System-wide Profiling of RNA-Binding Proteins Uncovers Key Regulators of Virus Infection

Abstract

Summary The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed “comparative RIC” and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.

CryoSIM: super resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultra-structural imaging

“…Further samples were prepared from stable and inducible HeLa cells expressing mov10-YFP [30] to label p-bodies. HeLa cells were induced to produce mov10-YFP by supplementation of the medium with 1 mg/ml doxycycline and then infected with sindbis virus (SINV-mCherry, see below [31]) and then incubated with MitoTracker Deep Red (Thermo Fisher) at 125 nM for 30 minutes before being plunge frozen as above for imaging.…”

Viral cross-linking and solid-phase purification enables discovery of ribonucleoprotein complexes on incoming RNA virus genomes

“…As a result, peptide intensity ratios cannot be calculated for all proteins because of the low complexity of the -4SU samples. For these proteins one can use a semiquantitative approach that assumes that peptides with zero intensity values are below the detection threshold 3,24 (Figure 3). After determining the candidate "VIR-CLASP RBPs", comparisons can be made between the conditions, timepoints, viruses, or cell lines tested.…”

Structural landscape of the complete genomes of Dengue serotypes and other viral hemorrhagic fevers

“…Previous studies already showed that genomes of the Dengue viruses interact with multiple human proteins during the infection and that the protein binding can enhance or inhibit the virulence 31 . Furthermore, RNA binding proteins tend to exhibit an altered activity during viral infection, in some cases due to the presence of highly abundant viral RNA, which can compete for the interaction with cellular RNA 32 . To study the relationship between human proteins and the viral RNA structures, we selected binding motifs from RNA Bind-n-Seq (RBNS) data of 78 human RNA-binding proteins 23 , and searched the complete viral genomes for these motifs (Materials and Methods: Protein-RNA interactions).…”