Analysis of novel NEFL mRNA targeting microRNAs in amyotrophic lateral sclerosis.
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.
Molecular Medicine Research Group, Robarts Research Institute, Western University, London, Ontario, Canada.
Muhammad
M
Ishtiaq
Molecular Medicine Research Group, Robarts Research Institute, Western University, London, Ontario, Canada.
Danae
D
Campos-Melo
Molecular Medicine Research Group, Robarts Research Institute, Western University, London, Ontario, Canada ; Department of Clinical Neurological Sciences, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.
Kathryn
K
Volkening
Molecular Medicine Research Group, Robarts Research Institute, Western University, London, Ontario, Canada ; Department of Clinical Neurological Sciences, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.
Michael J
MJ
Strong
D020413:3' Untranslated Regions; D000368:Aged; D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D055029:Inverted Repeat Sequences; D008297:Male; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D008958:Models, Molecular; D009154:Mutation; D016900:Neurofilament Proteins / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D020218:Response Elements; D013116:Spinal Cord / Q000378:metabolism
D020413:3' Untranslated Regions; D035683:MicroRNAs; D016900:Neurofilament Proteins; C044882:neurofilament protein L