Retention of hexanucleotide repeat-containing intron in C9orf72 mRNA: implications for the pathogenesis of ALS/FTD

Abstract

IntroductionThe most common forms of amyotrophic lateral sclerosis and frontotemporal dementia are caused by a large GGGGCC repeat expansion in the first intron of the C9orf72 gene. The repeat-containing intron should be degraded after being spliced out, however GGGGCC repeat-containing RNA species either accumulate in nuclear foci or are exported to the cytoplasm where they are translated into potentially toxic dipeptide repeat proteins by repeat-associated non-AUG-initiated (RAN) translation.ResultsIn order to determine the mechanisms of repeat-containing intron misprocessing, we have analyzed C9orf72 transcripts in lymphoblasts from C9orf72 expansion carriers (n = 15) and control individuals (n = 15). We have identified polyadenylated C9orf72 RNA species retaining the repeat-containing intron and in which downstream exons are spliced correctly resulting in a C9orf72 mRNA with an enlarged 5’-UTR containing the GGGGCC repeats. Intron-retaining transcripts are produced from both wild-type and mutant alleles. Intron-retaining C9orf72 transcripts were also detected in brain with a 2.7 fold increase measured in the frontal cortex from heterozygous expansion carriers (n = 11) compared to controls (n = 10). The level of intron-retaining transcripts was increased 5.9 fold in a case homozygous for the expansion. We also show that a large proportion of intron 1-retaining C9orf72 transcripts accumulate in the nucleus.ConclusionsRetention of the repeat-containing intron in mature C9orf72 mRNA can potentially explain nuclear foci formation as well as nuclear export of GGGGCC repeat RNA and suggests that the misprocessing of C9orf72 transcripts initiates the pathogenic process caused by C9orf72 hexanucleotide repeat expansions as well as provides the basis for novel therapeutic strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0289-4) contains supplementary material, which is available to authorized users.

Knocking out C9ORF72 Exacerbates Axonal Trafficking Defects Associated with Hexanucleotide Repeat Expansion and Reduces Levels of Heat Shock Proteins

“…We produced a complete set of isogenic human iPSC-derived MNs to assess the contributions of LOF and GOF to ALS phenotypes. Because C9ORF72 transcript levels are reduced by the HRE, which can also retain the repeat expansion in ALS patients (Niblock et al, 2016), it is possible that both mechanisms contribute to pathogenesis. To test the possibility that reducing C9ORF72 protein levels could exacerbate ALS pathogenesis in MNs with HRE, we created isogenic MNs with a C9ORF72 KO as well as HRE.…”

RAN translation—What makes it run?

“…3B), however, is not altered in C9 iPSC-derived neurons and patient brain tissue relative to controls, arguing against significant production of truncated transcripts or increased intron retention (Tran et al , 2015). However, a recent study suggests that intron retention occurs with some frequency in both control and C9 patient cells (Niblock M et al , 2016), and this may only have pathological consequences when the expanded repeat is present. Therefore, the lack of increased retention does not rule out the possibility that such transcripts undergo RAN translation in C9 patients.…”

SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits

“…A small proportion of C9ORF72 repeat transcripts retaining pathological repeat expansions in intron-1 escape nuclear retention mechanisms and were detected in the cytoplasm of patient-derived lymphoblasts28 where they can subsequently be translated into DPRs. Interestingly, nucleocytoplasmic transport defects of proteins and RNA were recently highlighted in Drosophila , yeast and human neuronal models of C9ORF72 -related ALS29303132.…”