Journal: bioRxiv

Article Title: Genome-Wide Interrogation of SARS-CoV-2 RNA-Protein Interactions Uncovers Hidden Regulatory Sites

doi: 10.1101/2025.05.26.656146

Figure Lengend Snippet: a) Enhanced crosslinking and immunoprecipitation resolves RNA bound to SARS-CoV-2 proteins in virus infected cells. After SDS-PAGE and membrane transfer, the region corresponding to the Immunoprecipitated (IP) protein and 75kDa above is excised for RNA extraction. Size-matched input (IN) is the same region in the cell lysate to determine background crosslinked RNA. b) Read density plots show RNA enriched by N, NSP8 and NSP12, distributed across the SARS-CoV-2 genome on the positive strand. Two biological replicates were performed for each sample and condition. c) Bar plot indicating the number of significantly enriched 20 nucleotide (nt) windows of IP vs IN samples for each replicate sample and condition. Significance, log2foldchange>1.5, qvalue<0.01. d) Bar plot showing the breakdown of the number of significant windows in each region. 5UTR, 5′ untranslated region; 3UTR, 3′ untranslated region. e) Scatter plot showing the mean read density of each 20 nt window across the positive sense virus genome in N IP samples at 6h and 48h. f) Heatmap showing the read density of N IP samples (colorbar) as 200 nt sliding window mean across the SARS-CoV-2 genome.

Article Snippet: Each sample of cell lysate was immunoprecipitated using 10 µg of primary antibodies that bind Nucleocapsid, NSP12 and NSP8: SARS-CoV / SARS-CoV-2 (COVID-19) NSP8 antibody Mouse monoclonal [5A10] (GeneTex GTX632696), SARS-CoV-2 (COVID-19) RdRp (nsp12) antibody rabbit polyclonal (GeneTex GTX135467), SARS-CoV-2 (2019-nCoV) Nucleocapsid rabbit polyclonal (Sino Biological 40143-R019).

Techniques: Immunoprecipitation, Virus, Infection, SDS Page, Membrane, RNA Extraction

Journal: bioRxiv

Article Title: Genome-Wide Interrogation of SARS-CoV-2 RNA-Protein Interactions Uncovers Hidden Regulatory Sites

doi: 10.1101/2025.05.26.656146

Figure Lengend Snippet: a) Secondary structure of the region from the 5′ untranslated region to the end of the conserved stem loop 5 (SL5). Genome coordinates use positive strand numbering. Highlighted in blue are regions bound by NSP8 and NSP12, corresponding to yellow highlighted regions in b . Conserved stem loop structures (SLs), start codons (green) and stop codon (maroon) are annotated for the upper open reading frame (uORF) and the beginning of ORF1ab . Structure is predicted using RNAfold. b) Read density plots showing RNA enriched by NSP8 and NSP12 eCLIP on the positive (left) and negative (right) strand near the 5′ end of the positive sense genome. c) Secondary structure of the region from the N protein stop codon to the 3′ end of the positive strand. Highlighted in red are regions bound by NSP8 and NSP12, corresponding with yellow highlighted regions in d. Structure predicted using RNAfold and pseudoknot annotation from ref . d) Read density plots showing RNA enriched by NSP8 and NSP12 on the positive (left) and negative (right) strand near the 3′ end of the positive sense genome.

Article Snippet: Each sample of cell lysate was immunoprecipitated using 10 µg of primary antibodies that bind Nucleocapsid, NSP12 and NSP8: SARS-CoV / SARS-CoV-2 (COVID-19) NSP8 antibody Mouse monoclonal [5A10] (GeneTex GTX632696), SARS-CoV-2 (COVID-19) RdRp (nsp12) antibody rabbit polyclonal (GeneTex GTX135467), SARS-CoV-2 (2019-nCoV) Nucleocapsid rabbit polyclonal (Sino Biological 40143-R019).

Techniques: