direct-RNA Nanopore Sequencing analysis of mammalian cells infected with SARS-CoV-2
SARS-CoV-2 is a positive single-stranded RNA virus that interacts with proteins of infected cells at different stages of its life cycle. These interactions are necessary for the host to recognize and block the replication of the virus. Yet, if cells fail to block the replication of SARS-CoV-2 , host proteins are recruited to translate, transcribe and replicate the genetic material of the virus. To identify the host proteins that bind to SARS-CoV-2 RNA, we adopted the RNA-Protein Interaction Detection coupled to Mass Spectrometry (RaPID-MS) technology, which allows MS-based proteomics the purification and identification by MS-based proteomics of the proteins associated with a specific RNA of interest expressed in mammalian cells. We used RaPID-MS to specifically investigate proteins associated with the 5' and 3' end regions of SARS-CoV-2 RNA. As associations might involve non-physical protein-RNA interactions, we defined a set of reliable protein-RNA interactions by exploiting the predictive power of the catRAPID algorithm, which assesses the binding potential of proteins to a given RNA region. Among these interactors, we identified the pseudouridine synthase PUS7 that binds to both the 5' and 3' ends of SARS-CoV-2 RNA, which harbor the canonical consensus sequence modified by PUS7. We corroborated our results through nanopore direct RNA sequencing, observing that the ionic current features of these sequences are significantly different between viral infected cells and the in vitro transcribed RNA. Overall, our data suggest that SARS-CoV-2 RNA could be recognized and modified by cellular pseudouridine synthases and point to a role for this post-transcriptional modification in the viral life cycle.