-miRNA hairpins vary widely in length and structure. Pri-miRNA processing is extensively regulated by RNA-binding proteins (RBPs) and the range of underlying mechanisms continues to expand. ResultsHere, we performed an unbiased screen to identify Arabidopisis thaliana RBPs that interact with pri-miRNAs, using pri-miRNA159a and pri-miRNA398b as paradigms. Nucleoplasmic proteins were significantly enriched in pulldowns with bead-immobilized in vitro transcripts of the stem-loop regions of these pri-miRNAs compared with pulldowns using empty beads. The enriched proteins included several members of the family of the serine/arginine-rich (SR) splicing factor family, including RS31 and SR34a. Mutant analysis indicated that selected candidate interactors affect pri-miRNA and/or mature miRNA accumulation. To validate interactions with pri-miRNAs in vivo, we performed individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). Whereas our conventional plant iCLIP procedure detected only a few crosslink sites on pri-miRNAs, likely because of the low abundance of these transcripts, our improved plant iCLIP2 protocol enabled the identification of binding sites on numerous pri-miRNAs. RS31 and SR34a bound overlapping sets of pri-miRNAs and contacted both stem-loop regions and flanking regions upstream and downstream of the stem-loop. RS31 and SR34 bound both overlapping and distinct regions of pri-miRNAs. ConclusionIn vitro pulldowns of nucleoplasmic proteins identified RBPs associated with pri-miRNA stem-loops, and the improved plant iCLIP2 protocol revealed in vivo binding of RS31 and SR34a to a subset of pri-miRNAs. Together, these findings expand the repertoire of RBPs implicated in plant miRNA biogenesis and support a role for SR proteins as candidate regulators of pri-miRNA processing.

plant biology↗

bioRxiv2025

Wuhan sharpbelly bornavirus (genus Cultervirus) can infect a wide range of host cells and establishes persistent infection in diverse cypriniform cells

Our recent study using in silico data mining identified novel culterviruses (family: Bornaviridae) in fish, including a variant of W[u]han sharpbelly bornavirus (WhSBV) in grass carp kidney and liver cell lines. Here, metagenomic sequencing of different fish cell lines revealed WhSBV in two cell lines from grass carp (Ctenopharyngodon idella; order: Cypriniformes). Using these cell lines, we investigated the ability of WhSBV to infect and establish persistent infection in other cell lines from bony fish (Cypriniformes, Chichliformes, Salmoniformes, Centrarchiformes and Spariformes), reptiles (Testudines and Squamata), birds (Galliformes) and mammals (Primates and Rodentia). WhSBV showed efficient replication and a time-dependent increase in viral RNA levels in cypriniform cells, whereas replication was limited, confined to single cells, and lacked a clear time-dependent increase in cells from other bony fish and reptiles. No replication was detected in avian and mammalian cells. In situ hybridisation and electron microscopy confirmed the presence of viral RNA and particles in infected cypriniform cells. Transcriptomic sequencing revealed minimal innate immune activation during early stages of infection and antiviral response only at later stages, suggesting that WhSBV establishes persistence by evading early immune recognition. In addition, we identified polycistronic viral mRNAs regulated by specific transcriptional start and termination sites and RNA splicing. Viral proteins were detected, confirming previous in silico predictions. These findings provide insights into the potential infectivity, persistence mechanisms and transcriptional strategies of WhSBV. This study validates previous findings from in silico data mining, further reinforcing its effectiveness as a powerful tool for discovering hidden viruses. IMPORTANCEUnderstanding the diversity and host range of viruses is crucial for assessing their ecological role, associated diseases and zoonotic potential. However, many newly discovered viruses are characterised using sequence data alone because isolates are often difficult to obtain. Using cell culture models, this study characterises W[u]han sharpbelly bornavirus (WhSBV), a member of the genus Cultervirus. Here we demonstrate its ability to establish persistent infection in cypriniform fish cell lines, while exhibiting restricted replication in certain non-cypriniform fish. The identification of polycistronic transcription, splicing events and immune evasion mechanisms advances our understanding of the molecular biology of WhSBV and culterviruses in general. By validating in silico predictions, this study highlights the power of computational approaches in uncovering viral diversity. As cypriniform fish include economically important species such as carp, understanding the dynamics of WhSBV host range and infection biology may be crucial for future aquaculture health management.

molecular biology↗

bioRxiv2021

The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei

The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.

microbiology↗

bioRxiv2020

The m6A reader Ythdf restricts axonal growth in Drosophila through target selection modulation of the Fragile X mental retardation protein

N6-methyladenosine (m6A) regulates a variety of physiological processes through modulation of RNA metabolism. The modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6A alters fly behavior albeit the underlying mechanism and the role of m6A during nervous system development have remained elusive. Here we find that impairment of the m6A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6A reader in the nervous system being required for limiting axonal growth. Mechanistically, we show that Ythdf directly interacts with Fragile X mental retardation protein to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6A pathway controls development of the nervous system by modulating Fmr1 target selection.

molecular biology↗