Fig 1.
α-synuclein-induced locomotion defects and shortened lifespan.
(A) Expression of A30P α-synuclein specifically in the nervous system shortened lifespan. Survival curves were compared using the log-rank test (P<0.01 between elav>w1118 and elav>α-synuclein A30P flies). (B) There is no difference for climbing ability for flies expressing A30P α-synuclein and control genotype at day 10. In contrast, PD flies showed significant age dependent locomotive impairments at days 30 and 40 (*P<0.05). Control flies: elav>w1118; PD flies: elav>α-synuclein (A30P).
Fig 2.
Length distribution of total small RNAs in PD and control flies.
The distribution of small RNAs in control (A) and PD flies (B) was randomly selected from the data of 3 biological repeats for each group. The horizontal axis means the total read counts and vertical means the read lengths for the complete adapter-trimmed read set.
Fig 3.
Frequency of different classes of RNA in small RNA libraries.
The pie-charts represent an overview of small RNA expression in control (A) and PD flies (B). Data were random selected from 3 biological repeats for each group. Small RNAs belonging to the miRNA constitute the majority as in control (89.5%) (top) and PD (88%) (bottom) samples.
Table 1.
The total numbers of the reads at the sequencing data processing stages.
Fig 4.
Differential expression analysis of miRNA.
(A) The differentially expressed miRNAs are graphed on thescatter plot to visualize variations in miRNA expression between control and PD flies (3 biological repeats for each genotype). The values on the X-axis and Y-axis of the scatter plot are the normalized values for control and PD flies (log2 scaled). The green lines are fold-change lines (default fold-change value: 1.2). (B) The Venn diagram shows the distribution of 184 unique miRNAs between PD (left, red) and control flies (right, green) libraries. The overlapping section represents 154 miRNAs coexpressed in both genotypes. The dashed circles indicated 6 miRNAs that were significantly differentially expressed (dme-mir-13b-1 and dme-mir-13b-2 shares the same mature sequence dme-miR-13b-3p).
Fig 5.
Validation analysis for dysregulated miRNA.
qRT-PCR were performed to validate the expression of dme-miR-13b-3p, dme-miR-133-3p and dme-miR-137-3p in control and PD flies. The results were consistent with sequencing data. (* p<0.05, ** p<0.01).
Table 2.
Differentially expressed miRNAs.
Fig 6.
GO annotation of predicted targets for differentially expressed miRNAs.
Functional annotations were performed using DAVID (count cutoff 10, EASE 0.01) to analyze predicted targets for differentially expressed miRNAs. The top 20 clusters in biological process and molecular function as well as top 10 terms in cellular component were shown (p<0.05).
Fig 7.
Pathway enrichment of predicted miRNA targets.
DIANA miRPath v.2.0 was used for pathway functional annotation. Significant affected pathways (p<0.05) were shown. The results were displayed as–log p values.
Table 3.
KEGG pathway analysis for differential expressed miRNAs.
Table 4.
Target genes for differential expressed miRNAs in neuroactive-ligand receptor interaction pathway.
Fig 8.
Validation analysis for targets in neuroactive ligand-receptor interaction pathway.
The mRNA levels for targets were validated using qRT-PCR in control and PD flies. The results showed that the targets were significantly inhibited in PD flies. (* p<0.05, ** p<0.01).
Table 5.
Target genes for hsa-miR-137-3p in GABAergic synapse and Glutamatergic synapse pathway in Homo sapiens.
Fig 9.
Luciferase reporter assays confirmed dme-miR-137 could inhibit the targets in neuroactive-ligand receptor interaction pathway.
(A) 3′UTRs of Nmdar2, D2R and GABA-B-R3 containing dme-miR-137-3p binding sites predicted by DIANA—microT (shown in square) were cloned into pGL3-promoter vectors. Arrows indicated the location of primers used for amplification. (B) The pGL3-promoter vector carrying Nmdar2, D2R and GABA-B-R3 3’UTR fragments flanking miR-137 targeting sites were co-transfected with Renilla plasmid pRL-TK as well as dme-miR-137-3p mimics into HEK 293 cells. The results showed that dme-miR-137-3p could inhibit the luciferase activities for all these vectors does dependently as compared with miR-negative control. (C) The inhibitory effects were abolished when all the miR-137 targeting sites within the amplified sequences in D2R 3’UTR were mutated. (* p<0.05, ** p<0.01).