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. 2020 Oct 13;59(40):3813-3822.
doi: 10.1021/acs.biochem.0c00534. Epub 2020 Sep 29.

The Human Fragile X Mental Retardation Protein Inhibits the Elongation Step of Translation through Its RGG and C-Terminal Domains

Youssi M Athar  1 , Simpson Joseph  1
Affiliations

The Human Fragile X Mental Retardation Protein Inhibits the Elongation Step of Translation through Its RGG and C-Terminal Domains

Youssi M Athar et al. Biochemistry. .

Abstract

The fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates the translation of numerous mRNAs in neurons. The precise mechanism of translational regulation by FMRP is unknown. Some studies have indicated that FMRP inhibits the initiation step of translation, whereas other studies have indicated that the elongation step of translation is inhibited by FMRP. To determine whether FMRP inhibits the initiation or the elongation step of protein synthesis, we investigated m7G-cap-dependent and IRES-driven, cap-independent translation of several reporter mRNAs in vitro. Our results show that FMRP inhibits both m7G-cap-dependent and cap-independent translation to similar degrees, indicating that the elongation step of translation is inhibited by FMRP. Additionally, we dissected the RNA-binding domains of hFMRP to determine the essential domains for inhibiting translation. We show that the RGG domain, together with the C-terminal domain (CTD), is sufficient to inhibit translation, while the KH domains do not inhibit mRNA translation. However, the region between the RGG domain and the KH2 domain may contribute as NT-hFMRP shows more potent inhibition than the RGG-CTD tail alone. Interestingly, we see a correlation between ribosome binding and translation inhibition, suggesting the RGG-CTD tail of hFMRP may anchor FMRP to the ribosome during translation inhibition.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

Figures

Figure 1.
Figure 1.
Human FMRP constructs. hFMRP is the full-length human FMRP isoform 1, spanning amino acids E2-P632, alongside the truncation constructs that were used. GST-hFMRP SGG is a fusion between the glutathione S-transferase and the RGG motif-containing sequence of hFMRP spanning amino acids G531-P632 where the 16 arginines spanning the RGG motif to the C-terminus were mutated to serines, illustrated using a different color for the C-terminus. Amino acids of hFMRP found within each construct are denoted to the right of the respective construct in parentheses.
Figure 2.
Figure 2.
Human FMRP inhibits translation of different mRNAs in RRL. (a) Diagram of Renilla luciferase (RLuc) mRNAs containing: both 5’-m7G cap and 3’-polyA25 tail (+/+, blue), only 3’-polyA25 tail (−/+, yellow), only 5’-m7G cap (+/−, green), or neither 5’-m7G cap nor 3’-polyA25 tail (−/−, black). (b) Relative luminescence units (RLU) from 10 nM of the four aforementioned Renilla luciferase mRNAs all as a percentage of the (+/+) RLuc mRNA. (c) RLU from 10 nM RLuc mRNAs (+/+) in blue and (−/+) in yellow upon the addition of 500 nM NT-hFMRP, all as a percentage of the (+/+) RLuc mRNA. (d) RLU from 10 nM Renilla luciferase mRNA (blue) and 10 nM NanoLuc luciferase mRNA (maroon) upon the addition of 0–1000 nM NT-hFMRP in RRL as a percentage of the respective control mRNA sample containing no NT-hFMRP. The standard deviations from three experiments are shown. P < 0.0001 is denoted with four asterisks (*) and P < 0.001 is denoted with three asterisks (*).
Figure 3.
Figure 3.
FMRP inhibits translation of canonical and IRES-driven Renilla luciferase mRNAs. (a) Relative luminescence units (RLU) from Renilla luciferase upon the addition of 0–400 ng of the indicated mRNAs in RRL. (b) RLU from canonical Renilla luciferase mRNA (blue), EMCV IRES-driven Renilla luciferase mRNA (red), and CrPV IRES-driven Renilla luciferase mRNA (green) upon the addition of 0–1000 nM NT-hFMRP in RRL as a percentage of the respective control mRNA sample containing no NT-hFMRP. The standard deviations from three experiments are shown. P value with no significant difference is denoted with NS.
Figure 4.
Figure 4.
The RGG domain and CTD of FMRP are required for inhibition of translation. (a) Relative luminescence units (RLU) from Renilla luciferase upon the addition of 0–1000 nM NT-hFMRP (blue), GST-hFMRP RGG (orange), GST-hFMRP SGG (orange with black diagonal stripes), GST-hFMRP KH0-KH1-KH2 domains (teal), and RGG peptide (black) in RRL as a percentage of the respective control mRNA sample containing no hFMRP. (b) 12% SDS-PAGE of GST-hFMRP RGG before (- TEV protease) and after (+ TEV protease) cleaving with TEV protease. (c) RLU from Renilla luciferase upon the addition of 0–1000 nM GST-hFMRP RGG (orange) and GST-hFMRP RGG pre-cleaved using TEV protease (orange with white horizontal stripes) in RRL as a percentage of the respective control mRNA sample containing no GST-hFMRP RGG. The standard deviations from three experiments are shown.
Figure 5.
Figure 5.
NT-hFMRP and GST-hFMRP RGG bind directly to the human 80S ribosome. (a) A representative 10% SDS-PAGE of 40% sucrose co-sedimentation assay showing NT-hFMRP (blue dot) co-sediments with human 80S ribosomes (h80S). Samples in lanes 4 to 6 were sedimented through the sucrose cushion. Lanes: 1, molecular weight ladder; 2, input h80S; 3, input NT-hFMRP; 4, NT-hFMRP in pellet; 5, h80S in pellet; 6, h80S+NT-hFMRP complex in pellet. NT-hFMRP migrated close to a ribosomal protein; however, quantification showed that the band intensity of the NT-hFMRP and the ribosomal protein in lane 6 increased by ~30% compared to the same ribosomal protein band in lane 5. (b) A representative 10% SDS-PAGE of 40% sucrose co-sedimentation assay showing GST-hFMRP RGG (orange dot) co-sediments with human 80S ribosomes, but GST-hFMRP KH0-KH1-KH2 (teal dot) does not. Samples in lanes 4 to 8 were sedimented through the sucrose cushion. Lanes: 1, molecular weight ladder; 2, input h80S; 3, input KH0-KH1-KH2; 4, KH0-KH1-KH2 in pellet; 5, h80S+KH0-KH1-KH2 in pellet; 6, h80S in pellet; 7, h80S+GST-hFMRP RGG in pellet; 8, GST-hFMRP RGG in pellet; 9, input GST-hFMRP RGG; 10, molecular weight ladder. GST-hFMRP RGG migrated close to a ribosomal protein; however, quantification showed that the band intensity of the GST-hFMRP RGG and the ribosomal protein in lane 7 increased by ~40% compared to the same ribosomal protein band in lane 6. (c) Model for the inhibition of translation by FMRP. RNA GQ structures are present in both the rRNAs and in the mRNA (yellow and black structures). Top: The RGG-CTD tail of FMRP binds to a GQ structure in an rRNA expansion segment and the KH domains interact with a functional site on the ribosome to inhibit translation. Bottom: The RGG-CTD tail of FMRP binds to a GQ structure in the mRNA and the KH domains interact with a functional site on the ribosome to inhibit translation. Labels: FMRP (red), 80S ribosome (blue), rRNA expansion segments (wavy dark blue lines), GQ structures (yellow and black), and mRNA (black).
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