Theiss, J. | bioRxiv Science

bioRxiv2024

An atlas of RNA-dependent proteins in cell division reveals the riboregulation of mitotic protein-protein interactions

Ribonucleoprotein complexes are dynamic assemblies of RNA with RNA-binding proteins (RBPs), which can modulate the fate of the RNA molecules from transcription to degradation. Vice versa, RNA can regulate the interactions and functions of the associated proteins. Dysregulation of RBPs is linked to diseases such as cancer and neurological disorders. RNA and RBPs are present in mitotic structures like the centrosomes and spindle microtubules, but their influence on mitotic spindle integrity remains unknown. Thus, we applied the R-DeeP strategy for the proteome-wide identification of RNA-dependent proteins and complexes to cells synchronized in mitosis versus interphase. The resulting atlas of RNA-dependent proteins in cell division can be accessed through the R-DeeP 3.0 database (R-DeeP3.dkfz.de). It revealed key mitotic factors as RNA-dependent such as AURKA, KIFC1 and TPX2 that were linked to RNA despite their lack of canonical RNA-binding domains. KIFC1 was identified as a new interaction partner and phosphorylation substrate of AURKA at S349 and T359. In addition, KIFC1 interacted with both, AURKA and TPX2, in an RNA-dependent manner. Our data suggest a riboregulation of mitotic protein-protein interactions during spindle assembly, offering new perspectives on the control of cell division processes by RNA-protein complexes. HighlightsO_LIDifferential R-DeeP screens in mitosis and interphase are provided as a resource in a user-friendly database at R-DeeP3.dkfz.de C_LIO_LIAn atlas of RNA-dependent proteins in cell division identifies a substantial number of unconventional RNA-binding proteins among mitotic factors C_LIO_LIInvestigation of protein-protein interactions reveals KIFC1 as a new AURKA and TPX2 interaction partner during spindle assembly C_LIO_LIKIFC1, AURKA and TPX2 interact with each other in an RNA-dependent manner and directly bind to RNA C_LIO_LIAURKA phosphorylates KIFC1 at residues S349 and T359 C_LI

cell biology↗

bioRxiv2020

High-resolution analysis of Merkel Cell Polyomavirus in Merkel Cell Carcinoma reveals distinct integration patterns and suggests NHEJ and MMBIR as underlying mechanisms

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses. Author summaryIntegration of viral DNA into the host genome is a key event in the pathogenesis of many virus-induced cancers. One such cancer is Merkel cell carcinoma (MCC), a highly malignant tumor that harbors monoclonally integrated and replication-defective Merkel cell polyomavirus (MCPyV) genomes. Although MCPyV integration sites have been analyzed before, there is very little knowledge of the mechanisms that lead to mutagenesis and integration of viral genomes. We used multiple sequencing technologies and interrogation of chromatin states to perform a comprehensive characterization of MCPyV integration loci. This analysis allowed us to deduce the events that likely precede viral integration. We provide evidence that the mutations which result in the replication defective phenotype are acquired prior to integration and propose that the cellular DNA repair pathways non-homologous end joining (NHEJ) and microhomology-mediated break-induced replication (MMBIR) produce two principal MCPyV integration patterns (simple and complex, respectively). We show that, although MCPyV integrates predominantly in open chromatin regions, viral oncogene expression is independent of host promoters and driven by the viral promotor region. Our findings are important since they can explain the mechanisms of MCPyV integration. Furthermore, our model may also apply to papillomaviruses, another clinically important family of oncogenic DNA viruses.

molecular biology↗