Dataset Information

SPIDR: a highly multiplexed method for mapping RNA-protein interactions uncovers a mechanism for selective translational suppression upon cellular stress

ABSTRACT: RNA binding proteins (RBPs) play crucial roles in regulating every stage of the mRNA life cycle and mediating non-coding RNA functions. Despite their importance, the specific roles of most RBPs remain unexplored because we do not know their specific RNA binding partners. Current methods, such as crosslinking and immunoprecipitation followed by sequencing (CLIP-seq), have expanded our knowledge of RBP-RNA interactions but are generally limited by their ability to map only one RBP at a time. To address this limitation, we developed SPIDR (Split and Pool Identification of RBP targets), a massively multiplexed method to simultaneously profile global RNA binding sites of dozens to hundreds of RBPs in a single experiment. SPIDR employs antibody-bead labeling coupled with split-pool barcoding to increase the throughput of current CLIP methods by two orders of magnitude. SPIDR identifies precise, single-nucleotide RNA binding sites for diverse classes of RBPs simultaneously. We identified several novel ribosomal RNA binders, including a novel interaction between LARP1 and 18S ribosomal RNA located within the mRNA entry site on the 40S small ribosomal subunit, and resolved this structure at 2.8 A using single-particle cryo-electron microscopy (cryo-EM). This structure provides a potential mechanistic explanation for the role of LARP1 in translational suppression. We next explored SPIDR ability to detect changes in RBP binding upon mTOR inhibition and identified that 4EBP1 acts as a dynamic RBP that selectively binds to 5-untranslated regions of translationally repressed mRNAs only upon mTOR inhibition. These observations provide a potential mechanism to explain the specificity of translational regulation controlled by mTOR signaling. SPIDR enables rapid, de novo discovery of RNA-protein interactions at an unprecedented scale and has the potential to transform our understanding of RNA biology and both transcriptional and post-transcriptional gene regulation.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Marko Jovanovic