UC San Diego

Fragile X syndrome (FXS) is a neurodevelopmental disorder and is the most common form of inherited intellectual disability. The multiple CGG repeats in the 5’ UTR of the Fragile X mental retardation gene 1(FMR1) lead to transcription silencing, the loss of FMRP production and ultimately leading to FXS. FMRP is an RNA-binding protein and the previous cryo-EM structure of the Drosophila ribosome-FMRP complex revealed that FMRP binds in the intersubunit space near the A-site; despite this structure, details of ribosome recognition by FMRP remain unclear.To better understand the interaction of FMRP with the ribosome, here we present a new biochemical technique to map the binding site of FMRP on the ribosome. Two fusion proteins were designed by either fusing Micrococcal nuclease (MNase) on NT-dFMRP’s N- or C- terminus (MNase-FMRP and FMRP-MNase). Each fusion protein was bound to Drosophila ribosomes, and the MNase was activated to cleave rRNAs in the vicinity of FMRP. The rRNA was sequenced using Oxford Nanopore, revealing the cleavage sites. The cleavage sites were in the 18S rRNA in the head domain of the 40S ribosomal subunit. The location of the cleavage sites in the 40S subunit head domain is consistent with the position of NT-dFMRP on the ribosome in the previous cryo-EM structure.