Biology subjects

Riepsaame, J.

Publications and source records attributed to Riepsaame, J..

6 recordsLinked to original sources

bioRxiv2026

Inhibition of CDK7, CDK9 or CDK12 affects phosphorylation of human SPT5.

SPT5, complexed with SPT4 in the DRB-sensitivity-inducing factor, DSIF, is a critical regulator of transcription elongation that acts negatively in the unphosphorylated form and positively when phosphorylated. In vivo, phosphorylation of multiple sites in the largely unstructured linker and C-terminal regions of the protein facilitates release from an early elongation checkpoint and processive transcription elongation. Transcription elongation factors, including the PAF complex (PAF1C), interact with phosphorylated SPT5 and phosphorylation of residues S666 and T806 are associated with the positive elongation functions of SPT5. Here, using in vitro phosphorylation of regions of the SPT5 C-terminus followed by GST-mediated pull-down we confirm that the RTF1 subunit of PAF1C interacts with residues 750-850 only when phosphorylated. Antibodies we raised to phospho-S666 or phospho-T806 are specific for these sites phosphorylated in vitro and in vivo. Western analysis of endogenous SPT5 in HEK293 cells using these antibodies before and after inhibition of analog-sensitive versions of CDK7, CDK9 and CDK12 indicate that all three kinases play roles in the phosphorylation of SPT5, although inhibition of CDK9 has the most marked effect. CDK9 inhibition also inhibits the association of PAF1C subunits with SPT5 in vivo and affects phosphorylation of chromatin-associated SPT5 as measured by chromatin immunoprecipitation (ChIP).

molecular biology↗

bioRxiv2025

Guide RNA library representation results in gene essentiality prediction bias in genome-wide CRISPR screens

Genome wide CRISPR-based perturbation screens are powerful discovery tools enabling the identification of novel gene dependencies through either gain or loss of function. While genome wide guide RNA (gRNA) libraries have advantages when using enAsCas12a, such as multiplex single gRNAs per gene, they may be subject to similar confounding factors that can affect the interpretation of large genome-wide datasets. Here, we examine the impact of these variables in over twenty enASCas12a multiple gRNA based perturbation screens performed using Humagne C, Humagne D and Inzolia libraries in human cells. We demonstrate that the choice of CRISPR library is often the most significant factor that influences genetic perturbation results, outweighing other variables such as either target cell lines or culture media conditions. A major contributor to this effect is gRNA representation bias within a given CRISPR library, where lower gRNA representation can lead to variable and more pronounced gene effect scores using either log fold change or Chronos analysis. These effects may be mitigated by using either multiple gRNA constructs per gene, by optimisation of CRISPR library production processes or by targeting with multiple independent gRNA libraries. Importantly, we also consider gRNA representation bias during CRISPR screen hit prioritisation. CRISPR library gRNA representation bias remains a major challenge in the interpretation of gene essentiality in perturbation screens.

genomics↗

bioRxiv2025

Remodelled cholesteryl ester enriched lipid droplets fuel flavivirus morphogenesis

Flaviviruses such as dengue and Zika viruses extensively remodel host cell membranes to create specialised replication organelles, but the role of lipid metabolism to generate them remain poorly understood. Through systematic screens of fatty acyl transferase enzymes (MBOAT and zDHHC families) and complementary approaches including CRISPR/Cas9 gene deletions, pharmacological inhibition, proteomics, and photo-crosslinkable cholesterol analogues, we identified cholesteryl ester-enriched lipid droplets (CE-LDs) as critical host components required for flavivirus infection. CE-LD formation is mediated by Sterol O-acyltransferases 1 and 2 (SOAT1/SOAT2), whose activities were upregulated early during infection, coinciding with increased CE-LD formation and transition to liquid crystalline phases. Genetic deletion or pharmacological inhibition of either enzyme resulted in a dramatic [~]100-fold reduction in viral production. Mechanistically, CE-LDs display distinct proteomic signatures, enriched in fatty acid remodelling enzymes, GTPases, and lipid transport proteins. Photo-crosslinking experiments demonstrated direct interactions between LD-derived cholesterol and viral prM, capsid and NS1. Disrupting CE-LD formation via SOAT1/2-deficiency resulted in defective, viral RNA-free replication organelles and complete absence of immature virions. Supporting the physiological and clinical relevance of viral LD exploitation, analysis in iPSC-derived macrophages mirrored findings in Huh7 cells, and dengue patients from a Sri Lankan cohort revealed that central obesity significantly increased the risk of severe dengue haemorrhagic fever. This study establishes CE-LDs as essential host metabolic hubs that enable flavivirus morphogenesis and identifies host LD metabolism as a promising therapeutic target for combating flavivirus infections.

cell biology↗

bioRxiv2024

Cerebellar organoids model cell type-specific FOXP2 expression during human cerebellar development

In this study, we demonstrate the potential of cerebellar organoids for studying features of early human cerebellar development. Forkhead box protein P2 (FOXP2) is a transcription factor associated with speech and language development that is highly expressed in the developing brain. However, little attention has been directed to the study of FOXP2 in the early developing cerebellum. We used CRISPR gene editing in human iPSCs to generate a fluorescent FOXP2-reporter line. By combining transcriptomic analysis of iPSC-derived cerebellar organoids with published cerebellar datasets, we describe the expression and identify potential downstream targets of FOXP2 in the early developing human cerebellum. Our results highlight expression of FOXP2 in early human Purkinje cells and cerebellar nuclei neurons, and the vulnerability of these cell populations to neurodevelopmental disorders. Our study demonstrates the power of cerebellar organoids to model early human developmental processes and disorders.

neuroscience↗

bioRxiv2023

PTCH1 mutant human cerebellar organoids are associated with altered neural development and early pathways of medulloblastoma oncogenesis

Patched 1 (PTCH1) is the primary receptor for Sonic Hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma (MB). As a result of differences between murine and human development, molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here, we employ cerebellar organoids differentiated from human induced pluripotent stem cells (iPSC) combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings support the occurrence of developmental mechanisms in cerebellar organoids that mirror in vivo processes of regionalisation and SHH signalling, and offer new insight into early pathophysiological events of MB tumorigenesis. HiglightsO_LIDifferentiation of human iPSC into cerebellar organoids C_LIO_LIHomozygous LOF of PTCH1 prevents cerebellar organoid differentiation C_LIO_LIPTCH1+/- cerebellar organoids display tissue-specific effects of SHH signalling C_LIO_LIEarly altered gene expression relevant for MB in PTCH1+/- cerebellar organoids C_LI

neuroscience↗

bioRxiv2018

Selective roles of vertebrate PCF11 in premature and full-length transcript termination

The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including: mNET-seq, 3 mRNA-seq, chromatin RNA-seq and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and downstream gene silencing. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript, and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.

molecular biology↗