Ribosome profiling reveals novel regulation of C9ORF72 GGGGCC repeat-containing RNA translation

Abstract

GGGGCC (G4C2) repeat expansion in the first intron of C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-containing RNA is translated into dipeptide repeat (DPR) proteins, some of which are neurotoxic. Using dynamic ribosome profiling, we identified three translation initiation sites in the intron upstream of (G4C2) repeats; these sites are detected irrespective of the presence or absence of the repeats. During translocation, ribosomes appear to be stalled on the repeats. An AUG in the preceding C9ORF72 exon initiates a uORF that inhibits downstream translation. Polysome isolation indicates that unspliced (G4C2) repeat-containing RNA is a substrate for DPR protein synthesis. (G4C2) repeat-containing RNA translation is 5’ cap-independent but inhibited by the initiation factor DAP5, suggesting an interplay with uORF function. These results define novel translational mechanisms of expanded (G4C2) repeat-containing RNA in disease.

Translation of dipeptide repeat proteins inC9ORF72-ALS/FTD through unique and redundant AUG initiation codons

“…In the latter case, it is thought that repeat-associated non-AUG (RAN) translation of poly-GA and poly-GR occurs via non-canonical CUG and AGG codons, respectively 9, 10, 11, 12, 14, 21 . However, this model of RAN translation for poly-GA and poly-GR has been recently challenged, as translation of these DPRs does not depend on the presence of GGGGCC repeats 13, 14, 15, 21 . Nevertheless, our findings merged with those of previous studies suggest that DPR synthesis involves at least two different modes of translation: near-cognate start codon (e.g., CUG, AGG) dependent translation for poly-GA and poly-GR from sense GGGGCC transcripts, as well as conventional AUG dependent translation for poly-PR and poly-PG synthesis from antisense CCCCGG transcripts.…”

“…Our finding of canonical AUG codons serving as translation initiation codons for antisense DPRs (poly-PR, poly-PG) differs from the proposed mode of translation of sense DPRs (poly-GA, poly-GR). In the latter case, it is thought that repeat-associated non-AUG (RAN) translation of poly-GA and poly-GR occurs via non-canonical CUG and AGG codons, respectively 9, 10, 11, 12, 14, 21 . However, this model of RAN translation for poly-GA and poly-GR has been recently challenged, as translation of these DPRs does not depend on the presence of GGGGCC repeats 13, 14, 15, 21 .…”

“…As previously shown, non-canonical codons (viz., CUG for poly-GA, AGG for poly-GR) initiate DPR synthesis from the sense strand, suggesting an unconventional form of translation, i.e., repeat-associated non-AUG (RAN) translation 6 . However, deletion analysis of cis -regulatory elements upstream of the GGGGCC repeats and ribosome profiling revealed that translation of the poly-GA and poly-GR frames is independent of the presence of G 4 C 2 repeats 13, 14, 15 . Moreover, a recent study reported that a canonical AUG initiation codon (194 nucleotides upstream of the repeat) is used for poly-PG synthesis from the antisense CCCCGG transcript, suggesting conventional translation is involved in the synthesis of at least one DPR.…”

RNA Dysmetabolism and Repeat-Associated Non-AUG Translation in Frontotemporal Lobar Degeneration/Amyotrophic Lateral Sclerosis due to C9orf72 Hexanucleotide Repeat Expansion

“…Conversely, another group has ruled out the production of poly-GP and poly-GR by frameshifting (18) . Additionally, the presence of an AUG-initiated upstream open reading frame (uORF) of 180 bases spanning exon 1 and intron 1 of the C9orf72 gene located in the poly-GP reading frame was reported (19) . This uORF ends just before the GGGGCC repeat and translation of the uORF suppresses RAN translation in poly-GA and poly-GP frames, adding further complexity to the regulation of C9orf72 RAN translation.…”

Eukaryotic translation initiation factor eIF4G2 opens novel paths for protein synthesis in development, apoptosis and cell differentiation

“…In this model, the translation of uORF was cap‐independent, and the involvement of eIF4G2 could not promote the translation of downstream ORF. 107 …”