1. Volume 62, Issue 2, Pages 314-322 (April 2016)
CGG Repeat-Associated Non-AUG Translation Utilizes a Cap-Dependent Scanning Mechanism of Initiation to Produce Toxic Proteins 
Michael G. Kearse, Katelyn M. Green, Amy Krans, Caitlin M. Rodriguez, Alexander E. Linsalata, Aaron C. Goldstrohm, Peter K. Todd 
Molecular Cell 
Volume 62, Issue 2, Pages (April 2016)
DOI: /j.molcel
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2016 62, 314-322DOI: (10.1016/j.molcel.2016.02.034)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 A CGG RAN Translation Reporter
(A) Schematic of nanoluciferase (nLuc)-3xFLAG reporters.
(B–E) Western blots of +1 CGG RAN translation reporters show a repeat length-dependent increase in molecular weight in HeLa cells (B) and in vitro (C). A smaller molecular weight protein (∗) derived from initiation within the nLuc coding sequence (Figure S1A) is detectable in vitro. The +2 CGG RAN reporters produce high molecular weight proteins that run at the top of the gel, suggesting insolubility or aggregation at expanded sizes in HeLa cells (D) and in vitro (E). To avoid overexposure on western blots, one-tenth of AUG control reporter was used to transfect HeLa cells and in vitro reactions were diluted 1:10 in sample buffer.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
CCG RAN Translation Exhibits Differential Kinetics across Repeat Reading Frames
(A–C) Relative expression of AUG control and CGG RAN translation reporters normalized to the GGG-nLuc control reporter (A) in vitro, (B) in HeLa cells, and (C) in rat primary cortical neurons.
(D) Representative immunostaining of control and CGG RAN translation reporters in neurons.
(E) RAN translation is 30%–40% as efficient in vitro as initiation from an AUG codon placed in optimal Kozak sequence context upstream of the repeat.
(F) Comparison of reading frames shows +1 CGG RAN translation is more efficient in vitro and in HeLa cells.
(G) Initiation at an AUG start codon placed in optimal Kozak context above CGG100 repeats translating the repeat in the +0, +1, or +2 frame in vitro suggests differences in both initiation rate and translated repeat codon dictate overall RAN translation levels. Graphs represent mean ± SD, n = 3. Student’s t test, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 RAN Translation Uses Canonical Translation Machinery
(A) Expression of the canonical translation reporter (AUG) and both CGG RAN translation reporters was reduced by 200- to 800-fold in vitro when A capped compared to when m7G capped. In contrast, expression of the cap-independent CrPV IRES reporter was similar for both A capped and m7G capped.
(B) m7G-cap dependency in mRNA-transfected HeLa cells.
(C) Expression of AUG control when m7G-cap analog was titrated in vitro as a competitive inhibitor of eIF4E. ApppG cap analog was included as a negative control.
(D) 250 μM m7GpppG (red line in C) reduced +1 and +2 CGG RAN translation to ∼30% of control levels.
(E) Dose response curve for the eIF4A-specific inhibitor hippuristanol on control reporters.
(F) The 4 μM hippuristanol (red line in E) inhibited CGG RAN translation. Graphs represent mean ± SD from three independent experiments. All differences are statistically significant (Student’s t test, p < 0.01 after Bonferroni correction).
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6. Figure 4
RAN Translation Is Repeat Dependent and Initiates at Separate Sites in Different Reading Frames
(A) HeLa cells transfected with equimolar in vitro transcribed CGG RAN translation reporter mRNA harboring zero, normal, and expanded repeats.
(B) Mutation analysis of near-cognate start codons in +1 CGG0 reporter identifies initiation at ACG and GUG codons upstream of the repeat.
(C) Mutation of ACG to AAA (mt3) does not preclude initiation upon repeat expansion. For each repeat length, expression of reporters harboring mt3 are shown as the percent signal of non-mutated (WT) reporters with the same repeat length, e.g., +1 CGG100-mt3(AAA) versus +1 CGG100-WT(ACG).
(D) A UAG stop codon at the GUG position in mt5 eliminates ≥90% of activity in vitro. This mt is less effective at expanded CGG100 repeats. Expression of reporters harboring stop codons are shown as the percent signal of non-mutated (WT) reporters with the same number of repeats.
(E) The +2 reading frame of the FMR1 5′ leader contains no near-cognate start codons between a native UAG stop codon and the CGG repeat. Insertion of a stop mt just upstream of the CGG repeat does not impede +2 CGG RAN translation.
(F) Western analysis of native and mutant +2 CGG RAN translation reporter in HeLa cells. Similar results are seen in vitro (Figure S4E). Student’s t test, ∗∗p < 0.01; one-way ANOVA, ∗∗∗p <
Molecular Cell  , DOI: ( /j.molcel )
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