The Fragile X Proteins Differentially Regulate Translation of Reporter mRNAs with G-quadruplex Structures

Edwards, Madison; Joseph, Simpson

doi:10.1016/j.jmb.2021.167396

Cited by 3 publications

(6 citation statements)

References 52 publications

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“…We refer to these reporters, which code for a 3X-FLAG tag sequence, as Microreporters 1 and 2. Within its 3′- UTR, Microreporter 1 (MR1) contains a GQ sequence from p21 mRNA to which we found all the FXPs can bind . Conversely, Microreporter 2 (MR2) was designed as a negative control.…”

Section: Resultsmentioning

confidence: 99%

“…Conversely, Microreporter 2 (MR2) was designed as a negative control. MR2 contains a 3′-UTR sequence, which does not contain any GQ sequences, to which the FXPs show poor binding . This negative control 3′-UTR sequence is not biologically relevant: it was created by substituting the guanines in MR1’s 3′-UTR to cytosines to remove GQ sequences.…”

Section: Resultsmentioning

confidence: 99%

“…Surprisingly, we saw similar binding patterns by the FXPs to both MR1 and MR2, even though we expected little to no binding to MR2 (Figure B). Next, we tested the FXPs’ influence on the translation of MR1 and MR2 in an in vitro translation system (IVTS) we previously developed . Translation of MR1 and MR2 in rabbit reticulocyte lysate was quantified through [35-S] methionine labeling of the resulting 3X-FLAG peptide.…”

Section: Resultsmentioning

confidence: 99%

“…Within its 3′-UTR, Microreporter 1 (MR1) contains a GQ sequence from p21 mRNA to which we found all the FXPs can bind. 51 Conversely, Microreporter 2 (MR2) was designed as a negative control. MR2 contains a 3′-UTR sequence, which does not contain any GQ sequences, to which the FXPs show poor binding.…”

Section: ■ Resultsmentioning

confidence: 99%

“…MR2 contains a 3′-UTR sequence, which does not contain any GQ sequences, to which the FXPs show poor binding. 51 This negative control 3′-UTR sequence is not biologically relevant: it was created by substituting the guanines in MR1's 3′-UTR to cytosines to remove GQ sequences. Surprisingly, we saw similar binding patterns by the FXPs to both MR1 and MR2, even though we expected little to no binding to MR2 (Figure 2B).…”

Section: ■ Resultsmentioning

confidence: 99%

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The Fragile X Protein Disordered Regions Bind a Novel RNA Target

2022

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The fragile X proteins (FXPs) are a family of RNA-binding proteins that regulate mRNA translation to promote proper neural development and cognition in mammals. Of particular interest to researchers is the fragile X mental retardation protein (FMRP), as its absence leads to a neurodevelopmental disorder: fragile X syndrome (FXS), the leading monogenetic cause of autism spectrum disorders. A primary focus of research has been to determine mRNA targets of the FXPs in vivo through pull-down techniques, and to validate them through in vitro binding studies; another approach has been to perform in vitro selection experiments to identify RNA sequence and structural targets. These mRNA targets can be further investigated as potential targets for FXS therapeutics. The most established RNA structural target of this family of proteins is the G-quadruplex. In this article, we report a 99 nucleotide RNA target that is bound by all three FXPs with nanomolar equilibrium constants. Furthermore, we determined that the last 102 amino acids of FMRP, which includes the RGG motif, were necessary and sufficient to bind this RNA target. To the best of our knowledge, this is one of only a few examples of non-G-quadruplex, non-homopolymer RNAs bound by the RGG motif/C-termini of FMRP.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: ■ Resultsmentioning

confidence: 99%

Section: ■ Resultsmentioning

confidence: 99%

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