The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS patient brains
- Columbia University, United States
- Columbia University Medical Center, United States
Abstract
An expanded GGGGCC hexanucleotide in C9ORF72 (C9) is the most frequent known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). It has been proposed that expanded transcripts adopt G-quadruplex (G-Q) structures and associate with proteins, but whether this occurs and contributes to disease is unknown. Here we show first that the protein that predominantly associates with GGGGCC repeat RNA in vitro is the splicing factor hnRNP H, and that this interaction is linked to G-Q formation. We then show that G-Q RNA foci are more abundant in C9 ALS patient fibroblasts and astrocytes compared to those without the expansion, and more frequently colocalize with hnRNP H. Importantly, we demonstrate dysregulated splicing of multiple known hnRNP H-target transcripts in C9 patient brains, which correlates with elevated insoluble hnRNP H/G-Q aggregates. Together, our data implicate C9 expansion-mediated sequestration of hnRNP H as a significant contributor to neurodegeneration in C9 ALS/FTD.
Data availability
-
Distinct brain transcriptome profiles in c9orf72-associated and sporadic ALSPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE67196).
-
Illumina mRNA-Seq of control and hnRNP H knockdown in 293T cellsPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE16642).
-
Analysis and design of RNA sequencing experiments for identifying mRNA isoform regulationPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE23694).
Article and author information
Author details
Funding
NIH Office of the Director (Training Grant)
- Erin G Conlon
NIH Office of the Director (RO1)
- James L Manley
NIH Office of the Director (R35 GM 118136)
- James L Manley
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Human subjects: Informed consent, including consent to publish, was obtained for human derived fibroblast and astrocyte lines used in this study by the IRB of Columbia University under protocols #AAAB0483 and #AAAC1257. For fibroblasts, written consent was given by the patients, and for astrocytes, written consent was given by the families of the deceased. Human tissue was donated for research purposes by the next of kin.
Copyright
© 2016, Conlon et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 7,479
- views
-
- 1,825
- downloads
-
- 238
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS patient brains
eLife 5:e17820.
https://doi.org/10.7554/eLife.17820
Further reading
-
- Biochemistry and Chemical Biology
In healthy cells, cyclin D1 is expressed during the G1 phase of the cell cycle, where it activates CDK4 and CDK6. Its dysregulation is a well-established oncogenic driver in numerous human cancers. The cancer-related function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G-Two and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unrecognized substrate of cyclin D1–CDK4/6 in tumor cells overexpressing cyclin D1 during G1 and subsequent phases. The phosphorylation of GTSE1 mediated by cyclin D1–CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 across all cell cycle phases. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1’s role in cell cycle control and oncogenesis beyond RB phosphorylation.
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.
