AUTOPHAGY 2017, VOL. 13, NO. 1, 3–23 http://dx.doi.org/10.1080/15548627.2016.1222992

REVIEW

Emerging connections between RNA and autophagy Lisa B. Frankel, Michal Lubas, and Anders H. Lund Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark

ABSTRACT

ARTICLE HISTORY

Macroautophagy/autophagy is a key catabolic process, essential for maintaining cellular homeostasis and survival through the removal and recycling of unwanted cellular material. Emerging evidence has revealed intricate connections between the RNA and autophagy research fields. While a majority of studies have focused on protein, lipid and carbohydrate catabolism via autophagy, accumulating data supports the view that several types of RNA and associated ribonucleoprotein complexes are specifically recruited to phagophores (precursors to autophagosomes) and subsequently degraded in the lysosome/vacuole. Moreover, recent studies have revealed a substantial number of novel autophagy regulators with RNArelated functions, indicating roles for RNA and associated proteins not only as cargo, but also as regulators of this process. In this review, we discuss widespread evidence of RNA catabolism via autophagy in yeast, plants and animals, reviewing the molecular mechanisms and biological importance in normal physiology, stress and disease. In addition, we explore emerging evidence of core autophagy regulation mediated by RNA-binding proteins and noncoding RNAs, and point to gaps in our current knowledge of the connection between RNA and autophagy. Finally, we discuss the pathological implications of RNA-protein aggregation, primarily in the context of neurodegenerative disease.

Received 29 April 2016 Revised 21 July 2016 Accepted 5 August 2016

Introduction Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved cellular degradation pathway involving sequestration of cytoplasmic components within a transient double-membrane vesicle, the so-called autophagosome. The outer membrane of the autophagosome fuses with the lysosome (in mammals) or the vacuole (in yeast and plants) resulting in degradation of autophagic cargo by acidic hydrolases and subsequent recycling of macromolecules.1,2 Autophagy occurs constitutively at basal levels but is further induced in response to various types of stress. Generally regarded as a cytoprotective mechanism, autophagy plays a critical role in the maintenance of cellular homeostasis, not only by nutrient recycling but also by enabling effective clearance of damaged or superfluous cytoplasmic material.2 Defective autophagy has been linked to diverse pathologies including infections, heart disease, cancer and neurodegeneration.3,4 Intriguing connections between the autophagy and RNA research fields have become evident in recent literature, where genome-wide screens and large-scale proteomics-based approaches have identified a substantial number of autophagy regulators with RNA-related functions.5-9 Moreover, recent elucidation of the RNA-binding proteome from several cell types has revealed a surprising number of unexpected proteins functionally linked to metabolic signaling pathways.10-13 In this review we provide and discuss evidence, intricately linking key aspects of RNA and autophagy research in several eukaryotic organisms spanning from yeast to humans. First, we review the

CONTACT Lisa B. Frankel Denmark. © 2017 Taylor & Francis

[email protected]

KEYWORDS

autophagy; granulophagy; noncoding RNA; ribophagy; RNA-binding proteins; RNA degradation

subject of RNA catabolism via autophagy, discussing i) how different types of RNA and RNA-related complexes are recruited to and degraded by the autophagy pathway, ii) how the mechanisms and dynamics of these processes are regulated, and iii) their biological importance in the regulation of RNA homeostasis and translational and genomic control in normal physiology, in response to stress and in the development of human disease. Highlighted examples include autophagy-mediated degradation of cytoplasmic RNA granules and retrotransposons, ribosomes and associated rRNA (rRNA), RNA-protein aggregates and viral RNA (Fig. 1). In the second part of this review we focus on how RNAbinding proteins (RBPs) and noncoding RNAs (ncRNAs) can act as regulators of the autophagy process. While the vast majority of studies on regulation of autophagy signaling focus on protein-protein interactions, post-translational modifications and transcriptional control,2,9, 14-16 here we explore regulation at the post-transcriptional and co-translational levels by highlighting how the fate of autophagyrelated (Atg) gene-encoding mRNAs can be regulated by a broad selection of RBPs and ncRNAs. With recently expanding functions for ncRNAs and RBPs in most, if not all, areas of fundamental biology, this topic has received increasing attention and enhances our comprehension of autophagy signaling. Finally, we discuss the contribution of RBPs and RNAs to cytoplasmic aggregate-formation and the importance of aggregate clearance via autophagy in disease, with a focus on neurodegenerative disorders.

Biotech Research & Innovation Center, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen,

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Figure 1. An overview of RNA-autophagy crosstalk in eukaryotic cells. The autophagy pathway degrades several different types of RNA and ribonucleoprotein complexes. RNAs and RNA-binding proteins (RBPs) can also act as regulators of the autophagy process. (A) Cytoplasmic RNA granules such as stress granules (SG) and processing-bodies (PB) are recruited to the autophagosome via SQSTM1 and CALCOCO2 receptors in the process of granulophagy. RNAs targeted for autophagic degradation via granulophagy include mRNAs and retrotransposon RNAs LINE 1 and Alu. VCP, or the yeast homolog Cdc48, are mediators of granulophagy. (B) LC3B can bind directly to RNA, although the significance of this with regard to autophagy is unknown. (C) During ribophagy, small (40S) and large (60S) ribosomal subunits are selectively recruited to the phagophore. In yeast, the mechanism for 60S recruitment involves de-ubiquitination via the Ubp3-Bre5 complex, whereas mechanisms for 40S recruitment remain unclear. (D) Aggrephagy involves selective recruitment of protein-RNA aggregates to phagophores. Disease-related aggregate-prone RNA-binding proteins include TARDBP and FUS. (E) RNA viruses can utilize phagophore and/or autophagosome membranes as viral replication hubs. RNA viruses can also be selectively degraded by xenophagy. (F) RNA degradation in the vacuole/lysosome can occur via the RNASET2 family. Additional recruitment of RNA directly to the lysosome may occur via an RNA-binding domain in the cytosolic region of the lysosomal membrane protein LAMP2C. (G) ATG mRNAs can be post-transcriptionally regulated by a large cohort of RNA-binding proteins and noncoding RNAs including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) (see text for details).

RNA as autophagic cargo Autophagy-dependent RNA catabolism Unlike the substantial body of knowledge on the autophagic processing of proteins, carbohydrates and lipids and their subsequent degradation by well-characterized lysosomal hydrolases, mechanisms responsible for the sequestration of RNA by autophagosomes and its subsequent degradation in lysosomes have not been extensively investigated. Nevertheless, recent studies are beginning to elucidate the biological impact of autophagy-dependent RNA catabolism in the maintenance of cellular RNA homeostasis, the regulation of translational fidelity, the evolutionary tempering of genomic change and in the protection against human disease.17-21 Canonical RNA decay pathways RNA metabolism, including the synthesis, processing, folding, modification and degradation of RNA molecules, is a complex and tightly controlled process, which ensures proper execution of gene expression and enables essential cellular functions in all aspects of biology. Indeed, disturbances in an RNA lifecycle can have fatal consequences for the cell; thus, RNA molecules are constantly examined by quality control pathways, and faulty molecules eliminated by RNA decay enzymes.22,23 These

canonical RNA decay pathways rely on ribonucleases (RNases), RNA helicases and other RBPs, which regulate specificities of decay. In the nucleus, the RNA exosome complex is the major RNase acting in the processing and decay of various RNA species, and serves as a checkpoint allowing only properly matured RNAs to be exported into the cytoplasm.24 Once in the cytoplasm, RNAs are further subjected to cytoplasmic quality control and decay pathways.25 General cytoplasmic mRNA decay is initiated by deadenylases and decapping enzymes, which remove stabilizing features of mRNAs including the poly(A) tail and 50 cap.26,27 These steps are followed by exoribonucleolytic decay mediated by Xrn1 and the RNA exosome. Moreover, decapping-independent decay pathways contribute significantly to cytoplasmic RNA decay by sensing premature stop codons (nonsense-mediated decay, NMD), ribosome stalling (no-go decay) or lack of termination codons (non-stop decay).28,29 While these RNA decay mechanisms have been studied in detail, RNA degradation by autophagy is far less explored. In the following sections we discuss mounting evidence suggesting that autophagy provides an additional mechanism of cytoplasmic RNA decay, the importance of which should not be underestimated. In fact, an enormous fraction of cellular RNA, including rRNA and tRNA, is highly structured and extensively bound within ribonucleoprotein (RNP) complexes, rendering them less accessible to the canonical RNA

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degradation machinery, and autophagy may be a more efficient mechanism for disposing of such RNAs. Moreover, considering the enormous amount of transcribed ncRNA, the existence and importance of which is becoming increasingly clear in recent years,30-32 it becomes imperative to understand additional mechanisms that control RNA turnover. Original evidence for RNA degradation by autophagy The subject of RNA decay via the autophagy-lysosome pathway has received increasing attention in recent literature, although it should be noted that pioneering studies from the 1980s– 1990s laid some initial groundwork in this research field.33-37 Early observations from human fibroblasts were among the first to establish that RNA turnover is markedly increased in the absence of serum though a lysosome-dependent pathway.33 This finding was followed up by studies using a rat liver perfusion system, which confirmed that bulk (i.e., nonselective) RNA degradation is induced under conditions of amino acid starvation in rat hepatocytes.34,35 Suppression of this degradation by lysosomal inhibitors or inhibitors of autophagosome formation effectively blocks starvation-induced RNA degradation, suggesting a clear role for autophagy in this process.36,37 Interestingly, early observations of stimulus-dependent differences in the rates of RNA vs. protein degradation by autophagy indicated an aspect of selectivity in this process.38 Lysosomal ribonucleases In line with these findings, there is longstanding evidence of the existence of acid RNases within mammalian lysosomes, initially purified from rat hepatocytes and HeLa cells.39,40 In fact, acid RNases comprise a group of highly conserved endonucleases found broadly in most organisms including plants, yeast and animals.41 These enzymes essentially comprise the RNase T2 family, which displays weak sequence specificity and an optimal pH of 4–5. RNase T2 family members catalyze the endonucleolytic cleavage of single-stranded RNA through 20 ,30 -cyclic

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phosphate intermediates, yielding mono- or oligonucleotides with a terminal 30 phosphate group.41 The cellular localization of T2 type RNases is mainly, but not exclusively, restricted to acidic cytoplasmic compartments, such as vacuoles in yeast and plants, and lysosomes in mammalian cells.41 Recent large-scale proteomic characterizations of lysosomes have confirmed the presence of at least 3 different RNases within mammalian lysosomes, including not only RNase T2 but also RNases with a higher pH optimum, RNASE1 and RNASE6/RNase K6.42,43 Since conditions of cellular stress, such as oxidative stress, can promote alkalinization of the lysosomal pH,44 it is possible that other RNases could come into play under such conditions. Conserved roles of the RNase T2 family in autophagic RNA degradation Yeast In a recent comprehensive study of RNA catabolism in yeast, Huang et al. demonstrated that nitrogen starvation triggers RNA decay in the vacuole through nonselective autophagy.17 Using mass spectrometry, the time-dependent changes of intracellular RNA-derived metabolites was examined and demonstrated remarkable transient changes in the concentrations of nucleotides, nucleosides and nucleobases resulting from autophagy-induced RNA degradation. Through the use of various deletion strains, the authors were able to systematically identify the enzymes responsible for the stepwise RNA degradation (Fig. 2). They showed that the RNA, which is sequestered to the vacuole via autophagosomes, is initially cleaved by the yeast vacuolar RNase T2, Rny1, resulting in the generation of 30 phosphorylated nucleotides.17 The authors further identified the vacuolar phosphatase Pho8, which acts as a nucleotidase by cleaving phosphate from the Rny1-generated nucleotides. The resulting nucleosides are subsequently exported from the vacuole to the cytoplasm where they are further processed by Pnp1 and Urh1 into purine and pyrimidine bases.17 Little is currently known about which RNA species are affected by this process,

Figure 2. RNA degradation in the vacuole/lysosome. tRNA, rRNA and likely additional types of RNA (RNA X) are degraded in vacuoles/lysosomes by the RNASET2 family. The yeast RNase T2, Rny1, cleaves RNA to 30 nucleotide monophosphates (30 NMPs), which are further converted to nucleosides by the vacuolar phosphatase Pho8. Both Rny1 and Pho8 expression levels are increased by starvation. Nucleosides are exported from the vacuole to the cytoplasm where Pnp1 and Urh1 further process them into nucleobases. Through as yet unknown mechanisms, nucleobases can be excreted from yeast cells. LAMP2C may mediate direct import of RNA to lysosomes through an ATP-dependent mechanism (see text for details).

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although the authors assumed rRNA as a likely major candidate since it comprises 80–85% of total cellular yeast RNA. This assumption is also supported by electron microscopy data and by the fact that Rny1 orthologs have been identified as key enzymes for rRNA degradation in plants and animals.18,45,46 Importantly, this degradation pathway is abrogated in cells defective for nonselective autophagy including atg7D, atg2D and atg17D mutants, whereas it occurs normally when genes for selective autophagy are deleted.17 Moreover, because deletion of the genes encoding the enzymes responsible for degradation do not have an impact on the autophagic process itself, this suggested their specificity as mediators of RNA degradation via autophagy but not as general effectors of autophagy.17 Ciliates and plants A family of 8 RNase T2 genes was described in the ciliate Tetrahymena thermophile, and mutant strains for these enzymes display distinct starvation-related profiles of tRNA and rRNA fragment accumulation.47 Although a definitive link to autophagy was not shown, the authors hypothesized that tRNA turnover is a consequence of autophagic targeting of either tRNAs themselves or tRNA-containing protein complexes, ultimately important for proper recycling and control of the protein translation machinery.47 In Arabidopsis thaliana, it was shown that the RNase T2, RNS2, is essential for the recycling of rRNA. By use of radioactive RNA-labeling experiments, the authors showed that 18S and 28S rRNAs display a substantially increased half-life in mutants lacking RNS2 relative to wild-type plants.46 In addition, RNA-specific staining indicated that much of this rRNA accumulates in the plant vacuoles; thus, the functional similarities between plant RNS2 and yeast Rny1 are striking, further substantiated by complementation studies showing that RNS2 can compensate for Rny1 inactivation in yeast.48 Interestingly, the RNS2 mutants show a general boost in the levels of autophagy, suggested by the authors to be a compensatory mechanism triggered by lack of proper RNA recycling.46 In a recent follow-up study, it was shown that RNS2 mutant plants display an increased formation of autophagosomes containing ribosomes and RNA.18 Although the mechanisms for such a compensatory effect remain unknown, it is plausible that the accumulated rRNA itself could serve as a feedback signal to induce bulk autophagy, or that the lack of RNA building blocks, perhaps due to inefficient scavenging of purines/pyrimidines or phosphate by RNS2, could serve as a triggering signal analogous to the lack of amino acids in autophagy-mediated protein degradation. The putative sensors of such mechanisms remain unidentified. Animals In line with the presumption that RNA-mediated degradation is a conserved mechanism with importance also in higher organisms, interesting roles for the T2 family of RNases in animals have also been found.45,49,50 Drosophila melanogaster has a single RNase T2, called RNaseX25, which is elevated in response to nutritional stress and correlates in expression with autophagy markers, supporting the idea that RNA recycling via autophagy also occurs in the fly.49 In addition, a role for the ortholog of yeast Rny1, Rnaset2 has recently been described in

zebrafish.45 This RNase localizes to the lysosomal compartment and its loss results in the accumulation of undigested rRNA within lysosomes, particularly in neurons, causing the appearance of white matter lesions in the zebrafish brain.45 Intriguingly, loss-of-function mutations in RNASET2 in humans leads to familial cystic leukoencephalopathy.50 Although the pathology of this disease is not fully understood, it is suggested to be a lysosomal storage disorder where rRNA accumulation in the lysosomes of neurons is a likely cause of disease symptoms.45 Lysosomal dysfunction caused by rRNA accumulation, would eventually impair the autophagy pathway, limit the cellular recycling capacity and ultimately be detrimental to the neurons. Indeed, the contribution of RNA aggregation to neuronal disease is well known and will be discussed later in this review. Physiological importance of the RNase T2 family in autophagic RNA degradation Both bulk and selective autophagy are highly regulated at multiple steps by a plethora of stimuli, which provide key signals affecting the specificity and dynamics of the pathway.51 Our present understanding of how RNA degradation by autophagy is regulated under basal conditions, during stress, and in the setting of human disease is poor. Indeed, if the RNA accumulation itself provides a triggering signal for the induction of autophagy, as suggested by Hillwig et al.,46 it will be interesting to identify the RNA sensors and investigate whether the amount and/or types of RNA could play any role in selectivity of this process. The fact that the expression of some of the RNA degrading enzymes described in yeast, including Pho8 and Rny1, is boosted upon starvation (Fig. 2), may provide some hints toward elucidating mechanisms of regulatory dynamics.17 Understanding how changes in localization and activity of the implicated enzymes are regulated upon various autophagic stimuli will provide additional insight into this subject. Indeed, the distribution of RNase T2 enzymes in other cellular compartments than lysosomes and vacuoles, such as nuclei and endoplasmic reticulum, has been described.41 In response to oxidative stress Rny1 is reported to be released from the vacuole to the cytoplasm where it induces tRNA cleavage.52 In addition, RNAse T2 enzymes can be secreted to the extracellular space where they can influence the surrounding microenvironment.53 These findings clearly indicate the existence of additional functions for this RNase family other than lysosomal/ vacuolar RNA degradation. Given the broad recognition of autophagy as the core cellular recycling capacity, the potential reuse of the generated RNA metabolites for de novo RNA synthesis or other purposes is a plausible scenario. However, the observation that autophagic degradation of RNA in yeast results in excretion of nucleobases into the environment,17 suggests the lack of an efficient RNA recycling system, at least in this particular experimental setup. Instead, it seems that the cells have alternative means of getting rid of potentially harmful RNA digestion products, a mechanism which could be important for maintenance of metabolic balance. Regardless of the fate of RNA metabolites, the clear roles of T2 type RNases, including plant RNS2, yeast Rny1 and its metazoan orthologs, in vacuolar/lysosomal rRNA decay, indicate a likely role for these enzymes as key downstream players in the selective autophagic turnover of ribosomes, also

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known as ribophagy.20 Indeed, not only ribosomal proteins, but also a substantial amount of rRNAs must be dealt with upon arrival at the vacuole/lysosome via ribophagy, a process that we discuss in detail later in this review. Autophagic digestion of retrotransposon RNA affects genomic stability In a recent study by Guo et al., it was shown that autophagy can specifically target retrotransposon RNA for degradation, thereby preventing retrotransposon insertion in the genome.19 By use of biochemical and microscopy-based approaches, they showed that long interspersed element 1 (LINE 1) and short interspersed nucleotide elements such as Alu retrotransposon RNAs both colocalize and copurify with autophagosomes in HeLa and 293T cells, leading to their subsequent degradation. Indeed, the decay of these RNAs is impeded upon treatment with bafilomycin A1 or an siRNA against ATG5, indicating that the degradation is specifically mediated by autophagy.19 Importantly, a retrotransposition reporter assay suggested that degradation of LINE 1 and Alu RNA by autophagy can restrict their degree of genomic insertion. Moreover, it was shown that LINE 1 RNA colocalizes with processing bodies (PBs) and stress granules (SGs) and, in line with recent finding from yeast, worms and mammals,54-56 these cytoplasmic RNA granules are targeted for autophagic degradation by the autophagy receptors CALCOCO2/NDP52 and SQSTM1/p62, respectively19 (Fig. 1). Interestingly, certain tissues of Becn1/Vps30/Atg6C/¡ mice display increased levels of LINE 1 RNA as well as a small but significant accumulation of genomic insertions, indicating that autophagy may play a role in tempering evolutionary change in mice and perhaps also in humans.19 Autophagy-mediated protection against genetic instability is a well-known phenomenon and regarded as a contributor to autophagy-mediated tumor-suppression in early tumor development.57 In line with this idea, compromised autophagy by allelic loss of Becn1 promotes DNA damage and chromosome instability.58 Interestingly, LINE 1 and Alu retrotransposition events are known frequent causes of translocations, deletions, inversions and amplifications, and have been linked to tumorigenesis.59,60 Additionally, the LINE 1-encoded ORF2 endonuclease generates double-strand DNA breaks in great excess of those required for LINE 1 reinsertion into the genome.59 Thus, a potential tumor suppressive role for autophagy via protection from genomic retrotransposition is an intriguing possibility. Autophagy and viral RNA Autophagy has a well-established role as an intracellular pathogen defense mechanism, where phagophores (the precursors to autophagosomes) can engulf infectious pathogens such as virus and bacteria. This involves a selective type of autophagy coined ‘xenophagy’, because it causes removal of foreign material.61 The relationship between autophagy and RNA viruses is particularly interesting, because not only can autophagosomes contribute to viral RNA decay, but conversely, RNA viruses including polio, dengue, measles, influenza and hepatitis C viruses, can utilize autophagosomes or similar cytoplasmic double-membrane vesicles (DMVs) to enhance their own RNA replication62-65 (Fig. 1).

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One of the most well-studied class of RNA viruses, the single-stranded (C) RNA viruses, which possess an RNA genome of positive polarity and can infect mammalian, plant or insect cells, manipulate their host cells by inducing intracellular DMVs, which can serve as “replication hubs,” greatly enhancing viral RNA replication efficiency.63-67 Although the precise origin and mechanism of formation of virus-induced DMVs is unclear, biochemical, genetic and cell imaging-based approaches provide evidence suggesting that DMVs are strikingly similar to, and in some cases identical to, autophagosomes.62,65-68 Perhaps similar to some autophagosomes, DMVs seem to originate from the ER.66,69 Intriguingly, disruption of autophagy through genetic silencing of essential autophagy genes in mammalian cells clearly reduces the viral replication efficiency for several RNA viruses,62,66,70 and has led to the proposal that RNA viruses can subvert the host cell machinery for autophagosome formation to favor their own replication.67,70 Interestingly, the correlation between proficient autophagy and increased viral yield is seemingly inconsistent with the established role for autophagosomes in antiviral defense through viral clearance,71 since the close vicinity of viral RNA and replication complexes to autophagosomes should ultimately also promote their degradation via delivery to lysosomes. The proand anti-viral roles for autophagy in RNA virus infection will ultimately be influenced by differences in host cell and virus types; whereas some RNA viruses may be degraded by autophagy, others may disrupt lysosomal function, prevent autophagosome-lysosome fusion or perhaps, as poliovirus, acquire some resistance to low pH and lysosomal hydrolases.65

The concept of RNautophagy A novel type of RNA degradation named “RNautophagy” was characterized by Fujiwara and colleagues, in which LAMP2C (lysosomal-associated membrane protein 2C) serves as an RNA receptor allowing direct uptake of RNA into lysosomes72 (Fig. 1 and 2). In vitro interaction studies of LAMP2C in HeLa cells and rat brain lysates revealed numerous RBPs bound to the cytoplasmic, evolutionarily conserved C terminus of LAMP2C. The nearly complete abolishment of these interactions in the presence of RNase indicates they are mediated via RNA.72 The interaction with RNA seems to be relatively nonspecific because a wide range of RNAs bind to a cytoplasm-exposed region of LAMP2C. Moreover, experiments using a cell-free system with isolated lysosomes indicated that LAMP2C is responsible for the uptake and subsequent degradation of RNA by lysosomes in an ATP-dependent manner (Fig. 2).72 The impact on global RNA degradation involves 10–20% of total RNA in living cells, although more obvious differences might be expected for specific RNA subtypes and/or during various conditions of stress. Extending these findings, a protein localized at the lysosome, SIDT2 (SID1 transmembrane family member 2), was recently shown to bind LAMP2C and contribute to lysosomal RNA uptake.73 Alluding toward aspects of RNA selectivity, the cytoplasmic region of LAMP2C was shown to possess some binding preference for stretches of consecutive guanosines.74 Indeed, the potential selectivity for RNA subtypes, which could be mediated via interaction with the identified RBPs, would be interesting to study further.

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As an example of a direct lysosomal RNA uptake mechanism, which bypasses the requirement for phagophore incorporation, RNautophagy resembles chaperone-mediated autophagy (CMA), a specific type of autophagy occurring in mammalian cells, where proteins are recognized one-by-one by cytosolic chaperone HSPA8/HSC70 (heat shock protein family A [Hsp70] member 8), via a pentapeptide amino acid sequence, and delivered across the lysosomal membrane.75,76 As such, RNautophagy may represent a CMA-like mechanism, contributing to general maintenance of RNA homeostasis. The lack of identification of LAMP orthologs in yeast suggests that this mechanism has evolved in higher eukaryotes, perhaps as an additional regulatory layer of protection against disease. Indeed, LAMP2 deficiency causes Danon disease, a lysosomal storage disorder characterized by cardiomyopathy, myopathy and mental retardation.77 Although additional LAMP2 isoforms have broad functions in the endolysosomal system, it would be intriguing to investigate the potential contribution of intracellular RNA accumulation to the pathogenesis of Danon and other diseases. Interestingly, recent studies globally characterizing the RNAbinding proteome have identified some lysosomal/vacuolar proteins as putative RNA binders. This includes the CMA effector itself, HSPA8, which targets CMA substrates to the lysosomal membrane,10,78,79 and the yeast HC-translocating vacuolar-type ATPase subunit Vma1.80 These and potentially other lysosomal proteins with suggested RNA-binding capacity would be conceivable candidates for novel RNautophagy receptors. Novel roles for RNA in key autophagy steps Remarkably, other autophagy-related proteins have also been suggested to possess RNA-binding capacity including the mammalian Atg8 homolog and key autophagy protein MAP1LC3B/ LC3B, which can directly interact with RNA through its arginine-rich motif.81,82 In smooth muscle cells, a pool of cellular LC3B copurifies with ribosomes and interacts with FN1/fibronectin mRNA, as shown by polysome fractionation and UV crosslinking experiments.81 In fact, the binding of LC3B to an AU-rich region in the 30 untranslated region (30 UTR) of the FN1 mRNA plays a role in the translational regulation of FN1.81,83 Moreover, the triple arginine motif of LC3B contributes to its interactions with nuclear and nucleolar proteins, and the nuclear localization of LC3B seems dependent on RNA and ribosomal proteins.82 These independent studies propose interesting properties of the LC3B protein, which may itself regulate and/or be regulated by RNA molecules (Fig. 1). In recent literature, novel regulation and functions of LC3B have been elucidated, including its nuclear/cytoplasmic shuttling via deacetylation84 and its role in chromatin rearrangement and oncogene-induced senescence.85 These and other studies suggest the existence of several as yet unknown LC3B functions, perhaps including those related to RNA binding. There are 2 unique ubiquitin-like protein conjugation reactions that are indispensable for autophagosome formation: conjugation of ATG12 to ATG5, and conjugation of LC3 and other Atg8 homologs to phosphatidylethanolamine (PE).86-88 The ATG12–ATG5 conjugation reaction is dependent on the E1like and E2-like enzymes, ATG7 and ATG10, respectively.89,90 ATG12–ATG5 conjugates are further associated with

ATG16L1 to form a large multimeric complex required for phagophore elongation.91,92 Somewhat surprisingly, it was recently shown by reconstitution of ATG12–ATG5 conjugation in vitro, that the presence of RNA can boost the ATG12–ATG5 conjugation reaction in a dose-dependent manner.93 These stimulatory RNAs encompass a broad group of RNA species including rRNAs and tRNAs; however, the existence of potential common sequence motifs and their biological relevance in vivo remains to be determined. Ribophagy The discovery of ribophagy Since the discovery of autophagy, ribosomes have been detected inside autophagosomes by electron microscopy.94 As autophagy was for a long time considered a nonselective bulk degradation pathway, it was assumed that these autophagosome-engulfed ribosomes were the result of bulk cytoplasmic degradation. However, along with a growing number of studies revealing the selective nature of autophagy signaling,95,96 it has been shown that ribosomes can be selectively turned over in a process called ribophagy.20 The mechanisms of ribophagy Examination of ribosome fate in yeast upon starvation revealed that 40S and 60S ribosomal subunits are more rapidly degraded than other cytoplasmic components through a mechanism that is dependent on essential autophagy genes including Atg7.20 Through a genetic screen, Kraft and colleagues identified the ubiquitin protease Ubp3 and its cofactor Bre5 to be specifically required for 60S but not 40S ribophagy. Starvation in ubp3D and bre5D cells triggers a clear accumulation of 60S ribosomal proteins, while these cells remain proficient in sensing starvation and triggering general autophagy, demonstrating that this selective form of autophagy is distinct from bulk autophagy.20 The fact that a catalytically inactive Ubp3 cannot rescue the ribophagy defect, combined with the observed increased ubiquitination of 60S ribosomal proteins in ubp3D cells, were important initial indications that this process is ubiquitin dependent, likely through direct conjugation of ubiquitin to the ribosomal subunits themselves.20 Following these initial findings, subsequent studies have led to the identification of additional players in this process, including the E3 ligase Rsp5 and the Ubp3-Bre5 binding partners, Cdc48 and Doa1/Ufd3, which are required for Ubp3-Bre5-dependent starvation-induced ribophagy97,98 (Figs. 1 and 3). More recently, it was found that the E3 ligase Rkr1/Ltn1 can inhibit 60S ribosomal subunit ribophagy by ubiquitination of Rpl25, and that its action is antagonized by Ubp399 (Fig. 3). A specific ubiquitination site in Rpl25, K74/75, was identified as a target of Rkr1 and importantly, expression of a ubiquitinationresistant Rpl25 protects the 60S subunit from starvationinduced degradation.99 Since the expression of Rkr1 is severely decreased during nitrogen starvation, possibly via auto-ubiquitination, this provides some insight into how the dynamics of this process are regulated. Taking these findings into account, a likely scenario is that specific ribosomal subunits or associated factors are protected from autophagic degradation by ubiquitination, and that upon

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Figure 3. Mechanisms of ribophagy in yeast. Ubiquitin ligases identified to have an impact on ribophagy include Rsp5 and Rkr1/Ltn1. Expression of Rkr1 is inhibited by nitrogen starvation. Rkr1 ubiquitinates Rpl25 within the large (60S) ribosomal subunit. The Ubp3-Bre5 complex, together with its binding partners Cdc48 and Doa1/Ufd3, is required for selective de-ubiquitination of the 60S ribosomal leading to its subsequent turnover by ribophagy. The small (40S) ribosomal subunit is also recruited to the phagophore through a distinct, unknown mechanism. Potential receptors at the phagophore responsible for ribosome recognition remain unknown (see text for details).

starvation de-ubiquitination serves as a signal to allow their degradation by ribophagy. The potential existence of additional players, regulating the marking of selected ribosomes or perhaps acting as receptors or recruiters of ribosomes to the phagophore is likely, but at present remains unconfirmed. In relation to this, a curious aspect of these studies is the fact that mechanisms contributing to 60S and 40S ribophagy in yeast are mechanistically distinct, underscoring the existence of cargo-specific regulation. Interestingly, independent studies in yeast and mammalian cells have shown specific localization of the small but not the large ribosomal subunit to SGs,100,101 suggesting that the 40S subunit route to phagophores may occur via SG intermediates, through the process of granulophagy54 discussed later in this review. At present, the mechanisms of ribophagy in mammalian cells remain unknown, although ribosomes are also observed in the interior of mammalian autophagosomes, where their degradation by autophagy occurs with different kinetics than that of other cytoplasmic proteins and organelles.94,102,103 A study on murine Purkinje cells showed that the disassembly of actively translating polysomes to nontranslating monosomes, is followed by the sequestering of free monosomes into autophagosomes.102 Combined with the evidence for well-conserved mechanisms of rRNA degradation by autophagy17,18,45,46 (as discussed earlier), there is thus every reason to think that ribophagy is an evolutionarily conserved pathway, which also occurs in mammals. Indeed, the mammalian homologs of Ubp3 and Bre5, namely USP10 and its modulator G3BP1, interact in HeLa and U2OS cells.104 The role of G3BP1 in SG formation combined with recent findings describing SG clearance by autophagy,54-56 presents obvious links to closely related processes in mammalian cells, which are worthy of further investigation. Finally, it should be mentioned that the Ubp3-Bre5 complex is not uniquely devoted to ribophagy, but likely also to other types of selective autophagy. A high-throughput screen in S. cerevisiae recently identified Ubp3-Bre5 as a regulator of mitophagy,105 and the ability of this complex to regulate Atg19 ubiquitination status suggests a role in the cytoplasm-to-vacuole targeting (Cvt) pathway.106 Ubp3 was recently also assigned a role in proteasome degradation via autophagy, through a process called proteophagy.107

Physiological importance of ribophagy Ribosome biogenesis and protein translation are among the most energy-consuming cellular processes,108 and it is therefore not surprising that these and closely related pathways are tightly controlled upon nutrient limitation, where it becomes imperative to effectively shut down protein synthesis.109 Not only do ribosomes constitute about 50% of all cellular proteins along with the vast majority of cellular RNA, namely rRNA,108 but they are also highly stable with a half-life of up to several days.110 This assigns an especially important role for ribophagy in rapid adjustment of both the number and quality of ribosomes during conditions of stress and upon changing metabolic needs of the cells. Indeed, since inhibiting starvation-induced ribophagy accelerates cell death, ribophagy has been assigned a physiologically important role in maintaining cell viability during nutritional stress.20 The evidence of constitutive rRNA decay in plants, occurring even in nutrient-rich conditions, suggests that ribophagy may additionally serve a housekeeping function through the recycling of both amino acids and nucleotides under nonstressed conditions.46 The possibility that ribophagy can contribute to protein quality control through the selective removal of damaged, nonfunctional, wrongly assembled or stalled ribosomes to ensure translational fidelity is an attractive theory. Indeed, ‘clearing the system’ to avoid pathological consequences is also important owing to the high abundance and stability of ribosomes, combined with the fact that several diseases have been associated with specific mutations in ribosomal proteins and rRNAs.111-113 Interestingly, autophagy may also act as a response mechanism to promote survival in the face of ribosomal stress, as demonstrated in a zebrafish model where genomic mutations leading to defects in rRNA processing and ribosome biogenesis cause severe defects in intestinal, liver, pancreas and craniofacial development.114 Interestingly, these larvae display a concurrent upregulation of autophagy, allowing intestinal cells to evade cell death and prolonging larvae life span. In another study, it was shown that in yeast exposed to various conditions of stress or aging, the 25S and 5.8S rRNAs are extensively degraded and that the degradation intermediates differ slightly dependent on the conditions.115 Although the mechanisms

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behind this observation are not fully understood, this finding substantiates the important point that cells are appropriately equipped with ribosome degradation response mechanisms, allowing them to eliminate stress-induced RNA-damage with potential pathological consequences. Along these lines, it is of interest to further investigate links between autophagy and different types of RNA damage, such as RNA oxidation, implicated in the process of neurodegeneration and demonstrated in several human disorders including Parkinson and Alzheimer disease (AD), and amyotrophic lateral sclerosis (ALS).116,117 A recent study shed light on a role for Ubp3-dependent selective depletion of a subset of translation and RNA turnover factors during nitrogen starvation in yeast by both proteasomal and autophagy pathways, emphasizing again the existence of complex and selective degradation mechanisms.118 Importantly, although yeast cells seem to lose a substantial fraction of their ribosomes upon starvation, not all ribosomes are lost. Indeed, selection mechanisms exist to maintain some ribosomes, either important for translation of selected mRNAs throughout the starvation period, or required to facilitate renewed growth upon replenishment of nutrients.119 Although little is known about the selection mechanisms governing such decisions of ribosome maintenance, a recent study by Van Dyke and colleagues identified a ribosome-associated protein in Saccharomyces cerevisiae, Stm1, which allows ribosome preservation in quiescent cells.119 In addition, their data suggest that the preserved ribosomes are translationally competent and can efficiently reassemble onto mRNA upon exit from quiescence by nutrient readdition.119 The mechanism for the observed ribosome preservation remains unknown. Finally, additional mechanisms contributing to ribosome turnover exist, including the RNA exosome and other nucleases.115,120 Interestingly, even in response to the MTOR (mechanistic target of rapamycin [serine/threonine kinase]) inhibitor rapamycin, a potent inducer of autophagy, a rapid decrease in the existing ribosome pool is in fact not appreciably affected by autophagic inhibition. Instead this seems to be dependent on the RNA exosome function, which is important for the degradation of rRNA from the large ribosomal subunit.120 This observation clearly emphasizes the existence of independent pathways contributing to ribosomal turnover, the individual contributions of which will likely depend on environmental cues and species/cell-type differences. Clearance of ribonucleoprotein granules by autophagy Stress granules and processing bodies. Eukaryotic cells contain organelles usually enclosed within lipid membranes, yet other membrane-less organelles exist in the cell. These assemblies are often enriched in RNA and RNA-binding proteins forming RNP granules. Processing bodies and SGs are the bestdescribed cytoplasmic RNP granules.22,121,122 PBs contain mostly mRNA decay factors and translation repressors, and serve both in RNA decay and RNA storage. SGs contain translation initiation factors and form during cellular stress where they are used for temporary storage of RNA. During cellular stresses cells can employ RNA granules to temporarily prevent production of surplus proteins by storing mRNAs away from the active translation machinery.122-124 Moreover, it has been

shown that repressed mRNAs can re-enter the translation cycle, if needed, allowing cells to rapidly respond to changing conditions.124 Granulophagy. SGs and PBs, together with their associated RNA, are cleared by autophagy in a process coined granulophagy54-56 (Fig. 1). Analysis of multiple yeast atg mutants shows increased accumulation of cytoplasmic RNA granules when blocking late steps of autophagy.54 Similarly, autophagy is required for efficient removal of P granules in the C. elegans germline and is dependent on the adaptor protein SEPA-1, which mediates interaction between P granules and LGG-1 (an Atg8 homolog).56 In humans, SGs are also targeted to the lysosome by autophagy, but the involvement of autophagy in PB clearance is less evident, perhaps due to their highly dynamic nature.54,125,126 Depletion of the yeast ATPase Cdc48, or its mammalian homolog VCP (valosin-containing protein), results in increased accumulation of SGs indicating its role in targeting of SGs to phagophores.54 It is noteworthy that Cdc48, as described above, has also been implicated in starvation-induced ribophagy through its binding to the Ubp3-Bre5 complex98 (Fig. 3). Interestingly, genetic impairment of autophagy using atg mutants, drug-mediated inhibition of lysosome function or siRNA-based depletion of VCP, all lead to defects in SG formation.126 SGs in such cells have a disturbed morphology and composition and contain noncanonical components, such as the 60S ribosomal subunit, normally absent from SGs.126 Hence, this study suggests a role for autophagy and VCPrelated signaling not only in SG clearance, but also in proper SG formation. A more recent study adds to the mechanism of SG clearance in human cells by describing a tyrosine kinase, SYK, which together with its binding partner GRB7 associates with SGs and promotes granulophagy.127 SYK is not needed for SG formation, but is recruited to SGs upon its phosphorylation, in turn causing the phosphorylation of proteins at or near SGs and promoting their clearance via autophagy once the stress is relieved. This process was further shown to promote cellular survival.127 What clearly pans out from such studies is that eukaryotic cells are equipped with a robust means of SG clearance via autophagy, which must be kept finely tuned due to its physiological importance for cellular viability.127,128 RNA as autophagic cargo—general considerations. Interpreting the effects of autophagy-modulated changes on RNA, whether it be via ribophagy, granulophagy or other routes, is complicated by the fact that many types of cellular stress, including autophagy-inducing stress, can modulate translation and thereby also RNA metabolism through several mechanisms.129,130 Proteotoxic stress, for instance, strongly inhibits translation initiation by phosphorylation of EIF2A/eIF2a, inducing SG assembly.129 In addition, the translation of mRNAs harboring a terminal oligopyrimidine (TOP) tract at their 50 end, which encode for components of the translational machinery including ribosomal proteins, are particularly sensitive to MTOR inhibition and thereby highly affected by various autophagic stimuli.130 Such aspects are critical to consider during data interpretation of the above sections describing RNA as autophagic cargo. For this reason, it becomes important to

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emphasize the strongest evidence for RNA degradation via the autophagy-lysosome system, which likely includes the direct visualization of RNA, ribosomes and RNA granule components within autophagosomes and lysosomes/vacuoles.17,19,20,54,72 A final consideration concerning RNA as autophagic cargo, is that RNA resides in most cellular compartments, and therefore bulk autophagy and various types of selective autophagy not elaborated upon in this review, including mitophagy,131 reticulophagy and nucleophagy132 will ultimately also account for some RNA degradation via this route.

RNA-binding proteins and RNA in autophagy regulation Emerging roles for RNA-binding proteins in autophagy Independent studies in yeast and mammalian cells aiming to disclose the RBP proteome applied stringent UV crosslinking-based methodology to identify proteins directly interacting with polyadenylated RNA.10,78,80 At present, the human RBP proteome is estimated at 1542 RBPs, comprising 7.5% of all human proteins, equal to 20% of the mass of the human proteome.133 Importantly, RNP complexes are often highly dynamic structures with changing properties depending on cellular processes, localization and environmental cues. This is exemplified in a recent study where global changes in RNA-protein networks were observed upon nitrogen and glucose starvation, indicating that nutrient stress causes dynamic rearrangement of post-transcriptional regulatory mechanisms via differential occupancy of RBPs on RNA targets.13 Importantly, it was demonstrated that this rearrangement has major effects on metabolic pathways,13 a finding which is supported by the fact that numerous metabolic enzymes have been ascribed previously unrecognized RNA-binding activity.11 An intriguing example of an unexpected RNA binder, identified in yeast, is the HC-translocating vacuolar-type ATPase subunit Vma1, which, as discussed earlier in this review, could be involved in targeting RNAs for vacuolar decay.80 These recent revelations underscore the importance of understanding new roles for RBPs across multiple cellular processes including metabolic-related pathways such as autophagy. The inverse scenario, where well-studied RBPs have been assigned newly identified roles in autophagy, also exists. One example is LRPPRC (leucine rich pentatricopeptide repeat containing), an abundant RNA-binding protein associated with the neuro-metabolic disorder Leigh syndrome.134,135 LRPPRC is a mitochondrial protein, containing multiple pentatricopeptide repeat motifs, implicated in post-transcriptional regulation of the mitochondrially-encoded COX (cytochrome c oxidase) mRNAs.136,137 Interestingly, LRPPRC has been reported to suppress autophagy via binding to BECN1 and BCL2, which prevents BECN1 from interacting with PIK3C3 and initiating autophagy.138 Upon mitophagic stress, the interaction between LRPPRC and BECN1 can be impaired by BST2/tetherin, which itself is degraded by autophagy.139 Additionally, LRPPRC interacts with the ubiquitin ligase and mitophagy regulator PARK2, affecting its stability and localization, thereby regulating mitophagy.140 Whether the autophagy-related functions of

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LRPPRC are linked to its RNA-binding capabilities remains unknown. Post-transcriptional regulation of autophagy Much of the described regulation of the core autophagy machinery lies at the post-translational level, where key posttranslational modifications such as phosphorylation and ubiquitination govern aspects of selectivity and enable rapid response to changing metabolic needs of the cell.141 Moreover, in recent years, our understanding of autophagy regulation at the transcriptional level has grown, in part due to the elucidation of key conserved transcription factors, particularly the helix-loop-helix transcription factor TFEB and the FOXO (forkhead box protein) family members, which control the expression of several autophagy-related genes.14,142,143 Importantly, as reviewed below, the mRNA transcripts encoding core autophagy proteins are subjected to regulation at the post-transcriptional and co-translational level by RBPs and by ncRNAs (Fig. 4). Post-transcriptional regulation of autophagy by RNA-binding proteins A recent study performed in yeast and mammals provided evidence that the TOR kinase, apart from its well-described role in autophagy inhibition,144,145 can also negatively affect autophagy by promoting degradation of several ATG mRNAs.146 In yeast, this regulation depends on the DEAD-box RNA helicase Dhh1,147 which recruits ATG mRNAs to the decapping enzyme Dcp2, initiating their rapid cytoplasmic decay (Fig. 4A). In nonstarved conditions, TOR increases decapping via phosphorylation of Dcp2, triggering degradation of ATG mRNAs and subsequent autophagy suppression. Conversely, upon starvation, TOR inactivation prevents Dcp2 phosphorylation causing dissociation of Dhh1 from its RNA substrates, allowing their stabilization. This mechanism is conserved, as the mammalian Dhh1 homolog DDX6 suppresses autophagy through a similar mechanism in mouse embryonic stem cells and human HeLa cells.146 Both Dhh1 and DDX6 target multiple RNA substrates for downstream RNA decay;147,148 thus, it would be of interest to identify common sequence/structure determinants of these RNA substrates. Importantly, this study suggests a dynamic model for controlling ATG mRNA stability shown not only to affect autophagy but also to have consequences for inflammation and microbial pathogenesis.146 At the co-translational level, modulation of autophagy gene expression can be mediated by the cytoplasmic RBP orb, as evidenced by studies in Drosophila ovaries.149 Similar to its vertebrate homolog, CPEB1 (cytoplasmic polyadenylation element binding protein 1), orb binds cytoplasmic polyadenylation elements (CPEs) in mRNAs to repress their translation149,150 (Fig. 4B). In the Drosophila germline, orb can repress Atg12 mRNA translation through interaction with the RNA deadenylase CCR4, which shortens the mRNA polyA-tail and initiates mRNA decay. Interestingly, half of Drosophila Atg mRNAs are found to contain CPE regions and associate with orb, suggesting that orb-driven regulation of autophagy may be more general. Notably, orb associates with PBs that respond to nutrient

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Figure 4. Post-transcriptional regulation of autophagy mRNAs. Multiple RNA-binding proteins (RBPs) and noncoding RNAs (ncRNAs) are involved in post-translational regulation of autophagy-relevant mRNAs. (A) Downregulation of a number of Atg mRNAs is mediated via Dhh1/DDX6 and Dcp2/DCP2, causing subsequent 50 –30 exonucleolytic decay both in yeast and humans. (B) In Drosophila the orb protein recognizes cytoplasmic polyadenylation elements (CPEs) in RNA and recruits the major cellular deadenylase complex CCR4-NOT to dampen Atg mRNA levels. (C) Proteins from the ELAV/Hu family, ELAVL4/HuD and ELAVL1/HuR, can upregulate SQSMT1 and ATG5 mRNAs. Their recruitment is mediated by AU-rich elements (ARE) in RNAs. (D) Stabilization of the Rptor mRNA via binding to TARDBP can lead to increased levels of RPTOR protein. (E) microRNAs (miRNAs) are well studied activators of RNA-induced silencing complex (RISC)-mediated mRNA silencing and are involved in the regulation of multiple autophagy-relevant mRNAs. (F) Long non-coding RNAs (lncRNAs) can act as molecular sponges by antagonizing miRNAs and thereby stabilizing mRNA targets of the miRNA (left). lncRNAs can also bind directly to proteins. The lncRNA NBR2 binds to and activates AMPK (right). Up- or downregulated mRNAs are indicated in green or red, respectively (see text for details).

stresses123 and could itself be regulated in response to environmental conditions, as is its human homolog CPEB1, involved in insulin signaling.151 RBPs from the ELAV/Hu protein family are positive regulators of mRNA stability and translation,152 also involved in the post-transcriptional regulation of autophagy components. A recent study demonstrated that ELAVL1/HuR is a potent proteasomal target, which is stabilized upon proteasome inhibition, resulting in increased SQSTM1 mRNA and protein levels in ARPE-19 cells153 (Fig. 4C). This regulation is likely due to the presence of the ELAVL1 binding AU-rich motif in the 30 UTR of the SQSTM1 mRNA. Another ELAV protein, ELAVL4/ HuD, associates with the AU-rich 30 UTR of the ATG5 mRNA acting to control its expression in pancreatic b cells.154 ELAVL4 depletion dampens ATG5 mRNA translation, whereas its overexpression enhances ATG5 protein expression and elevates both autophagosome number and LC3 lipidation. Although ELAV proteins are clearly implicated in the regulation of autophagy-relevant gene expression levels, further studies are needed to fully understand the observed biological effects on autophagy. TARDBP/TDP-43 (TAR DNA binding protein) is a highly promiscuous RNA-binding protein that binds >6000 RNA targets in the brain and is involved in many aspects of RNA metabolism, including splicing, transport, stability, translation and microRNA (miRNA) biogenesis.155 TARDBP contains 2 RNA recognition motifs and a protein-protein interaction glycine-rich domain important for most of these functions. Interestingly, TARDBP regulates splicing of neuron development- and neurological disease-associated mRNAs, including autophagy-related ATG7 and SQSTM1 transcripts.156-159 Recently, Rptor/Raptor mRNA has also been reported as a TARDBP target160 (Fig. 4D). Loss of TARDBP can, via regulation of RPTOR levels, affect

MTOR complex 1 (MTORC1) activity and cause subsequent activation of TFEB and increased expression of autophagy/lysosomal genes.143,160 The reverse is observed when TARDBP levels are low, resulting in a decrease of the motor protein DCTN1/ Dynactin 1, and a consequential block in autophagosome-lysosome fusion. Although TARDBP broadly affects multiple mRNAs, this regulation might contribute to TARDBP-linked pathologies caused by the loss of TARDBP function. In a recent study by Park et al.,161 it was shown that a recurring oncogenic mutation in the U2AF1/U2AF35 splicing factor causes abnormal processing of the ATG7 pre-mRNA by inducing selection of a distal cleavage and polyadenylation site. Interestingly, this leads to inefficient ATG7 translation and a consequent reduction in the ATG7 protein level, causing defective autophagy, mitochondrial dysfunction, genomic instability and secondary mutations, which result in cellular transformation.161 Post-transcriptional regulation of autophagy by noncoding RNA MicroRNA Posttranscriptional regulation of autophagy via miRNAs has been widely documented to have an impact at several stages of the autophagy pathway.162,163 miRNAs constitute a class of small ncRNAs that act as target recognition modules for the RNA-induced silencing complex (RISC). Through miRNA-mediated base pairing, predominantly at 30 UTRs, RISC induces posttranscriptional repression of target mRNAs via direct translational repression and transcript destabilization through deadenylation164,165 (Fig. 4E). In the following text, we highlight a few examples of well-characterized miRNAs affecting core autophagy components and some additional regulatory proteins (see Table 1). miRNAs

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affecting upstream or indirect regulators of autophagy signaling are not covered in this review. Concerted regulation of an autophagy/lysosomal transcriptional program is mediated via TFEB, which translocates to the nucleus upon nutrient stress and activates a large set of genes related to lysosome and autophagosome biogenesis.166 Interestingly, TFEB is targeted by Mir128, which thus has an impact on a cohort of key autophagy proteins.167 This was further shown using an in vivo model of SNCA/a-synuclein neuronal toxicity, where Mir128 was found to aggravate the effects of SNCA-mediated toxicity.168 Early studies demonstrated both direct regulation of key autophagy components, such as the regulation of BECN1 by MIR30A,169 and more indirect effects, shown by the overexpression of MIR196 in Crohn disease leading to reduced expression of IRGM (immunity related GTPase M) and resulting in compromised xenophagy.170 Using a more comprehensive high throughput screening approach, we have previously reported several miRNAs affecting autophagic flux and identified MIR101 as an inhibitor of early stages of the autophagy pathway via repression of ATG4D, RAB5A and STMN1.171 Subsequently, numerous papers have demonstrated the impact of miRNA on the autophagy pathway both in early regulatory complexes such as the ULK complex172,173 and the BECN1 complex169,174-176 as well as in the LC3 conjugation system, where, for instance, ATG12, ATG7 and ATG5 are all regulated by several miRNAs.177-185 Also, in relation to LC3 processing, both ATG4C and ATG4D have been identified as targets for MIR376b174 and MIR101,171 respectively. Additionally, genes encoding Atg8 homologs themselves have been identified as miRNA targets; LC3A and LC3B are both targets of MIR204,186,187 as, for instance, shown in renal clear cell Table 1. MicroRNAs regulating key autophagy components. Gene Autophagy induction ULK1 RB1CC1/FIP200 STMN1 TFEB Phagophore formation BECN1 ATG14 ATG4 RAB5A Cargo recruitment and phagophore elongation ATG5 ATG12 ATG7 ATG3 ATG16L1 SQSTM1/p62 LC3A/LC3B GABARAPL1 ATG2 ATG9 Lysosomes, fusion and endosomal crosstalk LAMP1, LAMP2 RAB11A SUMF1

MicroRNA

Reference

Mir20a, Mir106b MIR133b MIR101 Mir128

172

MIR17-5p, MIR30a, MIR129-5p, MIR144, MIR376b MIR152 MIR24-3p, Mir34a, MIR101, MIR376b MIR101

169,174-176

173 171 168

230 171,174,231,232

171

MIR30a, MIR181a, MIR224-3p MIR23b, MIR30b, MIR200b MIR17, Mir188-3p, MIR375 MIR155, Mir495 Mir20a, MIR106a, MIR142-3p Mir17/¡20/¡93/¡106, MIR372 MIR204 MIR143 MIR30d, MIR130a, MIR143 Mir34a

177-179

Mir207, MIR320a Mir21 MIR95

243,192

180-182 183-185 233,234 235-237 188,189 186,187 238 239-241 242

244 190

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carcinoma, where MIR204 is downregulated due to loss of VHL (von Hippel-Lindau tumor suppressor).187 Lastly, the autophagy receptor SQSTM1 is also regulated by several miRNAs, including MIR372188 and the MIR17 family.189 While a large bulk of evidence documents miRNA regulation at early stages of the autophagy pathway, fewer studies have unveiled important miRNA functions at later stages. Among these, we recently identified the nonconserved miRNA MIR95 as a potent regulator of lysosomal function.190 Repression of SUMF1, the sole activator of all cellular sulfatases,191 by MIR95, leads to the accumulation of unprocessed sulfated molecules, such as glycosaminoglycans, in the lysosomes. This disrupts lysosomal function and in turn blocks autophagymediated degradation. Through this study we demonstrated the potential use of miRNA inhibition as a therapeutic strategy to enhance autophagy-mediated clearance in a lysosomal storage disorder.190 Additional miRNA function at the lysosome was shown in a model for ischemic stroke, where Chen and coworkers identified Mir207 as a regulator of LAMP2 and found Mir207 overexpression to result in an accumulation of autophagosomes and reduced numbers of lysosomes and autolysosomes.192 Interestingly, not only do miRNAs affect autophagy, but the opposite regulatory route is evident as well. As such, the miRNA profiles in Atg5 knockout and wild-type cells differ widely following starvation,193 and both the key miRNA processing enzyme DICER1 and the main effector molecule AGO2 (argonaute 2, RISC catalytic component) are targeted for degradation by the autophagy-lysosomal pathway via the selective autophagy receptor CALCOCO2.194-196 Long noncoding RNAs More recently other classes of ncRNAs have also been shown to affect autophagy. Whereas the majority of these studies are mainly descriptive in nature and devoid of direct mechanistic links to autophagy, a couple of reports deserve mentioning here. Long ncRNAs (lncRNAs), arbitrarily defined as transcripts longer than 200 nucleotides, which do not encode protein, are emerging as important regulators of biological pathways and act through a very diverse set of mechanisms.32 One such mechanism is buffering of miRNA-mediated repression, where a lncRNA competes for binding to a particular miRNA or set of miRNAs, thereby relieving repression of cognate mRNA targets197 (Fig. 4F). Seeking to unveil novel pathways of autophagy regulation in cardiomyocytes following myocardial injury, Li and coworkers identified Mir188-3p as a regulator of ATG7 expression.185 Interestingly, the level of Mir188-3p was downregulated during myocardial injury and the lncRNA APF (autophagy-promoting factor) was identified and characterized to act as a molecular ‘sponge’ for Mir188-3p, and hence de facto upregulate the levels of ATG7. Along similar lines, the lncRNA FLJ11812 derived from the 30 UTR of the TGFB2 gene binds MIR4459 and in a competitive manner relieves ATG13 from repression by this miRNA.198 Antisense transcripts constitute a class of lncRNAs, which modulate the expression of their sense strand protein-coding partners.31 The lncRNA Chast is upregulated in a mouse model for cardiac hypertrophy, and Chast negatively regulates

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expression of PLEKHM1 (pleckstrin homology and RUN domain containing M1) situated on the opposite strand overlapping the Chast locus.199 PLEKHM1 regulates autophagosome-lysosome fusion by acting as an adaptor protein through direct interactions with several LC3 family members and the lysosomal HOPS complex.200 Besides post-transcriptional regulatory mechanisms, lncRNAs can influence cellular pathways by direct binding to effector proteins as exemplified by the lncRNA NBR2, recently found to bind and activate the 50 AMP-activated protein kinase (AMPK)201 (Fig. 4F). As NBR2 itself is regulated via the STK11/LKB1-AMPK pathway, these molecules engage in a positive feedback loop, which is activated under conditions of energy stress. AMPK is a well-characterized regulator of autophagy144 and NBR2 deficiency results in lowered ULK1 phosphorylation, compromised GFP-LC3 puncta formation and reduced SQSTM1 degradation. The field of ncRNA is currently in rapid progress as numerous research groups worldwide aim to harness new biological insight into ncRNA-regulated pathways. Undoubtedly, additional ncRNA molecules with important functions in autophagy will soon emerge. Connecting autophagy and nonsense-mediated decay Recent studies have shed light on the interconnection between the canonical RNA degradation pathway, NMD and autophagy.29,202,203 NMD is a cellular RNA surveillance system, which serves to eliminate faulty mRNAs containing premature stop codons, thus preventing production of potentially toxic proteins.29,204 Interestingly, many of the cellular stresses, which inhibit NMD, including amino-acid starvation, viral infection and hypoxia, are strong inducers of the autophagy pathway. In relation to this, it has been shown that direct inhibition of NMD components UPF1 and UPF2 results in increased autophagic flux, implicating a role for autophagy as an adaptive/protective response mechanism, which aids in the clearance of mutated, misfolded and aggregated proteins that otherwise accumulate when NMD is inhibited.202 In agreement with this hypothesis, simultaneous inhibition of NMD and autophagy leads to cell death. Additionally, NMD equips mammalian cells with a dynamic response mechanism by targeting a specific subset of mRNAs, including the stress-responsive ATF4 (activating transcription factor 4). In normal conditions, low levels of the ATF4 mRNA are observed, but upon stress the production of a longer NMD-resistant isoform of ATF4 mRNA is favored, thus promoting expression of ATF4-inducible genes and stimulating the unfolded protein response and autophagy.203 These observations highlight a noteworthy interconnection between mRNA and protein quality control systems through 2 mechanisms: i) via autophagy serving as a compensatory clearance response during defective NMD, and ii) via post-transcriptional regulation of the stress-responsive transcription factor ATF4. RNA-binding proteins, aggrephagy and disease TARDBP and FUS: Aggregate-prone RNA-binding proteins in neurodegeneration Several canonical RBPs are linked to neurological diseases, including the previously mentioned TARDBP, as well as FUS

(FUS RNA binding protein), HNRNPA2B1, HNRNPA1, ATXN1 (ataxin 1), ATXN2, TIA1 (TIA1 cytotoxic granuleassociated RNA binding protein) and FMR1 (fragile X mental retardation 1).133,205,206 A striking feature of these proteins is the presence of both RNA-binding domains and prion-like domains, which contribute strongly to aggregate formation.133,205 Normally, prion-like or low-complexity domain interactions in RNP granules are dynamic and reversible, but the loss of these properties results in formation of aberrant and persistent pathological inclusions, which may cause neurodegenerative diseases if not efficiently cleared.121,207,208 Well-studied examples of such RBPs include TARDBP and FUS, which are not only linked to multiple steps in cellular RNA metabolism, but are key components of pathological inclusions observed in ALS and frontotemporal dementia (FTD) patients.159 Importantly, several independent studies provide evidence that errors in RNA processing linked to defective TARDBP and FUS function, are central to ALS and FTD pathogenesis.208-210 In a yeast model of FUS-induced toxicity, a genome-wide screen identified proteins that could compensate for loss of FUS activity, including the yeast or human RNA helicases Ecm32 and UPF1, which function in RNA quality control.210 Moreover, cytoplasmic, disease-associated mutant FUS binds an altered set of RNA targets211 and the use of serially deleted FUS expression constructs demonstrated that both Nand C-terminal regions, including RNA-binding domains, are required for toxicity.208 Likewise, the RNA-binding function of TARDBP is required for mediating neurotoxicity in vivo.209 Importantly, these data suggest that FUS and TARDBP-mediated pathogenicity is established through deregulation of RNA homeostasis. Aggregate clearance by autophagy: Aggrephagy Besides mutations in RNA-binding and aggregate-prone proteins, such as TARDBP and FUS, the impairment of cytoplasmic aggregate clearance via autophagy, better known as aggrephagy, is suggested to be a fundamental contributor to the pathology of proteinopathies such as ALS and FTD21,212 (Fig. 1). SQSTM1 and OPTN (optineurin), both well-known autophagy receptors, which bind to LC3 and polyubiquitinated proteins,213-215 are found mutated in ALS where they localize to TARDBP inclusions.216,217 Moreover, the ATPase VCP, aside from key roles in SG dynamics, is also implicated in aggregate clearance via autophagy, and its depletion causes accumulation of immature autophagosomes and insoluble ubiquitinated proteins.218,219 Interestingly, TARDBP is mislocalized to the cytosol upon VCP-mediated autophagic misfunction220 and VCP also plays a role in clearance of damaged mitochondria.221 Consistent with the neuropathology of ALS and FTD, it seems clear that failure to remove protein-RNA aggregates can largely be explained by dysfunction of proteins important for aggrephagy. Interestingly, some of the above-mentioned clearance-related proteins such as VCP and SQSTM1 have been recently identified as putative RBPs10,78 although the contribution of these newly described RNA-binding functions to aggrephagy and disease has not been investigated. Since TARDBP and FUS can bind to and regulate the mRNAs of multiple autophagy-related genes including SQSTM1, VCP and ATG7,21,158,211 this is suggestive of complex regulatory feedback mechanisms where

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these proteins affect the very pathways that govern their own cellular fate. Stress-granules and aggregation Evidence has suggested that SGs themselves may transition, over time, to larger ubiquitinated aggregates.222 In line with this observation, both TARDBP- and FUS-positive aggregates in post-mortem tissue colocalize with markers of SGs including TIA1, EIF3 and PABPC1/PABP1.223,224 Moreover, the ALSlinked FUSR521C mutant causes accumulation of FUS-positive SGs under oxidative stress, which colocalize with LC3-positive autophagosomes and accumulate in autophagy-deficient cells.225 Considering the recent finding that SGs themselves are cleared via recruitment to autophagosomes,54,126 and the close connections to proteins such as VCP with roles in both SG dynamics and aggrephagy,54,218 these studies emphasize the physiological importance of links between aggregate clearance, autophagy, RNA-binding proteins and the maintenance of RNA quality control. Therapeutic targeting of aggrephagy Based on the evidence discussed above, aggrephagy, and more particularly the identification of molecules that selectively enhance aggrephagy, may in some contexts represent a valid therapeutic strategy for treatment of ALS, FTD and additional proteinopathies.21,212,226 However, results in this area of research are conflicting, because while induction of autophagosome formation has indeed shown promising results in several cell culture and in vivo models,227,228 other models suggest that autophagy induction may exacerbate pathological phenotypes, for instance as shown for rapamycin treatment in a mouse model of ALS.229 Caution must be taken with cases where the defect lies at the level of autophagosome maturation or lysosomal degradation. In such situations, boosting autophagosome formation would, on the contrary, have highly unfavorable consequences. It is therefore imperative to first determine the main cause of dysfunctional autophagy in the different types of proteinopathies when considering therapeutic strategy options.

Perspectives Traditionally viewed as a protein degradation pathway, regulated by protein-protein interactions and post-translational modifications, the focus of the autophagy research field has been somewhat protein-centric. However, emerging knowledge has now established many interesting connections between the RNA and autophagy research fields. Still at an early phase, these findings provoke several as yet unanswered questions. While many types of RNA are recruited to the phagophore and degraded by well-conserved mechanisms in the vacuole/lysosome, little is currently known regarding the stimuli and sensors of this process, and how they govern aspects of selectivity. RNA serves not only as autophagic cargo, but exerts regulatory functions on the autophagy pathway; it may act in bridging protein interactions and improving protein complex stability, as exemplified by ATG12–ATG5 conjugation, or it may mediate post-transcriptional regulation of autophagy genes, as seen for several long and short ncRNAs. Also RBPs have clear roles

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as autophagy regulators, mainly as post-transcriptional and cotranslational regulators of various ATGs and related mRNAs, and, perhaps surprisingly, key autophagy proteins such as LC3B and LAMP2C, can themselves bind to RNA, the precise implications of which will require further investigation. Other questions concern more global biological effects. For instance, how does the process of ribophagy affect the ribosome pool of a cell? Can it influence protein quality control and proteome composition by promoting the removal of selected ribosomes? And could this in turn play a role in ensuring translational fidelity? Undoubtedly, major ongoing quests within both the RNA and autophagy research fields will provide further insight into many of these unanswered questions, and further exploration of these links will increase our biological knowledge, potentially laying the foundation for novel therapeutic approaches.

Abbreviations AD ALS AMPK ATG CMA CPEs DMVs FTD FUS HSPA8/HSC70 LAMP2 LINE 1 miRNA MTOR ncRNA NMD PBs RBP RISC RNase RNP rRNA SG TARDBP/TDP-43 TFEB tRNA UTR VCP

Alzheimer disease amyotrophic lateral sclerosis 50 AMP-activated protein kinase autophagy related chaperone-mediated autophagy cytoplasmic polyadenylation elements double-membrane vesicles frontotemporal dementia FUS RNA binding protein heat shock protein family A (Hsp70) member 8 lysosomal-associated membrane protein 2 long interspersed elements 1 microRNA mechanistic target of rapamycin (serine/ threonine kinase) noncoding RNA nonsense-mediated decay processing bodies RNA-binding protein RNA-induced silencing complex ribonuclease ribonucleoprotein ribosomal RNA stress granule TAR DNA binding protein transcription factor EB transfer RNA untranslated region valosin containing protein

Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.

Acknowledgement The authors wish to thank Christian Kroun Damgaard for critical reading of the manuscript.

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Funding Work in the authors’ laboratory is supported by funding from the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program FP7/2007-2013/ under REA grant agreement 607720, the EU COST action Transautophagy (CA 15138), the Danish Council for Independent Research (Sapere Aude program), the Novo Nordisk Foundation, the Lundbeck Foundation, and the Danish Cancer Society. ML is supported by a personal stipend from the Danish Cancer Society.

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