Fig 1: MiR-b2122 alters protein levels of TDP-43, FUS/TLS and RGNEF within a human neuronal cell line. Changes in protein levels of TDP-43, FUS/TLS and RGNEF were studied when SH-SY5Y cells were transfected with either miR-b2122, miR-b2122 plus its anti-miR, or the anti-miR alone. Transfection of let-7a was used as a negative control. a Western blot showing expression of TDP-43, FUS/TLS, RGNEF, and GAPDH (b) Quantification of Western blots for TDP-43, FUS/TLS and RGNEF protein levels. TDP-43 and FUS/TLS show small reductions in protein levels when transfected with miR-b2122 alone, while their protein levels increased when the anti-miR is added. Differences in protein levels when miR-b2122 or the anti-miR are added alone are significantly different for both TDP-43 and FUS/TLS. RGNEF has increased and decreased protein levels when either miR-b2122 or its anti-miR are added alone, respectively, and these differences are significantly different from one another. Protein levels were normalized to GAPDH. Data was expressed as the mean ± SEM, and significance was determined using a one-way ANOVA followed by a Tukey’s post-hoc (** = p < 0.01, * = p < 0.05)
Fig 2: MiR-b2122 reduces firefly luciferase activity when it contains either the TARDBP, FUS/TLS or RGNEF 3’UTR. HEK293T cells were transfected with a pmirGLO plasmid containing the 3’UTR of one of the RNA-binding proteins of interest either with or without miR-194 or miR-b2122. PmirGLO plasmid without any 3’UTRs were also transfected with or without miRNAs of interest to determine the miRNAs effect on the plasmid itself. a Reporter gene assay revealed miR-b2122 reduced firefly activity when it contained either the TARDBP, FUS/TLS, or RGNEF-short/long 3’UTR, whereas miR-194 down-regulated firefly levels when it contained either TARDBP, FUS/TLS, but not RGNEF-long. MiR-194 has no MRE in RGNEF-short, and thus, the interaction between the two was not examined. b RT-qPCR results showed similar suppression of mRNA levels as seen to the luciferase activity observed in the reporter gene assay. Let-7a was used as a negative control for these experiments. Firefly was normalized to renilla luciferase, and then further normalized to account for the effect of each miRNA on the pmirGLO vector to determine the exact effect that each miRNA has on each 3’UTR. Each miRNA was compared to its own individual control based on the normalization of the data. Data is expressed as sample mean ± SEM, and significance was determined using a Student’s t-test (*** = p < 0.001, ** = p < 0.01, * = p < 0.05, NS = p > 0.05)
Fig 3: MiR-b2122 and miR-194 directly interact with their 3’UTR targets. HEK293T cells were co-transfected with either the pmirGLO plasmid containing the wild-type 3’UTR, or the 3’UTR mutant, and either with or without the miRNA of interest. Mutations within the MRE of miR-b2122 in the TARDBP, FUS/TLS, and RGNEF 3’UTRs, and the MRE of miR-194 in the TARDBP and FUS/TLS 3’UTRs abolished each miRNAs ability to reduce firefly activity. Firefly was normalized to renilla, and then further normalized to the effect of each miRNA on the pmirGLO vector to determine the miRNAs exact effect on the 3’UTR. Data is expressed as sample mean ± SEM, and significance was determined using a student’s t-test (*** = p < 0.001, ** = p < 0.01, * = p < 0.05)
Fig 4: MiR-b2122 regulates mRNA expression of TARDBP, FUS/TLS and RGNEF in a human neuronal cell line. SH-SY5Y cells were transfected individually with miR-194 or miR-b2122, or co-transfected with miR-194 or miR-b2122, and their anti-miRs. Transfection of let-7a was used as a negative control. a Transfection of miR-b2122 significantly down-regulates the mRNA levels of TARDBP, FUS/TLS and RGNEF, while co-transfection of miR-b2122 with its anti-miR led to a recovery in the mRNA levels. Transfection of the anti-miR alone lead to increased mRNA expression of all three RNA-binding proteins (b) Transfection of miR-194 alone resulted in reduction of TARDBP and FUS/TLS, which was abolished when it was co-transfected with its anti-miR. No effect was observed on RGNEF when miR-194 and/or its anti-miR were transfected. Data was expressed as the mean ± SEM, and significance was determined using a one-way ANOVA followed by a Tukey’s post-hoc. (*** = p < 0.001, ** = p < 0.01 * = p < 0.05)
Fig 5: A small group of miRNAs have MREs within the 3’UTRs of TARDBP, RGNEF and FUS/TLS. a 3’RACE PCR identified the 3’UTRs of TARDBP, FUS/TLS and RGNEF that are expressed in the human spinal cord tissue within three different control samples. One isoform of both TARDBP and FUS/TLS were identified with a length of 1398 and 150 bases, respectively. Two isoforms of RGNEF, which we have termed RGNEF-short (RGNEF-S) and RGNEF-long (RGNEF-L) 3’UTRs, were identified in all three subjects running at 177 and 981 bases, respectively. Bands in figures appear higher than actual 3’UTR size, as primers were designed upstream from stop codon. b Five miRNAs were identified to have binding sites within the TARDBP, FUS/TLS and RGNEF mRNA 3’UTRs. MiR-548d-3p in previous work has shown to have no dysregulation in sALS, and thus, four miRNAs (outlined in the black box) went under further study. c Schematic of all TARDBP, FUS/TLS and RGNEF 3’UTRs identified within human spinal cord, and location of MREs for miRNA candidates
Supplier Page from Abcam for Anti-RGNEF antibody