Identification and enrichment of SECReTE cis-acting RNA elements in the Coronaviridae and other (+) single-strand RNA viruses

Gal Haimovich; Tsviya Olender; Camila Baez; Jeffrey E Gerst

The ER is the primary site for the translation and translocation of soluble secreted and membrane (secretome) proteins. Thus, vRNA interactions with ER-associated RNA-binding proteins (RBPs) likely constitute a critical rate-limiting step in VP production. In our work on RNA trafficking and association with intracellular organelles as a means to regulate protein translation and localization, we recently identified a cis RNA element present in nearly all secretome proteins from bacteria to humans 6 . This motif, entitled "secretion-enhancing cis regulatory targeting element" (SECReTE), is based upon extended (>10 consecutive) triplet nucleotide repeats whereby a pyrimidine nucleotide is present every third base, whether in coding regions (as NYN or NNY; where N is any nucleotide and Y = U or C) or in the UTR regions. Mutational analyses performed using several yeast genes encoding secreted proteins (e.g. SUC2, CCW12, HSP150) revealed that the addition or removal of SECReTE motifs in yeast mRNAs could enhance or inhibit mRNA stability and association with the ER, respectively, thereby . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.20.050088 doi: bioRxiv preprint affecting protein secretion and cell physiology. Thus, SECReTE is important for mRNA-protein interactions at the level of the ER that facilitate protein production. We now identify numerous SECReTE motifs encoded in many of SARS-CoV-2 VPs, particularly in those encoding membrane-associated proteins. Viral SECReTE motifs (vSECReTEs) were found in the genes of all ssRNA viruses inspected and its abundance (i.e. SECReTE/kb) correlates overall with the percentage of C and T nucleotides (%CT) in the genomes (we use "T" instead of "U" henceforth for the convenience of sequence analysis). However, when looking at specific %CT levels we see a wide variability in SECReTE score between viral families/genera. Importantly, we found that (+)ssRNA viruses (e.g. Coronaviridae and Hepaciviruses) that utilize ER-derived DMVs for replication centers [3] [4] [5] 7 contain more SECReTE motifs per kb as compared to (-)ssRNA viruses (e.g. Rhabdoviridae, Orthomyxoviridae, and Paramyxoviridae), which replicate in the nucleus or the cytoplasm [8] [9] [10] [11] [12] [13] , or other (+)ssRNA viruses which use different organellar membranes and do not form ER-derived DMVs (e.g. Enteroviruses, Nodavirridae, and Flaviviruses) 4, 5, 7, 14 . Interestingly, the position of some vSECReTE motifs along the length of the Spike gene of all seven human coronaviruses is quite similar. This co-occurrence may indicate the possibility of conservation/convergence of motif position. Thus, we predict that SECReTE motifs may be important for the association of viral RNA with the ER, as well as for the efficient translation of viral membrane proteins and creation of VRCs at ER membranes. Thus, continued studies of SECReTE function and the identification of SECReTEbinding proteins could provide new drug targets to treat COVID-19, and other (+)ssRNA related diseases.