Figure 1.

FMRP inhibits translation independent of mRNA G-quadruplexes in the CDS.A, schematic of full-length (residues 1–632) and MBP- and His6-tagged WT N-terminally truncated human FMRP isoform 1 (NT-hFMRP). The Agenet 1 (Ag1), Agenet 2 (Ag2), and KH0 domains are absent in WT NT-hFRMP. Ag1 and Ag2 are also referred to as Tudor domains in some previous literature. WT NT-hFRMP harbors residues 218 to 632 of full-length human FMRP isoform 1. B, Coomassie stain of recombinant WT NT-hFMRP. C, schematic of custom nLuc reporters either lacking a G4 (control reporter) or harboring a G4 in the coding sequence (G₁₅ and (GGGU)₄ reporters). A P2A ribosome skipping motif was included immediately upstream of the nLuc coding sequence to ensure equal nLuc function between reporters. D, denaturing PAGE of control, G₁₅, and (GGGU)₄ reporters stained for total RNA with SYBR Green II or for G4 structures with NMM. E, in vitro translation of control, G₁₅, and (GGGU)₄ reporter mRNA preincubated with protein buffer or 1 μM WT NT-hFMRP. Data are shown as mean ± SD. n = 3 biological replicates. Comparisons were made using a two-tailed unpaired t test with Welch’s correction. F, in vitro translation of control nLuc reporter mRNA with protein storage buffer as a negative control, with 1 μM WT NT-hFMRP and nLuc mRNA preincubated together and with 1 μM WT NT-hFMRP without a preincubation step. Data are shown as mean ± SD. n = 3 biological replicates. Comparisons were made using a two-tailed unpaired t test with Welch’s correction. CDS, coding sequence; KH, K homology.