Overview of multiomics data
The DE genes and proteins in Drosophila between day 7 and day 42 were screened, and their networks were analysed (Table 1). A total of 537 DE mRNAs, 348 DE proteins, and 43 DE lncRNAs were obtained from a previous study in our laboratory (Dataset S1). A total of 6003 circRNAs and 226 miRNAs were identified at day 7 and day 42 (Dataset S2). Ultimately, 29 DE circRNAs and 30 DE miRNAs were found (Dataset S1). The merged sequences of novel circRNAs (Dataset S3, Dataset S4, and Dataset S5) and lncRNAs (Dataset S6) are shown in supplementary files.
Table 1. DE mRNA, proteins, lncRNAs, circRNAs, and miRNAs at day 42 compared to day 7
|
mRNA
|
Proteins
|
LncRNAs
|
CircRNAs
|
miRNAs
|
Total
|
537
|
348
|
43
|
29
|
24
|
Upregulated
|
194
|
134
|
15
|
21
|
11
|
Downregulated
|
343
|
214
|
28
|
8
|
13
|
DE circRNAs and miRNAs in Drosophila between day 7 and day 42
The DE circRNAs and miRNAs between 7 and 42-day-old Drosophila were analysed. Between day 7 and day 42, 29 DE circRNAs in Drosophila were identified, including 21 upregulated and 8 downregulated circRNAs at day 42 (Table 2). The circRNAs were derived from different source genes. Furthermore, 24 DE miRNAs (11 upregulated and 13 downregulated) were screened between day 7 and day 42 (Figure 1A). Dme-miR-9a-3p and dme-miR-985-3p were identified as canonical specific fruit fly miRNAs. Then, dme-miR-956-3p, dme-miR-284-3p, and dme-miR-289-5p were identified as non-canonical miRNAs. The rest of 19 DE miRNAs were canonically conserved miRNAs.
Furthermore, functional annotation was carried out for the source genes of DE circRNAs and DE miRNAs. These source genes were found to be involved in multiple molecular functions (Table 2). Evidently, the biological processes of the short lifespan-related source genes arm and pan were involved in the Wnt signalling pathway and had similar molecular functions, such as binding, protein binding, transcription factor binding, and transcription regulator activity. In addition, different circRNAs were observed to originate from the same mRNA transcript. For example, dme_circ_0008175 and dme_circ_0008173 were originated from Nlg1 gene, and dme_circ_0009514 and d me_circ_0009500 were derived from pan gene. The Gene Ontology (GO) annotations of ageing-related DE miRNAs were investigated, and 17 DE miRNAs were clustered with 20 GO terms (Figure 1B), including cell fate commitment, programmed cell death, developmental growth, and regulation of circadian rhythm terms.
Table 2. DE circRNAs of Drosophila between day 7 and day 42
ID
|
CircBase ID
|
Fold change
|
P value
|
Source gene
|
Portion of biological process term(s) from Flybase database
|
Dme_circ_0009372
|
Dme_circ_0005033
|
4.5034↑
|
0.007635
|
Asator
|
Protein serine/threonine kinase activity; ATP binding
|
Dme_circ_0006708
|
Dme_circ_0005241
|
4.2791↑
|
0.012415
|
Dad
|
TGF-beta signalling pathway; negative regulation of BMP signalling pathway
|
Dme_circ_0004259
|
Dme_circ_0002098
|
4.2083↑
|
0.014401
|
shot
|
Cytoplasmic microtubule organization; wound healing; branching involved in open tracheal system development; cilium organization
|
Dme_circ_0002070
|
Dme_circ_0002195
|
3.9032↑
|
0.026301
|
Scp1
|
-
|
Dme_circ_0010408
|
Dme_circ_0003710
|
3.8118↑
|
0.031385
|
CoRest
|
Negative regulation of transcription by RNA polymerase II; positive regulation of DNA methylation-dependent heterochromatin assembly; negative regulation of histone H4-K16 acetylation; negative regulation of histone H3-K27 methylation
|
Dme_circ_0003904
|
Dme_circ_0003738
|
3.5948↑
|
0.045349
|
CG33144
|
Ubiquitin-dependent protein catabolic process
|
Dme_circ_0008175
|
Dme_circ_0001709
|
3.5948↑
|
0.045412
|
Nlg1
|
Neuromuscular junction development; cellular process
|
Dme_circ_0005030
|
Dme_circ_0002884
|
3.5765↑
|
0.046714
|
Ccn
|
Negative regulation of cell death; signal transduction; cell adhesion
|
Dme_circ_0006667
|
Dme_circ_0001321
|
3.5765↑
|
0.046714
|
gish
|
Positive regulation of Wnt-TCF and hedgehog signalling pathways; negative regulation of Hippo signalling pathway
|
Dme_circ_0010536
|
Dme_circ_0000629
|
3.559↑
|
0.048096
|
slgA
|
Arginine and proline metabolism
|
Dme_circ_0008383
|
Dme_circ_0004519
|
2.6054↑
|
0.043863
|
mura
|
Protein ubiquitination; long-term memory
|
Dme_circ_0010134
|
Dme_circ_0002087
|
1.1067↑
|
0.039466
|
Stim
|
Developmental process; cellular homeostasis
|
Dme_circ_0009358
|
-
|
0.74476↑
|
0.002481
|
CaMKI
|
Protein phosphorylation
|
Dme_circ_0000626
|
-
|
3.7656↑
|
0.03384
|
CG17646
|
Triglyceride metabolic process; transmembrane transport
|
Dme_circ_0011075
|
-
|
3.7618↑
|
0.03415
|
Trf2
|
Post-embryonic development; response to organic cyclic compound; programmed cell death; respiratory system development; response to oxygen-containing compound
|
Dme_circ_0006956
|
-
|
3.5884↑
|
0.045804
|
GluClalpha
|
Cellular process; transport; localization; establishment of localization; biological regulation
|
Dme_circ_0009667
|
-
|
3.559↑
|
0.048096
|
dlg1
|
Hippo signalling pathway-fly
|
Dme_circ_0009514
|
-
|
4.0161↑
|
0.021197
|
pan
|
Canonical Wnt signalling pathway
|
Dme_circ_0006334
|
-
|
3.8925↑
|
0.026799
|
CG42402
|
-
|
Dme_circ_0008173
|
-
|
3.881↑
|
0.027405
|
Nlg1
|
Neuromuscular junction development; cellular process
|
Dme_circ_0004404
|
-
|
0.61768↑
|
0.020721
|
Dbp80
|
Poly(A)+ mRNA export from nucleus
|
Dme_circ_0003891
|
Dme_circ_0001623
|
-3.8065↓
|
0.033572
|
psq
|
Anterior/posterior axis specification, embryo; DNA binding
|
Dme_circ_0006619
|
Dme_circ_0003501
|
-4.3746↓
|
0.010954
|
srp
|
Autophagy; cell fate commitment; midgut development
|
Dme_circ_0004913
|
Dme_circ_0004913
|
-0.52833↓
|
0.047568
|
CG34347
|
Actomyosin structure organization
|
Dme_circ_0004843
|
-
|
-0.48662↓
|
0.02228
|
CG15715
|
|
Dme_circ_0010498
|
-
|
-0.73454↓
|
0.04614
|
CG1304
|
Proteolysis
|
Dme_circ_0006913
|
-
|
-2.7169↓
|
0.042802
|
Cyp12a5
|
Oxidation-reduction process
|
Dme_circ_0010310
|
-
|
-3.8065↓
|
0.033572
|
arm
|
Wnt signalling pathway
|
Dme_circ_0009500
|
-
|
-4.0543↓
|
0.021183
|
pan
|
Canonical Wnt signalling pathway
|
Notes: “↑” and “↓”mean upregulation and downregulation on day 42 compared to day 7, respectively.
DE circRNA/miRNA/mRNA networks in Drosophila ageing
In this section, the DE circRNA/miRNA/mRNA networks were analysed. Through the ceRNA mechanism, miRNAs can negatively regulate mRNA expression. Overall, 12 DE circRNAs, 21 DE miRNAs, and 30 DE mRNAs had interactions (Figure 2A). According to the trends in expression quantity changes, three DE circRNAs (dme_circ_0006913, dme_circ_0008173, and dme_circ_0009500) targeted the DE miRNAs with opposite expression trends (Figure 2B). Based on qPCR results, the expression patterns of two circRNAs (dme_circ_0008173 and dme_circ_0009500), four miRNAs (dme-miR-289-5p, dme-miR-985-3p, dme-miR-286-3p, and dme-miR-14-5p), and four mRNAs (frizzled, CG31064, Abl, and SERCA) were consistent with RNA-seq data (Figure 2C). Importantly, the expression trend of dme_circ_0009500/dme-miR-14-5p/SERCA, dme_circ_0009500/dme-miR-289-5p/frizzled, and dme_circ_0009500/dme-miR-289-5p/CG31064 conformed to the ceRNA mechanism.
DE lncRNA/miRNA/mRNA networks in Drosophila ageing
According to the functional patterns of lncRNAs competing with mRNAs for binding to miRNAs, the interaction networks of DE lncRNAs/miRNAs/mRNAs were identified. In addition, DE miRNAs targeted DE mRNAs with opposite expression trends. Based on the DE genes in our database, 15 lncRNAs, 15 miRNAs, and 32 mRNAs had interactions (Figure 3A). Several specific lncRNA/miRNA/mRNA networks were found, such as XLOC_100429/dme-miR-14-5p/SERCA, XLOC_100429/dme-miR-14-5p/Vha100-4, and XLOC_073604/dme-miR-985-3p/Abl (Figure 3B). In XLOC_027736/dme-miR-985-3p/Abl and XLOC_189909/dme-miR-985-3p/Abl networks, the expression trend of these genes by qPCR analysis is consistent with the RNA-seq data from our study (Figure 3C).
Functional annotation of DE circRNA/lncRNA-associated networks
GO functional annotation of DE circRNAs/lncRNAs/mRNAs was carried out based on 74 target mRNA genes (Dataset S7). The first 30 GO terms based on the lowest P values are listed (Figure 5). In circRNA/mRNA GO terms, there were just two major GO categories in circRNA-associated networks, including biological processes (29 GO terms) and cellular components (1 GO term, perinuclear region of cytoplasm). Similarly, GO annotations of lncRNA-associated networks consisted of 25 GO terms in biological processes and 5 GO terms in cellular component.
In GO annotations of circRNA-associated networks, there were several aging-related biological processes in GO terms, such as homeostatic process (10 DE genes), cellular homeostasis (7 DE genes), cation homeostasis (5 DE genes), ion homeostasis (5 DE genes), cell-cell signalling (9 DE genes), cell fate determination (5 DE genes), and developmental growth (7 DE genes) (Figure 4A). Furthermore, the 13 out of 30 first GO terms of lncRNA-associated networks were related to homeostatic process, including cellular homeostasis, retina homeostasis, ion homeostasis, calcium ion homeostasis, cellular cation homeostasis, etc (Figure 4B).
mRNA/protein correlation in Drosophila ageing
To reveal the post-transcriptional regulatory network of gene expression in Drosophila at day 7 and day 42, the expression trends between proteins and mRNA were analysed. A relatively weak correlation (Pearson value 0.049) between mRNA and proteins was observed. A total of 2753 mRNAs and mapped proteins were found in Drosophila at day 7 and day 42 (Dataset S8). 16 pairs of interacting DE mRNAs/proteins were identified, including 10 pairs with the same expression trends and 6 pairs with the opposite expression trends (Figure 5A, Dataset S8). GO annotation was performed for the 16 pairs of interacting mRNAs/proteins (Figure 5A). FBgn0035542 (Diabetes and obesity regulated, DOR) was enriched for the RNA processing term, FBgn0012042 (Attacin-A, AttA) was enriched for the extracellular region term, and FBgn0036948 (CG7298) was enriched for the carbohydrate derivative metabolic process term; the rest of the proteins/mRNAs were enriched for the response to biotic stimulus and organonitrogen compound metabolic process terms. Evidently, the mRNA levels of FBgn0001624 (discs large 1, dlg1), FBgn0052699 (lysophosphatidylcholine acyltransferase, LPCAT), FBgn0002707 (meiotic 9, mei-9), FBgn0086907 (cytochrome c distal, Cyt-c-d), and FBgn0265101 (suppressor-of-G2-allele-of-skp1, Sgt1) were not changed between day 7 and day 42; however, the proteins encoded by these mRNAs were DE between day 7 and day 42. Furthermore, dlg1, LPCAT, mei-9, Cyt-c-d, and Sgt1 were clustered into different KEGG pathways (Figure 5B).
10 mRNA genes were randomly selected for expression level detection via qPCR at day 7 and day 42. FBgn0034335 (Glutathione S transferase E1, GstE1), FBgn0034480 (CG16898) and FBgn0035542 (DOR) exhibited the same expression patterns as observed in the RNA-seq data (Figure 5C). When compared to 7 day-old fruit flies, GstE1 and CG16898 were upregulated in 42 day-old fruit flies, while DOR was downregulated at day 42.