J. Biochem. 2017;161(2):145–154
doi:10.1093/jb/mvw085
JB Special Review—Recent Topics in Ubiquitin-Proteasome
System and Autophagy
Lysosomal degradation of intracellular nucleic acids—multiple
autophagic pathways
Received September 21, 2016; accepted October 20, 2016; published online December 23, 2016
Department of Degenerative Neurological Diseases, National
Institute of Neuroscience National Center of Neurology and
Psychiatry, Kodaira, Tokyo, Japan
*Tomohiro Kabuta, Department of Degenerative Neurological
Diseases, National Institute of Neuroscience, National Center of
Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo
187-8502, Japan. Tel: +81-42-346-1715; Fax: +81-42-346-1745;
e-mail: [email protected]
Cell metabolism can be considered as a process of
serial construction and destruction of cellular components, both of which must be regulated accurately. In
eukaryotic cells, a variety of cellular components are
actively delivered into lysosomes/vacuoles, specialized
compartments for hydrolysis of macromolecules. Such
processes of ‘self-eating’ are called autophagy. Despite
a wide variety of lysosomal/vacuolar hydrolases, much
of the interest has been focused on the proteolytic functions of autophagy and less attention has been devoted
to the degradation of other macromolecules such as
nucleic acids. In this review, we focus on delivery and
degradation of endogenous nucleic acids by autophagic
systems, and discuss their molecular mechanisms and
physiological/pathophysiological roles.
Keywords: autophagy/DNA/lysosome/nucleic acid/
RNA.
Cells maintain their homeostasis through continuous
synthesis and degradation of their components.
Impairment in the balance within such cycles can be
responsible for the pathogenesis of various human diseases. One major site for intracellular degradation is
the lysosome. Lysosomes contain a wide range of
hydrolases in their lumen and can degrade virtually
all kind of cellular component such as nucleic acids,
proteins, lipids, carbohydrates and even organelles.
Lysosomes were first described by de Duve et al. in
1955 through tissue fractionation of rat liver, and characterized as membrane-bound vesicles containing multiple acid hydrolases (1). The process through which
intracellular components are delivered into lysosomes
(or vacuoles in yeasts or plants) and degraded is called
autophagy. The term ‘autophagy’ was also coined by
de Duve in the context of comparison with internalization of exogenous components into lysosomes,
‘heterophagy’ (2). The proteolytic aspects of autophagy have been studied extensively for the last few
decades. However, there have been limited studies on
the precise mechanisms and significance of autophagic
degradation of other biological macromolecules. This
review focuses on our present knowledge on delivery
and degradation of endogenous nucleic acids by autophagic systems, and their biological significance.
Overview of the Multiple
Autophagic Pathways
To date, at least three kinds of autophagy have been
described, namely, macroautophagy, microautophagy
and chaperone-mediated autophagy (CMA) (3) (Fig. 1).
In macroautophagy, cytosolic components are sequestered by double membrane structures called autophagosomes and delivered into lysosomes by membrane fusions
of autophagosomes and lysosomes. The compartments
produced by the fusions of autophagosomes and lysosomes are termed autolysosomes. In microautophagy,
substrates are entrapped in lysosomes by invagination
of the lysosomal membrane. Both macro- and microautophagy are thought to be essentially non-selective
‘bulk’ systems. However, a number of studies have also
revealed selective pathways in these systems. Conversely,
CMA is a highly selective form of autophagy, which does
not involve rearrangement of membrane structures. In
CMA, specific cytosolic proteins containing a sequence
called the ‘KFERQ-like motif’ are unfolded by a chaperone complex consisting of hsc70 and other co-chaperones,
and directly imported into lysosomes via a lysosomal
membrane receptor, LAMP2A. In addition to these
kinds of autophagy, we have recently identified a novel
type of autophagy that directly delivers RNA (4) and
DNA (5) into lysosomes, which we have termed
RNautophagy/DNautophagy (RDA) (Fig. 2).
Delivery
An advantage of macro- and micro-autophagy is that
they can carry large cargos, such as molecular complexes
and organelles, by sequestering them into membranebound structures. To date, a selective type of macroand micro-autophagy that directly targets nucleic acids
per se has not been identified. However, both RNA and
DNA can be selectively delivered into lysosomes/vacuoles
as a component of larger structures such as ribosomes,
mitochondria, a portion of the nucleus and RNA granules. We first focus on advances in studies on selective
macro- and micro-autophagy for delivery of nucleic acidcontaining structures to lysosomes/vacuoles.
Delivery of Ribosomes to Lysosomes/Vacuoles
Ribosomes are giant molecular complexes consisting
of multiple ribosomal proteins and ribosomal RNA
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Featured Article
Yuuki Fujiwara, Keiji Wada and
Tomohiro Kabuta*
Y. Fujiwara et al.
Fig. 1 Common pathways of autophagy. In macroautophagy, cytosolic components are sequestered by double membrane structures
called autophagosomes and delivered into the lysosomal/vacuolar
lumen through membrane fusion of lysosomes/vacuoles and autophagosomes. In microautophagy, cytosolic cargo proximal to lysosomes/vacuoles is engulfed into the lumen by invagination of the
lysosomal/vacuolar membrane. Chaperone-mediated autophagy
(CMA) is a process through which specific cytosolic proteins are
unfolded by a chaperone complex consisting of Hsc70 and other
cochaperones, and directly transported into lysosomes via a lysosomal membrane receptor, LAMP2A.
(rRNA). Approximately 80% of total RNA in live
cells is rRNA (6). In 2008, Kraft et al. proposed selective degradation of mature ribosomes by macroautophagy upon nitrogen starvation in yeast (7). They
termed this selective pathway as ‘ribophagy’.
Conversely, non-selective ‘bulk’ autophagy is also
induced by nitrogen starvation, and ribosomes are delivered into lysosomes/vacuoles as a major component
of the cytoplasm (8). Both selective and non-selective
processes are based on same basic macroautophagy
machinery, because vacuolar accumulation of ribosomal proteins under nitrogen starvation is completely
abrogated in mutants lacking genes necessary for
basal macroautophagy (7).
Ubp3 and Bre5 have been reported as genes responsible for degradation of 60S, but not 40S, subunits of
ribosomes by ribophagy in yeast (7). Ubp3 is a deubiquitylating enzyme, and Bre5 is its cofactor. The fact
that the impaired vacuolar localization of ribosomal
proteins seen in ubp3 yeast cannot be rescued by
expression of the catalytically inactive form of Ubp3
reinforces that the deubiquitylation activity of Ubp3 is
important for ribophagy. In 2014, Ossareh-Nazari et
al. reported that ubiquitylation of the ribosomal protein Rpl25 by the ubiquitin ligase Ltn1 protects the 60S
ribosomal subunit from ribophagy, which is reversed
by Ubp3 (9). These reports suggest that the balance
between ubiquitylation and deubiquitylation of ribosomal proteins by Ltn1 and Ubp3/Bre5 regulates the selective degradation rate of ribosomes by ribophagy.
However, a recent study by Huang et al. showed that
the levels of 30 -monophosphate nucleotides (NMPs),
metabolites of RNA degradation, produced by
ubp3 and bre5 yeasts during nitrogen starvation
are comparable with those of wild-type yeast (8).
Although nucleoside production in the mutant yeasts
was lower than that in wild-type yeast, this difference
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Fig. 2 A model for the processes of RNautophagy/DNautophagy.
Nucleic acids are directly translocated into the lysosomal lumen
through an interaction with lysosomal membrane receptors and
transporters. LAMP2C and SIDT2 function as at least one of the
receptors and putative transporters, respectively. The requirement of
a receptor and the existence of other factors involved in
RNautophagy and DNautophagy warrant further investigation.
would be the consequence of the fact that the processing and catalytic activity of Rho8, a vacuolar nucleotidase that further converts 30 -NMPs to nucleosides, is
partially attenuated in both ubp3 and bre5 yeasts
(8). Further investigations are needed to clarify the
degree of contributions of both selective and nonselective types of macroautophagy to ribosome degradation during starvation. In addition, most selective
types of autophagy identified to date require the involvement of specific receptor or adaptor proteins to
link substrates to the membranes of autophagosomes
or lysosomes (10). Exploration of receptor proteins
involved in ribophagy is an important future issue. In
mammals, machinery of ribophagy has not been elucidated to date, although mammals possess orthologs of
both Ubp3 and Bre5.
Delivery of Mitochondria to Lysosomes/Vacuoles
Mitochondria are sites of ATP production by aerobic
respiration, and consequently generate large amounts
of reactive oxygen species (ROS) as byproducts (11).
As a result, mitochondrial DNA (mtDNA) is frequently exposed to oxidative stress, leading to higher
DNA damage compared with nucleic DNA (12).
Because accumulation of damage in mitochondria
can cause mitochondrial dysfunction and lead to
the production of even more ROS, elimination of
damaged mitochondria is critical for cell survival.
Selective autophagy targeting mitochondria is called
‘mitophagy’. Mitophagy has been reported in both
macro- and micro-autophagy of yeasts and mammals.
In terms of macroautophagy in yeast, Atg32 has
been identified as a receptor protein necessary for
mitophagy based on two independent studies by
Okamoto et al. (13) and Kanki et al. (14). Atg32 is a
membrane protein localized to the mitochondrial outer
membrane, which interacts with two autophagyrelated proteins, Atg8 and Atg11. Atg8 is one of core
factors for basic macroautophagy machinery localized
to the autophagosomal membrane, and Atg11 is a scaffold protein required for various selective types of
autophagy but not non-selective autophagy (10).
Through interactions between Atg8 and Atg11,
Atg32 functions in mitophagy as a receptor that joins
Lysosomal degradation of intracellular nucleic acids
mitochondria to autophagosomes. Many other receptor proteins involved in selective types of macroautophagy also interact with both Atg8 and Atg11 through
sequences similar to those in Atg32 (10). In addition,
Mao et al. have reported that Atg11 recruits mitochondrial fission machinery to substrate mitochondria, and
mitochondrial fission is required for the process of
mitophagy (15). Although their sequence homology is
low, Atg32 is well conserved among yeast species (10).
Conversely, a mammalian homolog of Atg32 has not
been found according to sequence similarity. However,
a recent study by Murakawa et al. based on database
searches according to the molecular features of Atg32
revealed that Bcl2-L-13 is a mammalian homolog of
Atg32 (16). Both mitochondrial fission and mitophagy
are induced by Bcl2-L-13 overexpression and attenuated by knockdown of the gene. Importantly, Bcl2-L13 can partially restore mitophagy activity upon expression in atg32 yeast, suggesting that Atg32 and
Bcl2-L-13 induce mitophagy via an exchangeable
mechanism.
In mammals, the machinery for mitophagy by
macroautophagy, which is mediated by the Ser/Thr
kinase PINK1 and E3 ubiquitin ligase Parkin, has
been studied extensively (17). Upon mitochondrial
damage and loss of their membrane potential,
PINK1 accumulates in the outer membrane of the
mitochondria (18, 19). PINK1 is a kinase that phosphorylates ubiquitin (20—24) and the ubiquitin-like
domain of Parkin (25, 26). Because Parkin is an ubiquitin ligase that preferentially interacts with phosphorylated ubiquitin (p-Ub), and both p-Ub and
phosphorylation of Parkin itself activate the E3 ubiquitin ligase activity of Parkin, PINK1 can therefore
trigger a positive feedback loop generating poly-p-Ub
chains on the surface of the mitochondrial outer membrane via Parkin (20—26). ‘Labeling’ of substrate mitochondria with ubiquitin chains appears to be an
important process for PINK1/Parkin-mediated mitophagy, because two deubiquitinases, USP15 (27) and
USP30 (28), have been shown to inhibit mitophagy by
presumably removing ubiquitin chains from mitochondria. Furthermore, a recent study by Lazarou et al.
revealed that NDP52 and OPTN are principal receptors for PINK1/Parkin-mediated mitophagy, and their
binding to ubiquitin is necessary for the process (29).
These two receptors recruit core macroautophagy
machineries to mitochondria to initiate sequestration
of the substrate by the proceeding autophagosome
formation.
Because Bcl2-L-13 can induce mitophagy even in the
absence of Parkin, the Bcl2-L-13 mediated- and
PINK1/Parkin mediated-mitophagy are likely to be independent pathways (16). Biological significance of the
dual pathways in mammalian mitophagy is an intriguing question remains to be solved.
Compared with studies on macroautophagic mitophagy (macromitophagy), our knowledge on selective
degradation of mitochondria by microautophagy
(micromitophagy) is still limited. In yeast, a study
based on electron microscopic analyses suggested
that, after growth in the presence of lactate instead
of glucose, the majority of autophagy induced under
nitrogen starvation is microautophagy rather than
macroautophagy (30). Uth1 is reported to be responsible for micromitophagy in yeast (30). However, the
contribution of Uth1 to this process is still controversial (14, 31). Interestingly, a group of genes related to
macroautophagy (ATG genes) are also reported to be
necessary for this microautophagic process in lactategrown yeasts (30). In mammals, recent studies show
that glyceraldehyde-3-phosphate dehydrogenase mediates direct uptake of damaged mitochondria into lysosome-like structures by accumulating in mitochondria,
independently of its catalytic activity (32, 33). Unlike
in yeast, this pathway is likely to be independent of the
basic machinery of macroautophagy.
Delivery of the Nucleus to Lysosomes/Vacuoles
In 2009, Park et al. reported giant perinuclear autophagosomes and autolysosomes containing a part of the
nuclei in muscles of human patients and a mouse
model of nuclear envelopathy, and, to a lesser extent,
even in wild-type cultured cells (34). After this report
suggesting the existence of macroautophagy in selective degradation of the nucleus, or ‘nucleophagy’, there
have been reports on budding of a portion of the nucleus including DNA and its elimination by macroautophagy in mammalian cells under senescence and
genotoxicity (35, 36). A more recent study by Dou et
al. likely describes at least a part of the mechanistic
background of this elimination of nuclear buds (37).
A nuclear lamina protein, lamin B1, was found to
interact with LC3, a mammalian homolog of Atg8,
at lamina. Upon oncogenic insult, structures containing lamin B1 bud off from the nucleus with fragments
of heterochromatin and become substrates for macroautophagy. Although the precise mechanism of the induction of this process is still unclear, this selective
elimination of nuclear lamina by macroautophagy is
induced by oncogenic insult and not nitrogen
and serum starvation (37). Similar to this process, sequestration of micronuclei, a chromatin-containing structure observed in response to genotoxicity,
by macroautophagy in cells under cell cycle perturbation has also been reported (38). However,
whether this autophagic process is selective remains
elusive.
Conversely, a mechanism of selective macroautophagy targeting a part of the nucleus under nitrogen
starvation has been reported in yeast (39). Atg39 was
identified as a receptor in this pathway, although
whether nucleic acids are included in the substrates
of this pathway is still unclear. Similar to many other
receptors of selective macroautophagy, Atg39 interacts
with both Atg8 and Atg11. Atg39 localizes to the perinuclear endoplasmic reticulum (pnER) and functions
as a receptor for selective degradation of the
pnER (39). Although a mammalian homolog of
Atg39 has not been found according to sequence homology, a functional homolog of Atg39 might be
identified in the future, similar to Atg32 in mitophagy
(16).
‘Piecemeal microautophagy of the nucleus (PMN)’,
or more simply ‘micronucleophagy’, is a microautophagic process in the nucleus-vacuole (NV) junction
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of yeast (40). NV junctions are formed between a vacuolar membrane protein, Vac8 and an outer nuclear
membrane protein, Nvj1. Upon PMN, the surface
area of the NV junction increases and the nuclear envelope protrudes into the vacuole as the vacuolar
membrane invaginates and is then pinched off into
the vacuolar lumen. At the stage of scission and release
of the invaginated vesicle into the vacuolar lumen,
ATG genes are also required for PMN (41). PMN vesicles have not been reported to contain bulk chromatin, but include a granular nucleolus, which is enriched
with preribosomes (40).
Delivery of RNA Granules to Lysosomes/Vacuoles
RNA granules, such as stress granules and processing
bodies (P-bodies), are cytosolic complexes of various
proteins and RNAs involved in regulation of RNA
metabolism and translation (42). In 2013, Buchan et
al. reported that stress granules and P-bodies are targeted for macroautophagy in both yeast and mammalian cells (43). The fact that deletion of ATG genes
including atg8 and atg11 increases both stress granules
and P-bodies in yeast suggests that the elimination of
such RNA granules is based on machinery as observed
in other types of selective macroautophagy. A similar
defect in clearance of stress granules and P-bodies
upon deficiency and chemical inhibition of macroautophagy is observed in mammalian cells. In addition,
Cdc48 and its ortholog, VCP, are reported to be
required for this pathway in yeasts and mammals, respectively, although its mechanism is unclear. The authors termed this macroautophagic degradation of
RNA granules as ‘granulophagy’ (43).
A more recent study may have provided a further
insight into this mechanism. Guo et al. reported that
receptor proteins for various other types of selective
macroautophagy, p62 and NDP52, are recruited to
stress granules and P-bodies, and colocalize with
autophagosomes in mammalian cells (44). Because
knockdown of these proteins also causes an increase
of these RNA granules, p62 and NDP52 are likely receptors for granulophagy. It is noteworthy that
NDP52 also colocalizes to cytosolic puncta of transfected plasmid DNA, and the DNA puncta are targeted for macroautophagy (45). Because knockdown
of NDP52 attenuates colocalization of DNA puncta
with autophagosomes, autophagic degradation of
DNA may share a similar mechanism with granulophagy. Conversely, NDP52 is also reported to be a
receptor for selective macroautophagy involving
DICER and AGO2, a microRNA (miRNA)-processing enzyme and effector, respectively, but miRNA
itself is not targeted in this pathway (46).
Direct Uptake of Nucleic Acids by Lysosomes:
RNautophagy/DNautophagy
In addition to canonical types of autophagy, we have
reported a novel type of autophagy that directly delivers RNA and DNA into lysosomes (4, 5) (Fig. 2).
This pathway, which we termed ‘RNautophagy/
DNautophagy’ (abbreviated as RDA), was discovered
by an in vitro reconstitution assay using isolated lysosomes derived from mammalian cells or tissues. Direct
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import and degradation of nucleic acids by lysosomes
was observed only in the presence of ATP, indicating
that this system is based on ATP-dependent machinery. A lysosomal membrane protein, LAMP2C, can
function as a receptor in this pathway (4, 5). The cytosolic sequence of LAMP2C directly binds to both
RNA and DNA, and lysosomes derived from
LAMP2C-overexpressing cells show elevated levels of
RDA activity, whereas those derived from Lamp2
knockout mouse tissues show decreased levels of
RDA activity. An increase in the overall RNA degradation rate was also observed in LAMP2C-overexpressing cells, and the total amount of RNA observed in
the aged Lamp2 knockout mouse brain was higher
than that in wild-type mice (4). These data suggest
that LAMP2C-mediated RDA activity is responsible
for RNA metabolism even in live cells and tissues.
LAMP2C is the product of one of three splice variants of the gene encoding Lamp2 (47). LAMP2 is a
lysosomal membrane protein with a single transmembrane region, and the three variants, LAMP2A, B and
C, share an identical luminal region. Conversely, sequences of the 11—12 amino acid C-terminal ‘cytosolic
tail’ differ by variant. In CMA, another variant of the
protein, LAMP2A, binds to substrate proteins via its
cytosolic tail and functions as a receptor (48, 49)
(Fig. 1). From this viewpoint, RDA and CMA may,
at least in part, share a similar molecular basis.
However, unlike CMA, RDA activity is not affected
by addition of hsc70 in the in vitro reconstitution assay
(4). Because RDA has some sequence specificity for
substrate RNA and DNA in vitro (50), specific populations of nucleic acids may be selectively eliminated by
RDA in cells.
Although the rate of direct uptake of nucleic acids
decreases in lysosomes derived from Lamp2 knockout
mice, the RDA activity is not abolished completely,
indicating a limited effect of LAMP2C on RDA activity (4, 5). This finding suggests a LAMP2C-independent pathway in RDA. One possibility is that, in
addition to LAMP2C, other nucleic acid receptors
could exist. Our study revealed that the cytosolic sequence of LAMP2C binds to nucleic acids via a
common RNA-binding motif, the arginine-rich motif
(51). We found that cytosolic sequences of several
other LAMP family proteins are also enriched with
arginine residues, and some of them can also bind to
nucleic acids (51). Another possibility is that a more
important factor, other than receptors, may play a
more central role in RDA. Because transporter proteins are generally multipass membrane proteins (52),
it is unlikely that LAMP2C, a singlepass membrane
protein, could function as a transporter in RDA.
Recently, we found that SIDT2, a putative nucleic
acids transporter, mediates direct uptake of RNA
and DNA by lysosomes (53, 54). SIDT2 is a multipass
membrane protein and mammalian ortholog of a
C. elegans RNA transporter, SID-1 (55). SID-1 has
also been reported to transport plasmid DNA (56).
While the main distribution of SID-1 is at the plasma
membrane (55), SIDT2 predominantly localizes to
lysosomes (53, 57—59). Similar to LAMP2C, gainand loss-of-function studies using isolated lysosomes
Lysosomal degradation of intracellular nucleic acids
revealed that the level of SIDT2 significantly affects
the rate of direct RNA/DNA transport into the lysosomal lumen (53, 54). A putative hydrolase activity of
SIDT2 may be important for the translocation of nucleic acids, because substitution of S546, a residue reported to be responsible for the putative hydrolase
activity of the protein, abrogates the effect of SIDT2
overexpression on upregulation of lysosomal RNA/
DNA uptake activity (53, 54). Strikingly, knockdown
of SIDT2 in wild-type mouse embryonic fibroblasts
inhibits approximately 50% of total RNA degradation
(53). This result suggests that RNautophagy is an important system for constitutive RNA metabolism in
live cells, and that SIDT2 is the core factor of RDA.
Although a specific interaction between SIDT2 and
LAMP2C is observed at the endogenous level, overexpression of SIDT2 upregulates RNautophagy activity even in LAMP2 knockout cells (53), indicating that
SIDT2 is able to function independently of LAMP2C.
Degradation
After translocation into the lysosomal lumen, autophagic substrates undergo degradation by catabolic
enzymes. RNase T2 and DNase II are a well-characterized lysosomal RNase and DNase, respectively.
RNase T2 family ribonucleases are distributed in
almost all species across kingdoms from bacteria to
plants and animals, and even viruses (60). RNase T2
is an endonuclease that cleaves single-stranded RNA
into mono- or oligo-nucleotides with generally little
sequence specificity (60). However, some extent of
base specificity or preferences is reported in many of
them. For example, the human homolog of RNase T2,
RNASET2, cleaves polyA and polyU oligonucleotides,
but not polyG or polyC, at least in vitro (61). The
majority of T2 ribonucleases are glycosylated and
their optimal pH for hydrolytic activity is generally
around 5 (60). These features are consistent with the
lysosomal function of RNase T2, although extralysosomal and even non-hydrolytic functions of these proteins are also present (62). DNase II is an endonuclease
that cleaves double-stranded DNA with low sequence
specificity (63). Similar to RNase T2, the molecular
features of DNase II also adjust to the environment
of the lysosomal lumen, so that they are glycosylated
and their optimal pH is acidic (63). Another acid
DNase, referred to as DLAD or DNase IIb, which is
predicted to have evolved by gene duplication of
DNase II, is known in mammals. While the expression
pattern of DNase II is ubiquitous, DNase IIb is expressed in a limited number of tissues (63).
Recently, a well-defined study on degradation processes of RNA in yeast vacuoles under starvation was
reported by Huang et al. (8). The authors found that
degradation of RNA into nucleotides by autophagy is
completely dependent on a yeast homolog of RNase
T2, Rny1. Furthermore, a vacuolar phosphatase,
Pho8, appears to be responsible for processing nucleotides (30 -NMPs) into nucleosides. The resulting nucleosides are further converted to nucleobases by Pnp1 and
Urh1, a purine nucleoside phosphorylase and pyrimidine nucleoside hydrolase, respectively. While Rny1
and Pho8 localize to vacuoles, both Pnp1 and Urh1
are distributed in the cytosol, suggesting that nucleosides are converted to nucleobases in the cytosol after
export from vacuoles. Degradation processes of nucleic acids by mammalian lysosomes could share similar mechanisms with those in yeast, because
nucleosides are also likely the end product of both
RNA and DNA degradations in mammalian lysosomes, and the generation of nucleobases are not
observed in lysosomes (64). Because a lysosomal nucleoside transporter protein has been identified in
mammals, cytosolic export of nucleosides from lysosomes/vacuoles could occur through a specific transport system (65). Nucleobases derived from RNA
degradation via autophagy are mostly released out of
cells and not recycled, at least in yeast (8).
Physiological/Pathophysiological Roles
Although a large number of studies have revealed the
physiological and pathophysiological roles of autophagy, most of these studies are on lysosomal degradation of proteins or whole organelles, and there are
fewer studies focusing on lysosomal degradation of
intracellular nucleic acids per se. In addition, lysosomal degradation of nucleic acids involves
‘heterophagic’ degradation of extracellular components, such as endocytosis and phagocytosis, which
must be discriminated from autophagy. However, because lysosomes are possibly the main site of nucleic
acid degradation in cells under both steady state and
inducible conditions (66), its physiological significance
is promising. Deficiencies of various lysosomal proteins, including hydrolases, transporters and other soluble and membrane proteins, cause a group of genetic
diseases called lysosomal storage diseases (67). As its
name indicates, lysosomal storage diseases are characterized by aberrant accumulation of undigested macromolecules and monomeric metabolites in lysosomes.
The similarities of symptoms among different lysosomal storage diseases and the diversity of accumulated molecules, which is not necessarily related to
the function of causative genes, suggest that dysfunction of one lysosomal protein can perturb the function
of others (67). Lysosomes, as well as endosomes, can
also function as sites for induction of immune responses. Toll-like receptors (TLRs) are membrane proteins that initiate innate immune responses by
recognizing molecular patterns derived from infectious
agents. Interestingly, all TLRs that recognize nucleic
acids, but not others, localize to endosomes/lysosomes.
It has also been reported that the localization of TLRs
is important for discrimination of self and non-self-nucleic acids (68). Aberrant recognition of self-nucleic
acids by TLRs can be responsible for the pathogenesis
of autoimmune diseases.
In humans, loss-of-function mutations in the gene
encoding RNASET2 have been reported to cause familial cystic leukoencephalopathy (69). In rnaset2-deficient zebrafish, which exhibit white matter lesions as
seen in the brains of human patients, accumulation of
undigested rRNA in lysosomes of neuronal cells is
observed in the brain (70). These observations suggest
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that constitutive degradation of rRNA by autophagy is
important for homeostasis of neurons in animals.
Vacuolar accumulation of RNA, which must be exclusively rRNA, in response to RNase T2 deficiency is
also reported in yeast and plants (8, 71).
The contribution of DNase II to autophagic degradation of mtDNA, which prevents inflammation,
has also been reported (72). Because mitochondria
supposedly originate from bacteria, mtDNA harbours
features similar to bacterial DNA, including unmethylated CpG sequences. In endosomes and lysosomes,
TLR9 detects unmethylated CpG DNA as a marker
of bacterial invasion and induces innate immune responses (73). Cardiac-specific DNase II-deficient mice
show a higher rate of myocarditis and heart failure
compared with wild-type mice in response to pressure
overload (72). The fact that such symptoms are rescued
by inhibition or deletion of TLR9, and that mtDNA
accumulates in autolysosomes of the mice suggest that
aberrant detection of mtDNA in autolysosomes by
TLR9 induces inflammation and cardiac damage. A
similar detection of self-nucleic acids may be involved
in the pathogenesis of familial cystic leukoencephalopathy caused by RNASET2 mutations, because other
endosomal/lysosomal TLRs, TLR7/8, detect singlestranded RNA and are capable of responding to
endogenous RNA (74). Indeed, symptoms suggesting
inflammation around white matter lesions have been
observed in rnaset2-deficient zebrafish (70).
Because many of the studies on autophagic degradation of the nucleus in mammalian cells are related to
genotoxicity (35—38), nucleophagy may be responsible
for quality control of the genome in mammals.
Colocalization of gH2AX, a marker of DNA doublestrand breaks, in autophagosomes also reinforces this
view (34—36, 38). Analogous blebbing of the nucleus
and its elimination by macroautophagy are induced
upon cell cycle perturbation (38), cellular senescence
(35), DNA replication arrest (36) and oncogenic
insult (37), suggesting that autophagic elimination of
chromatin fragments could take place in a variety of
biological events such as development, aging and
tumourigenesis. Because Atg39-mediated nucleophagy
and PMN are induced by nutrient deprivation in yeast
(39, 40), the physiological significance of autophagy in
terms of the nucleus may be different between mammals and yeasts. In addition to autophagic elimination
of a portion of the nucleus, lysosomal degradation of
whole intracellular DNA appears to be important for
differentiation of the eye lens in mice (75). While
DNase II is generally expressed ubiquitously, another
lysosomal acid DNase, DNase IIb, is expressed exclusively in the eye lens but not in other tissues of mice.
Interestingly, a deficiency of DNase IIb causes cataract
due to intracellular accumulation of undigested DNA
in lens fiber cells, suggesting the importance of lysosomal clearance of DNA in lens development (75).
Although the precise mechanism still remains elusive,
this process has been reported to be independent of
macroautophagy (76, 77).
A recent study by Guo et al. focused on suppression
of retrotransposon insertion in the genome by macroautophagy degrading RNA granules (44). Their
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findings showed that retrotransposon RNA localizes
to stress granules and P-bodies, and is degraded by
macroautophagy that selectively targets these RNA
granules via receptor proteins p62 and NDP52 recruited to RNA granules. The increased levels of retrotransposon RNA and their genomic insertions in both
macroautophagy-inhibited cells and macroautophagydeficient mice suggest that this mechanism contributes
to maintenance of genome stability at the physiological
level (44). Because over 100 genomic insertions by
retrotransposons have been reported to cause a wide
range of diseases (78), degradation of retrotransposon
RNA by macroautophagy and its dysfunction could be
involved in a variety of genetic disorders. In addition,
autophagic degradation of other factors composing
RNA granules may also be important for maintenance
of biological homeostasis.
The physiological and pathophysiological significance of RDA is still largely elusive. However, the
fact that knockdown of SIDT2 halves intracellular
RNA degradation suggests that RNautophagy might
play a pivotal role in cellular RNA metabolism at least
in some cell types (53). An increase in the RNA degradation rate in LAMP2C-overexpressing cells and increases in the amount of total RNA in the brains
of Lamp2 knockout mice are also observed (4). In
humans, Lamp2 deficiency has been reported to cause
a lysosomal storage disease, Danon disease, whose
main symptoms are cardiomyopathy, myopathy and
mental retardation (79). It is intriguing that Lamp2c
is highly expressed in the brain, heart and skeletal
muscle (4). However, other Lamp2 variants, Lamp2a
and Lamp2b, are also deficient in Danon disease, and
one patient has been reported with a mutation in the
exon specific to Lamp2b (79). Therefore, the involvement of LAMP2C in Danon disease pathology is controversial. The effect of SIDT2 deficiency on nucleic
acid metabolism and physiology in vivo warrants further investigation. In addition, it has been reported
that mtDNA is released into the cytosol upon stress
conditions, which is mediated by the NALP3 inflammasome, and the cytosolic mtDNA induces inflammatory responses (80). Because mtDNA can be a
substrate for DNautophagy in vitro (5), mtDNA may
be transported into lysosomes by DNautophagy in
cells under such conditions.
The definition of the term ‘autophagy’ is limited to
the delivery and degradation of substrates. However,
the export of nucleic acid metabolites from lysosomes
following degradation is also an important process for
biological homeostasis. Multiple mutations in the gene
encoding human SLC29A3/ENT3, a nucleoside transporter protein localized to lysosomes, are reported to
cause several syndromes with divergent disorders
(81—83). Because all of the syndrome-related mutations in SLC29A3 analysed in a study by Kang et al.
resulted in reductions of nucleoside transport activity
(84), it is likely that dysfunctions in nucleoside transport activity are at least one of the direct causes of such
diseases. Further studies are needed to clarify how the
impairment in transport of nucleosides, presumably
from lysosomes to the cytosol, leads to the symptoms
of these diseases.
Lysosomal degradation of intracellular nucleic acids
Concluding Remarks
Most of the studies on autophagic degradation of nucleic acids began in the 21st century. This is in contrast
to the fact that ribonuclease and deoxyribonuclease
were identified in lysosomal fractions in 1955 when
de Duve first proposed the name of the organelle as
a lysosome (1). Further studies are required to delineate RNA and DNA elimination and metabolism by
autophagic systems.
Funding
This work was supported by Grants-in-Aid for Scientific Research
from the Japan Society for the Promotion of Science (24680038,
26111526, 16H05146 and 16H01211 to T.K.), Grants-in-Aid for
Scientific Research from the Japan Society for the Promotion of
Science (to K.W.), a grant from Japan Agency for Medical
Research and Development (AMED) (to K.W.) and a Grant in
Aid for JSPS Research Fellow (26-8223 to Y.F.).
Conflict of Interest
None declared.
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