Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by progressive motor neuron degeneration and neurofilament aggregate formation. Spinal motor neurons in ALS also show a selective suppression in the levels of low molecular weight neurofilament (NEFL) mRNA. We have been interested in investigating the role of microRNAs (miRNAs) in NEFL transcript stability. MiRNAs are small, 20–25 nucleotide, non-coding RNAs that act as post-transcriptional gene regulators by targeting the 3′ untranslated region (3′UTR) of mRNA resulting in mRNA decay or translational silencing. In this study, we characterized putative novel miRNAs from a small RNA library derived from control and sporadic ALS (sALS) spinal cords. We detected 80 putative novel miRNAs, 24 of which have miRNA response elements (MREs) within the NEFL mRNA 3′UTR. From this group, we determined by real-time PCR that 10 miRNAs were differentially expressed in sALS compared to controls. Functional analysis by reporter gene assay and relative quantitative RT-PCR showed that two novel miRNAs, miR-b1336 and miR-b2403, were downregulated in ALS spinal cord and that both stabilize NEFL mRNA. We confirmed the direct effect of these latter miRNAs using anit-miR-b1336 and anti-miR-b2403. These results demonstrate that the expression of two miRNAs (miRNAs miR-b1336 and miR-b2403) whose effect is to stabilize NEFL mRNA are down regulated in ALS, the net effect of which is predicted to contribute directly to the loss of NEFL steady state mRNA which is pathognomic of spinal motor neurons in ALS.
Highlights
Amyotrophic lateral sclerosis (ALS; Lou Gehrig’s disease) is an adult onset neurodegenerative disease characterized by progressive muscle weakness which leads to paralysis and death by respiratory failure [1,2]
This hypothesis is further supported by the observation that the stability of NEFL mRNA is regulated by several RNA binding proteins that have been linked to the pathogenesis of ALS, including mutant superoxide dismutase 1, TAR DNA binding protein of 43 kDa (TDP-43) and Rho guanine nucleotide exchange factor (RGNEF) [9,15,16,17]
Recognizing that such commercial arrays are limited to known miRNAs, we have generated a library of small RNAs from human spinal cord and analyzed the sequencing data from novel short RNA species that may be miRNAs
Summary
Amyotrophic lateral sclerosis (ALS; Lou Gehrig’s disease) is an adult onset neurodegenerative disease characterized by progressive muscle weakness which leads to paralysis and death by respiratory failure [1,2]. Motor neurons show a selective decrease in the steady state of low molecular weight NF (NEFL) mRNA levels altering the stoichiometry of NF expression [10,11,12,13] This is consistent with the hypothesis that alterations in RNA metabolism play a central role in ALS [14]. This hypothesis is further supported by the observation that the stability of NEFL mRNA is regulated by several RNA binding proteins that have been linked to the pathogenesis of ALS, including mutant superoxide dismutase 1 (mtSOD1), TDP-43 and RGNEF [9,15,16,17]. Both mtSOD1 and RGNEF destabilize NEFL mRNA, while TDP-43 stabilizes the transcript [9,16,18]
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