A hidden threshold in motor neuron gene networks for survival revealed by modulation of miR-218 dose
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ABSTRACT:
PROVIDER: PRJEB45888 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR6110560.fastq.gz | Fastqsanger.gz | |||
| ERR6110570.fastq.gz | Fastqsanger.gz | |||
| ERR6110580_1.fastq.gz | Fastqsanger.gz | |||
| ERR6110580_2.fastq.gz | Fastqsanger.gz | |||
| ERR6110590_1.fastq.gz | Fastqsanger.gz |
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Project description:miRNAs are post-transcriptional repressors with wide variation in cellular abundance across cell types and disease states. Yet, the transcriptomic and biological impact of altering miRNA levels (rather than binary gain or loss) has not been systematically investigated. By genetic combination, we generated an allelic series of mice expressing varying levels of miR-218, a motor neuron-specific miRNA associated with amyotrophic lateral sclerosis (ALS). Modulation of miR-218 dose causes threshold-like neuromuscular synaptogenesis and mouse viability phenotypes and revealed heterogenous dose-response curves of target mRNA repression. A dose-response network analyses unmasked a specific regulon exhibiting an inflection point in repression concomitant with the emergence of motor phenotypes. Furthermore, we find that miR-218 indirectly activates a coherent peripheral neuronal genetic signature and that the magnitude of miR-218 mediated effects varies in distinct motor subpopulations. This work reveals miRNA dose as a potent, non-linear modulator of in vivo mRNA target selection, suggesting how cellular dysfunction might abruptly arise when miRNA levels fall below a critical threshold. For the data uploaded here, motor neurons carrying an Hb9:gfp reporter were FACS isolated based upon GFP expression and used for either bulk or single cell RNA sequencing (10x genomics). Motor neurons were either mouse embryonic stem cell derived motor neurons (ESMNs) or mouse motor neurons from developmental stage E12. ESMN samples carry unique mutations of the miR-218-2 promoter of distinct sizes, or are wild type. E12 motor neurons carry combinations of mutations to either miR-218-1 or miR-218-2 or the promoter for miR-218-2. For single cell RNA sequencing, we have WT and miR-218 double knockout (DKO) motor neurons in duplicate. We also sequenced 4 samples of dorsal root ganglia from E12 mice (DRG1-4).
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Project description:Motor-neuron specific microRNA-218 (miR-218) was recently put in the spotlight because of its striking roles in mouse development. However, miR-218 relevance to human motor neuron disease was not yet explored. Here, we demonstrate by neuropathology that miR-218 is abundant in healthy human motor neurons. However, in amyotrophic lateral sclerosis (ALS) motor neurons miR-218 is downregulated and its mRNA targets are reciprocally upregulated (de-repressed). We further identify the potassium channel Kv10.1 as a new miR-218 direct target that controls neuronal activity. In addition, we screened thousands of ALS genomes and identified six rare variants in the human miR-218-2 sequence. Intriguingly, miR-218 gene variants fail to regulate neuron activity, suggesting the importance of this small endogenous RNA for neuronal robustness. The underlying mechanisms involve inhibition of miR-218 biogenesis and reduced processing by DICER. Therefore, miR-218 activity uncovers a previously unappreciated facet of motor neuron specificity that may be particularly susceptible to failure in human ALS, contributes to a view of ALS as a disease with a prominent RNA component and suggests that miR-218 is a potential therapeutic target for motor neuron disease.
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Project description:Disruption of homeostatic microRNA (miRNA) expression levels is known to cause human neuropathology. However, the gene regulatory and phenotypic effects of altering a miRNA's in vivo abundance (rather than its binary gain or loss) are not well understood. By genetic combination, we generated an allelic series of mice expressing varying levels of miR-218, a motor neuron-selective gene regulator associated with motor neuron disease. Titration of miR-218 cellular dose unexpectedly revealed complex, non-ratiometric target mRNA dose responses and distinct gene network outputs. A non-linearly responsive regulon exhibited a steep miR-218 dose-dependent threshold in repression that, when crossed, resulted in severe motor neuron synaptic failure and death. This work demonstrates that a miRNA can govern distinct gene network outputs at different expression levels and that miRNA-dependent phenotypes emerge at particular dose ranges because of hidden regulatory inflection points of their underlying gene networks.
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