Autophagy is a widely conserved process in eukaryotes that is involved in a series of physiological and pathological events, including development, immunity, neurodegenerative disease, and tumorigenesis. It is regulated by nutrient deprivation, energy stress, and other unfavorable conditions through multiple pathways. In general, autophagy is synergistically governed at the RNA and protein levels. The upstream transcription factors trigger or inhibit the expression of autophagy- or lysosome-related genes to facilitate or reduce autophagy. Moreover, a significant number of non-coding RNAs (microRNA, circRNA, and lncRNA) are reported to participate in autophagy regulation. Finally, post-transcriptional modifications, such as RNA methylation, play a key role in controlling autophagy occurrence.
1. Introduction
Macroautophagy, hereafter referred to as autophagy, is the main type of autophagy, which is characterized by the formation of autophagosomes. Autophagosome biogenesis involves a series of autophagy-related (Atg) proteins that accompany the different steps of the autophagic process. Autophagosome initiation is mediated by the activity of the ULK1/Atg1-ATG13/Atg13 protein kinase complex. Nucleation of the autophagosome requires the BECN1/Beclin 1/ATG6-PIK3C3/Vps34 (catalytic subunit of the class III phosphatidylinositol 3-kinase (PtdIns3K)) complex. Elongation and maturation of the autophagosome involves two ubiquitin-like systems, i.e., Atg5–Atg12-Atg16 and LC3/Atg8–phosphatidylethanolamine (PE). Finally, the mature autophagosome fuses with the lysosome, leading to cargo degradation and completing the autophagic flux.
Autophagy is a well-regulated physiological process that is implicated in development, metabolism, immunity, neurodegenerative diseases, and tumorigenesis
[1]. Induction of autophagy is accompanied by an increase in mRNA levels of certain
Atg genes,
WIPI1 (WD repeat protein interacting with phosphoinositides),
p62/SQSTM1, and
vacuolar (H+)-adenosine triphosphatases (V-ATPases), or by a variation of post-translational modifications of autophagy-associated proteins
[2][3][4][5]. Nutrient deprivation, endoplasmic reticulum (ER) stress, hypoxia, lipotoxicity, cholesterol, and insect molting hormone can all affect the transcription of
Atg genes. In response to starvation and circadian signals, FXR (farnesoid X receptor) and PPARα (peroxisome proliferator-activated receptor alpha) oppositely regulate the great variation of
Ulk1 transcription in mammalian liver cells, accompanied by limited changes in mRNA levels of
GABARAPL1,
Bnip3, and
LC3b [6]. ER stress and hypoxia increase the transcription of
ULK1/ULK2, Atg5/ATG5,
ATG4B, ATG13, LC3, and
GABARAPL1 in several mammalian cancer cells
[5][7]. In mouse liver fibrosis, insulin-like growth factor-binding protein-related protein 1 (IGFBPrP1) increases the expression of
Atg9a, which encodes the sole transmembrane protein and delivers membrane to the expanding phagophore, and thereby formation of the autophagosome
[8][9]. On the other hand, the transcription of
ULK1 and
ULK2 is repressed by the chromatin non-histone protein HMGA1 (high-mobility-group AT-hook 1) during the initiation and progression of malignant neoplasia such as skin cancer
[10]. In addition, the transcription of
V-ATPases from the V0 and V1 subunits are consistently unregulated during the induction of autophagy by upstream signals to facilitate the flux
[3][4][5]. Autophagy regulates the homeostasis of cholesterol, whereas cholesterol and its derivatives, such as the insect-molting hormone 20-hydroxyecdysone (20E) and 27-hydroxycholesterol, are able to induce autophagy by promoting the transcription of
Atg genes, as well as inducing the deacetylation of ATG proteins in both
Bombyx mori and mammals
[11][12]. Here, researchers summarize the most recent studies on the regulation of autophagy at the mRNA level, and provide a deep thinking and prospects in studies on autophagy.
2. Regulation of Autophagy by Transcriptional and Post-Transcriptional Modifications
2.1. Transcription Factors Regulate Autophagy at RNA Level
Several transcription factors play critical roles in regulating autophagy. The transcription factor EB (TFEB), a basic helix loop helix (b-HLH) leucine zipper protein from the microphthalmia-associated family (MiT/TFE), is one of the key transcription factors first identified to mediate autophagosome formation and autophagosome–lysosome fusion under starvation, in addition to its role in lysosomal biogenesis
[13]. In mice liver, the circadian pattern of
Atg gene expression depends on nutrient-sensitive activation of TFEB and TFE3: in the absence of nutrients (supplied with light), TFEB and TFE3 translocate to the nucleus and upregulate the expression of
Atg3, Atg5, Bnip3, and
LC3, which are involved in autophagy
[14]. TFEB positively regulates the expression of genes involved in lysosomal biogenesis and autophagy during starvation in mouse liver, so autophagy shuttles lipid droplets to the lysosome for hydrolysis. Moreover, TFEB overexpression rescues obesity syndrome and lipid metabolism in
Atg7 liver-KO mice, in which autophagy is blocked and lipids accumulate in the liver. Thus, TFEB is proven to prevent diet-induced obesity in mice by mediating autophagy
[15]. Interestingly, the zinc-finger-family DNA-binding protein (ZKSCAN3) inhibits starvation-induced autophagy, and knockdown of
ZKSCAN3 can promote TFEB-induced autophagy
[16].
Members of FOXO (forkhead Box O) family can regulate autophagy induction at the transcriptional level. Notably, adenovirus-mediated expression of constitutively active
FOXO3 (
ca-FOXO3) causes dramatic atrophy in mouse muscles and myotubes, since FOXO3 increases the autophagic flux by binding to the promoters of
LC3b, Atg12L, and
Gabarapl1 and directly increasing their transcription
[17][18]. In lung cancer cells, acetylated FOXO1 activates
ATG7 expression to enhance autophagy, and it is thus implicated in the suppression of tumor growth through autophagy activation
[19]. AMPK (AMP-activated protein kinase) is activated by glucose starvation. Subsequently, activated AMPK phosphorylates FOXO3a and leads to its nuclear translocation, followed by the upregulation of
CARM1 (
co-activator-associated arginine methyltransferase 1), which coactivates autophagy with TFEB by increasing the transcription of autophagy- and lysosome-related genes
[20].
Nuclear receptors sensitive to metabolism play key roles in autophagy occurrence. PPARα is activated by fatty acids to promote their oxidation under starvation conditions, whereas FXR is activated by bile acids returning to the liver under nutrient-rich conditions. Consistently, PPARα is required for the full induction of autophagy by starvation, whereas FXR is needed for the suppression of autophagy in the liver of fed mice. PPARα and FXR competitively bind to shared sites in the promoters of autophagy-associated genes and control the expression of
Atg7,
Beclin1,
Bnip3,
and
LC3 [6]. Small heterodimer partner (SHP), which is an orphan nuclear receptor responsible for maintaining the homeostasis of bile acids, is required for hFGF19 (bile acid-induced fibroblast growth factor-19, mFGF15)-mediated inhibition of hepatic autophagy, and plays a negative role in autophagy induction through FGF19-SHP-LSD1 axis by repressing the expression of most autophagy-associated genes, including
Atg3,
Atg5,
Atg7,
Atg10,
WIPI1,
Uvrag, and
Tfeb.
[21][22]. In preadipocyte 3T3-L1 cells, adipogenic transcription factors C/EBPβ (CCAAT/enhancer binding protein beta) and PPARγ (peroxisome proliferator-activated receptor gamma) directly bind to the promoter region of autophagy genes, leading to the expression of
LC3,
Beclin1, and
Atg4b, to facilitate autophagy. C/EBPβ and PPARγ directly bind to the promoters of
TFEB and
FOXO1, too, to indirectly control the expression of autophagy-associated genes
[23]. The transcription factor E2F1 (E2 transcription factor 1) not only mediates apoptosis, but also enhances autophagy by binding to the promoters of
LC3, ATG1, ATG5, and
DRAM (
damage-regulated autophagy modulator) to upregulate their expression,
showing a positive role for E2F1 in DNA damage-induced autophagy
[24]. In LNCaP and HeLa cells, the ER stressor tunicamycin induces the transcription of
ATG16L1, GABARAP, ATG12, ATG5, ATG3, and
BECN1 to upregulate autophagy through the activation of ATF4 (transcription factor 4)
[5]. Furthermore,
Atg gene expression is also linked to the status of histone acetylation: the inhibition of histone deacetylase sirtuin1/2 increases the expression of
ATF4 to induce autophagy, playing a pro-survival role in human NSCLC (non-small cell lung cancer) cells
[25].
In insects, several transcription factors have been documented to mediate autophagy. In
Drosophila melanogaster, E93, a downstream transcription factor of 20E signaling induces both autophagy and caspase activity by blocking PI3K-MTORC1 signaling
[26]. The transcription factor FOXO prevents the aggregation of damaged proteins by promoting the expression of
Atg1, Atg5, Atg6, and
Atg8 in
D. melanogaster [27]. Zika virus (ZIKV) triggers NF-κB-dependent inflammatory signaling in the fly brain and induces the expression of
Atg5 and
Atg7, leading to autophagy activation in neurons and limiting the infection and proliferation of ZIKV in this organ
[28]. In
B. mori, 20E upregulates the downstream transcription factors
BmBr-C, BmE74, BmHR3,
and
Bmβ-ftz-F1 and thus determines the transcriptional induction of most of
Atg genes to promote autophagy, which is essential for larval tissue remodeling during metamorphosis
[11][29]. Recent studies have shown that 20E and starvation are both able to activate BmTFEB in
B. mori to promote the transcription of
BmV-ATPases and the assembly of the subunits, thus triggering lysosomal acidification and the autophagic flux
[4]. Moreover, ACSS2 (acyl-CoA synthetase short-chain family member 2) forms a complex with TFEB, which facilitates the acetylation of histone using acetyl-CoA as an acetyl donor, and then promotes the transcription of TFEB-targeted genes in the nucleus, enhancing lysosomal biogenesis and autophagy
[30].
Transcriptional regulation of autophagy is evolutionarily conserved between insects and mammals
[12]. Notably, transcription factors have been reported to regulate autophagy in plants, too. In
Arabidopsis thaliana, the transcription factor TGA9 (TGACG motif-binding protein 9) is confirmed to be a positive regulator of autophagy. The overexpression of
TGA9 upregulates the mRNA levels of
Atg genes and induces autophagy
[31]. Transcription factors and their function in the regulation of autophagy are listed in
Table 1.
Table 1. Transcription factors and their function in autophagy.
Transcription Factor |
Function |
Leucine zipper transcription factors (MiT/TFE) |
MiT/TFE recognize promoters of lysosomal and Atg genes and represent transcriptional controllers of lysosomal biogenesis and autophagy [4][13]. |
Nuclear receptors PPARα and FXR |
PPARα and FXR oppositely control the expression of Atg7, Beclin1, Bnip3, and LC3 and autophagic vesicle formation [6]. |
Small heterodimer partner (SHP) |
SHP decreases mRNA levels of Atg genes and inhibits autophagy [21]. |
Transcription factors FOXO/FOXA |
Activation of FOXO/FOXA induces the expression of multiple Atg genes and lysosomal genes [17][32]. |
CCAAT/enhancer binding protein beta (C/EBPβ) |
C/EBPβ targets key Atg genes and induces the expression of Atg genes [23][33]. |
Activating transcription factor 4(ATF4) |
ATF4 is involved in the cellular stress response and autophagosome formation [5][34]. |
Nuclear factor-kappa B (NF-κB) |
NF-κB activates the expression of Atg genes and induces autophagy [28][35]. |
Zinc-finger-family DNA-binding protein, ZKSCAN3 |
ZKSCAN3 decreases mRNA levels of Atg genes and inhibits autophagy [16]. |
Tumor suppressor p53 |
In the nucleus, P53 transactivates Atg genes and induces autophagy by inhibiting mTOR; in the cytoplasm, P53 suppresses autophagy [36][37]. |
Signal transducer and activator of transcription (STAT) |
STAT3 phosphorylation upregulates BNIP3 expression; STAT1 suppresses the expression of Atg genes [38][39]. |
Transcription factor E2F |
Activation of E2F1 upregulates the expression of Atg genes [24]. |
TGA9 (TGACG motif-binding protein 9) |
TGA9 activates autophagy by upregulating the expression of Atg genes [31]. |
E93 |
Knockdown of E93 reduces the expression of several Atg genes in B. mori [40]. |
EcR-USP |
20E-EcR-USP upregulates the transcription of Atg genes to induce autophagy [11]. |
2.2. Regulation of Autophagy by Non-Coding RNAs
In addition to the transcription factors reported above, non-coding RNAs represent key regulators of autophagy. Non-coding RNAs mainly include microRNA, circRNA, and lncRNA. A series of non-coding RNAs are able to mediate the occurrence of human diseases and drug sensitivity in therapy by modulating autophagy
[41]. MicroRNAs (miRNAs), about 22 nucleotides long, are conserved in evolution and expressed in almost all eukaryotes. Interestingly they have been identified as sequence-specific post-transcriptional regulators of gene expression, including
Atg genes
[42].
miRNA-101 inhibits autophagy by targeting
RAB5A, a member of the RAS oncogene family, and
ATG4d, leading to the suppression of tumor formation
[43]. Moreover,
miRNA-101 and
miRNA-376b inhibit the expression of
ATG4c and
ATG4d, respectively
[44]. Finally,
miRNA-103a-3p directly targets
Atg5 to inhibit autophagy and protect cardiomyocytes
[45]. In
Caenorhabditis elegans,
miRNA-83 disrupts autophagy in multiple tissues by inhibiting
cup-5 (autophagy regulator), whereas
miRNA-34 inhibits the autophagic flux in vitro and affects the protein levels of Atg9, which is evolutionarily conserved in mammals
[46][47]. In summary, according to the current literature, all microRNAs negatively regulate autophagy by directly inhibiting the expression of
Atg genes, which are involved in the occurrence of diseases such as cancer and aging.
CircRNAs, formed by head-to-tail splicing of exons, are naturally generated from the family of non-coding RNAs, and show a regulatory role in gene expression at the post-transcriptional level
[48][49]. In astrocytes, circRNA
NF1-419 upregulates the expression of
ULK1, BECLIN1, ATG5, ATG12,
and
ATG13 by binding to Dynamin-1 and adaptor protein 2 B1(AP2B1)
[50], whereas circRNA
PABPN1 blocks the binding of human antigen R (HuR) to
ATG16L1 mRNA and thus inhibits autophagy in human intestinal epithelial cells
[51]. HuR is reported to upregulate
ATG7,
LC3II, and
ATG16L1 expression to enhance autophagosome formation
[52]. Thus, autophagy is differentially regulated by multiple circRNAs.
In mammals, genomic transcription produces a large number of long non-coding RNAs (lncRNA), which can regulate
Atg genes expression and thus mediate autophagy occurrence
[42]. In mouse,
lncRNA NEAT1 directly binds to
miR-29b and then upregulates
Atg9a expression to activate autophagy; similarly,
LncRNAXIST enhances ethanol-induced autophagy by binding to
miRNA-29b [8][53]. In human gallbladder cancer tissues,
lncRNA GBCDRlnc1 increases the expression of phosphoglycerate kinase 1 (PGK1), which upregulates
ATG5 and
ATG12 expression. Moreover, PGK1 phosphorylates BECLIN1 to induce autophagy
[54][55]. Antisense intronic l lncRNA
eosinophil granule ontogeny transcript (
Ai-lncRNAEGOT) enhances autophagosome formation, as well as paclitaxel sensitivity in human cancer
[56]. However,
lncRNA HOX transcript antisense RNA (HOTAIR) downregulates the expression of
LC3B, BECLIN1, ATG3,
and
ATG7 to inhibit autophagy, which suppresses the invasion of oral squamous cell carcinoma cells
[57]. Non-coding RNAs participating in autophagy are listed in
Table 2.
Table 2. Non-coding RNAs involved in autophagy.
Non-Coding RNAs |
Target Genes |
Species |
Impact on Autophagy |
miR30b |
Atg12, Beclin-1 |
Helicobacter pylori |
↓[58] |
miR-17 |
Ulk1 |
Mouse |
↓[59] |
miR-30a |
Beclin1, Atg12, Atg5 |
Mouse |
↓[44][60] |
miR-188-3p |
Atg7 |
Mouse |
↓[61] |
miR-93, miR106b, miR142-3p |
ULK1, ATG16L |
Human |
↓[44][62][63] |
miR-101 |
ATG4D, LC3 |
Human |
↓[43][44] |
miR-155 |
ATG3 |
Human |
↓[64] |
miR-214-3p |
ATG5, ATG12 |
Human |
↓[65] |
miR-216b |
BECLIN1 |
Human |
↓[66] |
miR-103a-3p |
ATG5 |
Human |
↓[45] |
miR-183, miR-204 |
LC3B1/LC3-II |
Human |
↓[44][67] |
miR-83, miR-29 |
atg-4.2 / ATG4D, ATG9a |
Caenorhabditis elegans/Human |
↓[8][46] |
miR-34 |
Atg9a/ATG9a |
Caenorhabditis elegans/Human |
↓[47] |
miR-4459 |
ATG13 |
Human |
↓[68] |
miR-23b |
ATG12 |
Human |
↓[69] |
miR-19a |
BECLIN1, LC3 |
Human |
↓[70] |
miR-376b |
ATG4C, BECLIN1 |
Human |
↓[44] |
miR-15a, miR-16 |
Rictor (mTORC1) |
Human |
↑[71] |
circNF1-419 |
Dynamin-1 |
Mouse |
↑[50] |
circHIPK2 |
ATG5, BECLIN1-1 |
Human |
↑[72] |
circPABPN1 |
ATG16l1 |
Human |
↓[51] |
lncRNA APF |
Atg7 |
Mouse |
↑[61] |
lncRNA NEAT1, lncRNA XIST |
Atg9a |
Mouse |
↑[8][53] |
lncRNA HAGLROS |
PI3K-AKT-NF-κB |
Human |
↑[73] |
lncRNA TGFB2-OT1 |
ATG3, ATG7, ATG13 |
Human |
↑[74] |
lncRNA CA7-4 |
AMPK |
Human |
↑[75] |
lncRNA GBCDRlnc1 |
BECLIN1, ATG5, ATG12 |
Human |
↑[54][55] |
lncRNA MALAT1 |
Beclin1, LC3 |
Mouse |
↓[76] |
lncRNA LINC00470 |
BECLIN1, ATG3, ATG7 |
Human |
↓[77] |
lncRNA CTA |
Unknown |
Human |
↓[78] |
lncRNA HOTAIR |
BECLIN1, LC3, ATG3, ATG7 |
Human |
↓[57] |
2.3. Regulation of Autophagy by RNA Methylation
N6-methyl-adenosine (m
6A) modification of mRNAs is pervasive and highly conserved in eukaryotic cells. m
6A modification is mediated by methyltransferases (writers) consisting of methyltransferase-like 3 (METTL3), METTL14, Wilms tumor 1-associated protein (WTAP), RNA-binding motif protein 15 (RBM15), and zinc-finger CCCH domain-containing protein 13 (ZC3H13)
[79]. The demethylases (erasers) reported in m
6A modification are represented by fat mass and obesity-associated protein (FTO), flavin mononucleotide (FMN), and α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5)
[80]. m
6A modification also indirectly affects RNA processing by recruiting reader proteins, which harbor the YT521-B homology (YTH) domain (
Figure 1)
[79].
Figure 1. Schematic diagram of m
6A modification and its regulation of autophagy. m
6A modification is mediated by the methyltransferases (writers) METTL3, METTL14, WTAP, RBM15, and ZC3H13 and the demethylases (erasers) FTO, FMN, and ALKBH5. m
6A indirectly affects RNA processing by recruiting reader proteins, which contain the YTH domain
[79][80]. FTO upregulates
UKL1,
ATG5,
and
ATG7 expression to induce autophagy by YTHDF2-dependent targeting of their mRNA. ALKBH5 demethylates
TFEB,
FOXO, and
AMPK mRNAs to activate autophagy.
FOXO,
TFEB, and
AMPK are the targets of m
6A reader protein YTHDF1. METTL3 increases m
6A levels of
UKL1,
ATG5, and
ATG7 to upregulate autophagy, whereas METTL3 and METTL14 negatively regulate autophagy through m
6A methylation of
TFEB,
FOXO, or
AMPK mRNAs, which are responsible for the expression of
Atg genes or ULK1 phosphorylation. P indicates phosphorylation.
METTL3 positively regulates autophagy by increasing the expression of
ATG5,
ATG7, and
LC3B through m
6A modification of their mRNA, whereas β-elemene reverses gefitinib resistance in gefitinib-resistant PC9GR and HCC827GR derived from NSCLC cells by inhibiting METTL3-mediated autophagy
[81]. Moreover, METTL3 suppresses autophagy by methylating
FOXO3 (in an 800 bp region of
FOXO3 3′UTR containing the m
6A modification site), which subsequently downregulates the expression of
ULK1,
ATG5,
ATG7, ATG12, ATG16L1, and
MAP1LC3B in human sorafenib-resistant hepatocellular carcinoma
[82]. Similarly, METTL3 methylates the 3’ UTR of
TFEB mRNA and thus inhibits autophagy
[83].
Demethylase FTO increases the autophagic flux in patients with chronic kidney disease
[84][85]. In particular, FTO upregulates
ULK1 expression by demethylating the adenine residues 3335, 3397, and 3784 at 3’ UTR of
ULK1, thus promoting autophagy in Hela cells. In a mouse preadipose cell line, mRNAs of
Atg5 and
Atg7 are the targets of m
6A reader protein YTHDF2, and FTO upregulates
Atg5 and
Atg7 expression in a YTHDF2-mediated manner to promote autophagy
[86]. There is a close interaction between demethylases or methyltransferases and the autophagic pathway. In melanoma, FTO is induced by metabolic starvation through autophagy, whereas knockdown of
ATG5 or
ATG7 in turn attenuates the expression of
FTO [87]. In Leydig cells, human chorionic gonadotropin (HsCG) promotes the binding of the transcriptional factor C/EBPβ and TFEB to the promoter of
ALKBH5, inducing its expression, whereas HsCG decreases
METTL14 expression, leading to the activation of AMPK-ULK1 axis and autophagy occurrence
[83][88] (
Figure 1).