CUG initiation and frameshifting enable production of dipeptide repeat proteins from ALS/FTD C9ORF72 transcripts

Abstract

Expansion of G₄C₂ repeats in the C9ORF72 gene is the most prevalent inherited form of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded transcripts undergo repeat-associated non-AUG (RAN) translation producing dipeptide repeat proteins from all reading frames. We determined cis-factors and trans-factors influencing translation of the human C9ORF72 transcripts. G₄C₂ translation operates through a 5'-3' cap-dependent scanning mechanism, requiring a CUG codon located upstream of the repeats and an initiator Met-tRNA^Met_i. Production of poly-GA, poly-GP, and poly-GR proteins from the three frames is influenced by mutation of the same CUG start codon supporting a frameshifting mechanism. RAN translation is also regulated by an upstream open reading frame (uORF) present in mis-spliced C9ORF72 transcripts. Inhibitors of the pre-initiation ribosomal complex and RNA antisense oligonucleotides selectively targeting the 5'-flanking G₄C₂ sequence block ribosomal scanning and prevent translation. Finally, we identified an unexpected affinity of expanded transcripts for the ribosomal subunits independently from translation.

Fig. 1

G₄C₂ RAN translation is length dependent and displays different efficiencies across reading frames. RNA transcripts with (G₄C₂)₃₀ or (G₄C₂)₆₆ repeats were transcribed in vitro with T7 RNA polymerase, capped or not capped and subjected to translation in rabbit reticulocyte lysate (RRL) system. Increasing RNA concentrations (100 and 200 nM) were used for translation in RRL. RAN translation was probed on immunoblot with antibodies to (a) HA tag in the +1 poly-GA frame, (b) His tag in the +2 poly-GP frame, and (c) FLAG tag in the + 3 poly-GR frame. Schematics of constructs with 30 repeats (#3) and 66 repeats (#4) are shown in Figure S1. (d) Efficiencies of RAN translation in the different frames were measured relatively to Renilla Luciferase with the corresponding tags driven by the intergenic region (IGR) IRES from the cricket paralysis virus. The efficiencies of RAN translation from capped RNAs were compared to uncapped RNAs at 100 nM for (e) poly-GA, (f) poly-GP, (g) poly-GR with 30 or 66 G₄C₂ repeats, relatively to the capped 66 repeats. Graphs represent mean ± SEM, n = 3. Student’s t-test, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001

Fig. 2

G₄C₂ RAN translation is cap-dependent and initiates with a methionine. (a) Schemes of the RNA with (G₄C₂)₃₀ (#3) or (G₄C₂)₆₆ (#4) repeats that were transcribed in vitro with T7 RNA polymerase, capped and subjected to translation in RRL. (b) Translation was performed in the presence of [³⁵S]-methionine and capped RNA #3 or #4 at 100 and 200 nM. RAN translation products were detected by autoradiography. Asterisk indicates bands in the stacking gel. (c) Translation was performed in presence of [³⁵S]-methionine and capped RNA #4 followed by immunoprecipitation with antibody against HA-tag and detection of immunoprecipitated [³⁵S]-methionine proteins by autoradiography. (d) Scheme of the canonical translation involving the cap-binding protein eukaryotic initiation factor 4E (eIF4E), the protein platform (eIF4G) and the helicase (eIF4A) that recruit the 40S ribosomal subunit. This pre-initiation complex scans the 5′ of the transcript for an appropriate start codon. Compounds used for the competition assay in (e) and (f) are represented by dark circles and squares for the cap analog (m⁷GpppG) and the inactive form (ApppG), respectively. (e–f) Translation was performed in presence of [³⁵S]-methionine, capped (G₄C₂)₆₆ RNA #4 and an increased concentration of inactive cap (control, ApppG) or cap analog (competitor of the cap, m⁷GpppG). [³⁵S]-methionine RAN translation products and poly-GA were detected by (e) autoradiography and (f) immunoblot with an antibody against HA-tag, respectively

Fig. 3

G₄C₂ RAN translation of all reading frames initiates at the same near-cognate CUG start codon in RRL. (a) Scheme of the pre-initiation complex loaded at the 5′cap and the 40S ribosomal subunit ready to scan toward the start codon. RAN translation occurs in absence of an AUG codon. (b) Schemes of the transcripts #4 to #11 showing mutations in the 5′ flanking sequence of (G₄C₂)₆₆ used in (c-f). Construct #4 contains the native sequence of 113 nucleotides upstream of the G₄C₂ repeat. Construct #5 has a CUG > CCG mutation (blue nucleotide) in a near-cognate start codon located in a perfect Kozak sequence 24 nucleotides upstream of the repeat. Construct #6 has CUG > CCG and GAG > GGG (blue nucleotides) mutations in two potential start codons located 24 and 13 nucleotides upstream of the repeat, respectively. Construct #7 contains a GAG > GGG mutation in a potential near-cognate start codon located 13 nucleotides upstream of the repeat. Constructs #8 and #9 harbor a deletion leaving 33 nucleotides (including CUG and GAG codons) and eight nucleotides (deleting both CUG and GAG codons) upstream of the repeat, respectively. Construct #10 has a CUG > AUG mutation in the near-cognate start codon. Construct #11 has GCUCUGG > UCUCUGC mutations in the Kozak sequence. (c–f) Translation was performed in presence of [³⁵S]-methionine using each RNA variant separately (#4 to #11). (c and e) [³⁵S]-methionine RAN translation products were detected by autoradiography. (d and f) Poly-GA, poly-GP, and poly-GR were detected by immunoblot using antibodies against HA-tag, His-tag, and FLAG-tag, respectively. Asterisk indicates unspecific proteins translated in the RRL system

Fig. 4

Poly-GA, poly-GP, and poly-GR RAN translation initiate at the near-cognate CUG start codon in cells. (a) Schematic representations of constructs #4 and #5 containing the near cognate start codon CUG or mutant CCG upstream of (G₄C₂)₆₆ repeats. These constructs are driven by a CMV early enhancer/chicken β actin (CAG) promoter. Human neural progenitor cells (b–c), mouse motor neuron like cells (NSC-34) (d–e) and human HEK293T cells (f–g) were co-transfected with the constructs #4 or #5 along with a GFP plasmid reporter. GFP, Hsp90 or β-Actin proteins were analyzed by immunoblot to control for the transfection efficiency and protein loading. Poly-GA, poly-GP, and poly-GR proteins were identified by immunoblot using antibodies raised against poly-GA, poly-GP, and poly-GR. Levels of the different DPR proteins were quantified and normalized to GFP, HSP90 or β-Actin. Error bars indicate SEM of three independent transfections. Student’s t- test, ** and *** indicate p < 0.01 and p < 0.001, respectively

Fig. 5

RAN translation of G₄C₂ repeats is down-regulated by a short upstream open reading frame (uORF). (a) Retention of intron 1 in C9ORF72 repeat-containing transcripts creates an uORF located 226 nucleotides upstream of the start CUG codon. This uORF may inhibit or enhance G₄C₂ RAN translation. (b) To interrogate the regulation of RAN translation by this uORF, RNAs harboring the 5′ full-length sequence including C9ORF72 exon 1A (#1) and a AUG > CCG mutation in the uORF start codon (#2) were compared to RAN translation from RNA without the uORF (#4). Black boxes represent exons 1a and 1b and the gray box represents the uORF overlapping exon 1a and intron 1. (c) Translation in RRL system was performed in presence of [³⁵S]-methionine and capped RNA (#1, #2, or #4) followed by detection of [³⁵S]-methionine proteins by autoradiography. (d) Poly-GA, poly-GP, and poly-GR were detected by immunoblots using antibodies against HA-tag, His-tag, and FLAG-tag, respectively

Fig. 6

Inhibition of RAN translation by eIFs inhibitors and RNA ASOs support a 5′–3′ scanning-dependent mechanism. (a) Illustration of translation inhibitors used to delineate the recruitment of the ribosome at the CUG start codon: 4EIRCat prevents the interaction between eIF4E (4E) and eIF4G (4G). FL3 inhibits RNA helicase eIF4A (4A). Edeine blocks the codon–anticodon interaction. Cycloheximide (CHX) blocks the translational elongation. (b) Translation was performed in presence of CHX, FL3, 4EIRCat, or Edeine in RRL followed by immunoblot detection of anti-HA (poly-GA) antibody. (c–e) HEK293T cells were transfected with the construct #4 expressing 66 G₄C₂ repeats and treated with FL3 or DMSO control. (c) Immunoblots using antibodies against poly-GA, poly-GP, poly-GR, and HSP-90 proteins. (d) Levels of poly-GA, poly-GP, and poly-GR after FL3 treatment were quantified and normalized to HSP90 and DMSO-treated cells. Graphs represent mean ± SEM, n = 5. Student’s t-test, *** indicate p < 0.001. (e) Levels of repeat-containing transcripts determined by quantitative RT-PCR and normalized to the Rplp0 transcripts and DMSO treated cells. (f) Schematic representations of construct #4 with sequences of sense (RNA-SO) and antisense (RNA-ASO) RNA oligonucleotides used to inhibit RAN translation. (g–h) Translation of capped (G₄C₂)₆₆ RNAs (construct #4) was performed in RRL in presence of two concentrations of sense or antisense RNA oligonucleotides. (g) [³⁵S]-methionine RAN translation products were detected by autoradiography. (h) Poly-GA and poly-GR were detected by immunoblot using anti-HA (Poly-GA) and -FLAG (Poly-GR) antibodies, respectively

Fig. 7

G₄C₂ containing transcripts have intrinsic ribosome binding capacity independently of their translation. (a) Scheme of the capped (G₄C₂)₆₆ RNA (#4) and uncapped (G₄C₂)₃₀ transcripts (#3) used for translation in RRL and polyribosome fractionation on sucrose gradients. As controls, capped β-globin and capped (C₂G₄)₆₆ antisense repeat RNAs were used in the same system. (b–d) Radiolabeled capped (G₄C₂)₆₆ RNA profile by polyribosome fractionation in RRL comparatively to capped β-globin mRNA and (C₂G₄)₆₆ antisense RNAs. Fractionation on sucrose gradients was performed without inhibitor (b), in presence of Edeine (c) or CHX (d). (e) Sucrose gradient fractionation of radiolabeled uncapped (G₄C₂)₃₀ transcripts (#3) was performed in presence of purified 40S or 60S ribosomal subunits

Fig. 8

Model of translation mechanisms associated with G₄C₂ expansions in C9ORF72 ALS/FTD. (a) Pre-Initiation ribosomal complex (PIC) assembles on the 5′ cap of mRNA by interacting with eIF4F complex formed by the cap binding factor eIF4E, the platform eIF4G and the RNA helicase eIF4A. The PIC complex scans the 5′ end for an appropriate AUG start codon, where the 60S ribosomal subunit joins the 40S to form a functional 80S ribosome ready to translate the coding sequence. (b) G₄C₂ RAN translation initiation shares the same pathway as the canonical one to translate poly-GA dipeptides, including the need of 5′ cap, eIF4E, eIF4G, eIF4A, initiator methionyl-tRNA, and the scanning mechanism. However, it initiates on a near-cognate CUG codon embedded in a perfect Kozak sequence, in frame with poly-GA, instead of a canonical AUG start codon. The ability of G₄C₂ expansions to form stable G-quaduplex structures forces the ribosome to occasionally undergo frameshifting to translate poly-GP and poly-GR in the +2 and +3 frames, respectively. (c) When G₄C₂ repeats are expanded, a subset of C9ORF72 mRNA is mis-spliced retaining intron 1 with the repeats. RAN translation from these RNAs is inhibited by a uORF that is translated canonically. (d) G₄C₂ expanded transcripts associate with ribosomal subunits independently from their translation