autophagy leads to iron-dependent ferroptosis by degradation of ferritin and induction of transferrin receptor 1 (TfR1) expression
Iron is essential for the survival of nearly all organisms, as it serves as a cofactor for a host of biochemical processes, including oxygen storage, oxidative phosphorylation, and enzymatic reactions required for cellular proliferation1. However, the levels of free iron in a cell must be tightly regulated to avoid the generation of reactive oxygen species (ROS) via the Fenton reaction2. Transferrin receptor 1 (TfR1) and ferritins are well-known regulators of cellular iron. TfR1 is a transmembrane glycoprotein responsible for internalizing the transferrin-bound iron, which is then released into the cytoplasm and stored in a non-toxic form inside metalloprotein complexes called ferritins3. Ferritin is a complex of 24 subunits, consisting of two basic subunits: a heavy subunit (FTH1) and a light subunit (FTL).
MHV (Mouse hepatitis virus, coronavirus)
https://jvi.asm.org/content/75/7/3352
ABSTRACT
Mouse hepatitis virus (MHV), a member of the Coronaviridae,
contains a polyadenylated positive-sense single-stranded genomic RNA
which is 31 kb long. MHV replication and transcription take place via
the synthesis of negative-strand RNA intermediates from a
positive-strand genomic template. A cis-acting element previously identified in the 3′ untranslated region binds to trans-acting
host factors from mouse fibroblasts and forms at least three
RNA-protein complexes. The largest RNA-protein complex formed by the cis-acting
element and the lysate from uninfected mouse fibroblasts has a
molecular weight of about 200 kDa. The complex observed in gel shift
assays has been resolved by second-dimension sodium dodecyl
sulfate-polyacrylamide gel electrophoresis into four proteins of
approximately 90, 70, 58, and 40 kDa after RNase treatment. Specific RNA
affinity chromatography also has revealed the presence of a 90-kDa
protein associated with RNA containing the cis-acting element
bound to magnetic beads. The 90-kDa protein has been purified from
uninfected mouse fibroblast crude lysates. Protein microsequencing
identified the 90-kDa protein as mitochondrial aconitase. Antibody
raised against purified mitochondrial aconitase recognizes the
RNA-protein complex and the 90-kDa protein, which can be released from
the complex by RNase digestion. Furthermore, UV cross-linking studies
indicate that highly purified mitochondrial aconitase binds specifically
to the MHV 3′ protein-binding element. Increasing the intracellular
level of mitochondrial aconitase by iron supplementation resulted in
increased RNA-binding activity in cell extracts and increased virus
production as well as viral protein synthesis at early hours of
infection. These results are particularly interesting in terms of
identification of an RNA target for mitochondrial aconitase, which has a
cytoplasmic homolog, cytoplasmic aconitase, also known as iron
regulatory protein 1, a well-recognized RNA-binding protein. The binding
properties of mitochondrial aconitase and the functional relevance of
RNA binding appear to parallel those of cytoplasmic aconitase.
The coronaviruses belong to the newly established order Nidovirales (5)
and have enveloped virions containing the largest known RNA virus
genome. Mouse hepatitis virus (MHV) is a prototypic member of theCoronaviridae family and contains a single-stranded, positive-sense RNA genome approximately 31 kb in length (28, 29,33, 44).
Viral proteins are translated from six to seven subgenomic mRNAs as
well as from the genome. These RNAs are produced in different
quantities, and their molar ratios remain constant during MHV
replication. The virus-specific subgenomic and genomic RNAs make up a
3′-coterminal nested set (33, 62) and contain a leader sequence of approximately 70 nucleotides (nt) at the 5′ end (27,60).
Coronaviruses perform their entire replication program in the cytoplasm
of infected cells. Following uncoating, coronaviruses express the
largest known replicase polyproteins, which in turn are proteolytically
processed to yield a large number of mature proteins, including
RNA-dependent RNA polymerase. The RNA-dependent RNA polymerase, perhaps
in association with host proteins, directs the synthesis of
negative-sense full-length and subgenomic RNA from the 3′ end of the
viral genome (40). Several alternative models have been described to explain the mechanism of MHV RNA synthesis (25, 54, 61).
In all of these models, the initial step in MHV RNA replication is the
synthesis of negative-sense RNA from a positive-strand genomic template.
Analysis
of the structure of defective interfering RNAs indicated that
approximately 470 nt at the 5′ terminus, 436 nt at the 3′ terminus, and
about 135 internal nt were required for defective interfering RNA
replication in MHV-infected cells and suggested that these sequences
retain signals necessary for RNA replication (21, 22, 35,36).
The cis-acting
signals for the synthesis of negative-strand RNA have been shown to be
contained within the last 55 nt plus the poly(A) tail at the 3′ end of
the MHV genome (34)
Evidence supporting the involvement of host proteins in the replication
of a number of RNA viruses has been reported previously (for reviews,
see reference 26).
Recently, the specific binding of host cellular proteins to two
distinct sites within the 3′ untranslated region (3′-UTR) of MHV-JHM
genomic RNA was reported (68).
One site, the 3′(+)42 protein-binding element [(3′(+)42], was mapped within the 3′-most 42 nt of the genomic RNA (68),
and the other was mapped between nt 154 to 129 upstream from the 3′ end of the viral genome (38).
In
the current work, we have attempted to identify and characterize the
host proteins that interact with the 3′-most protein-binding element of
3′-UTR in MHV-JHM. Our results indicate that the RNA-protein complex
formed at this region contains at least four proteins of 90, 70, 58, and
40 kDa. The 90-kDa protein has been identified as mitochondrial
aconitase (m-aconitase), the counterpart of a well-known RNA-binding
protein, the iron-regulatory protein (IRP), which is also known as
cytoplasmic aconitase. Although m-aconitase structural data have been
used extensively to model the RNA-binding site in cytoplasmic aconitase,
this is the first report to identify a target RNA for m-aconitase. We
present data suggesting that binding of m-aconitase to the MHV 3′-UTR
substantially increases the production of infectious virus and the
expression of MHV proteins during the early stages of infection.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727448/
Nucleic Acids Res. 2017 Dec 15; 45(22): 12974–12986.
Published online 2017 Oct 23. doi: 10.1093/nar/gkx969
PMCID: PMC5727448
PMID: 29069499
Biogenesis and iron-dependency of ribosomal RNA hydroxylation
DISCUSSION
Hydroxylation of biological molecules is a fundamental enzymatic reaction involved in various biological events (72,73). In the context of the epitranscriptome, hydroxylation is a major modification that modulates RNA functions (73) and as such is involved in several tRNA modifications (34,74–77). N6-methyladenosine (m6A) and 1-methyladenosine (m1A) are demethylated via hydroxymethyl formation (78).
The RNA hydroxylation events reported to date are catalyzed by Fe(II)-
and 2-oxoglutarate (2-OG)-dependent oxygenases, including ALKBH family
proteins (75,77,79), Tet family proteins (80,81), and JmjC-domain containing protein (34).
Fe(II)/2-OG-dependent RNA oxygenases use molecular oxygen as a
substrate for hydroxylation. RlhA does not have any of the
characteristic domains and motifs conserved in these RNA oxygenases,
suggesting that it represents a novel family of proteins responsible for
RNA hydroxylation.
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