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.
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).
- Erin G Conlon
- James L Manley
- James L Manley
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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.
- Douglas L Black, University of California, Los Angeles, United States
- Received: May 13, 2016
- Accepted: September 8, 2016
- Accepted Manuscript published: September 13, 2016 (version 1)
- Accepted Manuscript updated: September 14, 2016 (version 2)
- Accepted Manuscript updated: September 17, 2016 (version 3)
- Accepted Manuscript updated: September 21, 2016 (version 4)
- Version of Record published: October 4, 2016 (version 5)
- Version of Record updated: October 7, 2016 (version 6)
- Version of Record updated: October 10, 2016 (version 7)
© 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.
Profilin-1 (PFN1) is a cytoskeletal protein that regulates the dynamics of actin and microtubule assembly. Thus, PFN1 is essential for the normal division, motility, and morphology of cells. Unfortunately, conventional fusion and direct labeling strategies compromise different facets of PFN1 function. As a consequence, the only methods used to determine known PFN1 functions have been indirect and often deduced in cell-free biochemical assays. We engineered and characterized two genetically encoded versions of tagged PFN1 that behave identical to each other and the tag-free protein. In biochemical assays purified proteins bind to phosphoinositide lipids, catalyze nucleotide exchange on actin monomers, stimulate formin-mediated actin filament assembly, and bound tubulin dimers (kD = 1.89 µM) to impact microtubule dynamics. In PFN1-deficient mammalian cells, Halo-PFN1 or mApple-PFN1 (mAp-PEN1) restored morphological and cytoskeletal functions. Titrations of self-labeling Halo-ligands were used to visualize molecules of PFN1. This approach combined with specific function-disrupting point-mutants (Y6D and R88E) revealed PFN1 bound to microtubules in live cells. Cells expressing the ALS-associated G118V disease variant did not associate with actin filaments or microtubules. Thus, these tagged PFN1s are reliable tools for studying the dynamic interactions of PFN1 with actin or microtubules in vitro as well as in important cell processes or disease-states.
Activation of G protein-coupled receptors (GPCRs) is an allosteric process. It involves conformational coupling between the orthosteric ligand binding site and the G protein binding site. Factors that bind at non-cognate ligand binding sites to alter the allosteric activation process are classified as allosteric modulators and represent a promising class of therapeutics with distinct modes of binding and action. For many receptors, how modulation of signaling is represented at the structural level is unclear. Here, we developed FRET sensors to quantify receptor modulation at each of the three structural domains of metabotropic glutamate receptor 2 (mGluR2). We identified the conformational fingerprint for several allosteric modulators in live cells. This approach enabled us to derive a receptor-centric representation of allosteric modulation and to correlate structural modulation to the standard signaling modulation metrics. Single-molecule FRET analysis revealed that a NAM increases the occupancy of one of the intermediate states while a PAM increases the occupancy of the active state. Moreover, we found that the effect of allosteric modulators on the receptor dynamics is complex and depend on the orthosteric ligand. Collectively, our findings provide a structural mechanism of allosteric modulation in mGluR2 and suggest possible strategies for design of future modulators.