RNA Modifications: Reversal Mechanisms And Cancer - Biochemistry

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RNA Modifications: Reversal Mechanisms And Cancer Roopa Thapar, Albino Bacolla, Clement Oyeniran, Joshua Brickner, Naga Babu Chinnam, Nima Mosammaparast, and John Tainer Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00949 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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Biochemistry

RNA Modifications: Reversal Mechanisms And Cancer Roopa Thapar1*, Albino Bacolla1, Clement Oyeniran2, Joshua R. Brickner2, Naga Babu Chinnam1, Nima Mosammaparast2* and John A. Tainer1*

1Department

of Molecular and Cellular Oncology, University of Texas M.D. Anderson

Cancer Center, Houston, TX 77030, U.S.A and 2Department of Pathology and Immunology, Siteman Cancer Center, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, U.S.A

KEYWORDS RNA epitranscriptome, RNA repair, Alkylation damage, genome instability, AlkB, ASCC, ALKBH3

Corresponding

authors:

Roopa

Thapar

([email protected]),

John

A.

Tainer

([email protected]), Nima Mosammaparast ([email protected])

ACS Paragon Plus Environment

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ABSTRACT An emerging molecular understanding of RNA alkylation and its removal are transforming our knowledge of RNA biology and its interplay with cancer chemotherapy responses. DNA modifications are known to perform critical functions depending on the genome template, including gene expression, DNA replication timing, and DNA damage protection. Yet, current results suggest that the chemical diversity of DNA modifications pales in comparison to those on RNA. Over 150 RNA modifications have been identified to date, and their complete functional implications are still being unveiled. These include intrinsic roles such as proper processing and RNA maturation; emerging evidence has furthermore uncovered RNA modification ‘readers’, seemingly analogous to those identified for histone modifications. These modification recognition factors may regulate mRNA stability, localization, and interaction with translation machinery, affecting gene expression. Not surprisingly, tumors differentially modulate factors involved in expressing these marks, contributing both to tumorigenesis and responses to alkylating chemotherapy. Here we describe current understanding of RNA modifications and their removal, with a focus primarily on methylation and alkylation as functionally relevant changes to the transcriptome. Intriguingly, some of the same RNA modifications elicited by physiological processes are also produced by alkylating agents, thus blurring the lines between what is a physiological mark versus a damage-induced modification. Furthermore, we find that high gene expression of enzymes with RNA dealkylation activity is a sensitive readout for poor survival in four different cancer types, underscoring the likely importance of examining RNA dealkylation mechanisms to cancer biology and for cancer treatment and prognosis.


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INTRODUCTION The “Central Dogma” of molecular biology3 by F.H. Crick, posited that genetic information flows in a unidirectional manner, from the DNA sequence to messenger RNA to protein, and this information cannot be reversed. This seminal idea, which was the keystone of molecular biology, has been updated and transformed in the past two decades by groundbreaking discoveries of epigenetic modifications, such as the occurrence of 5-methyl cytosine (m5C)4 and the ensuing 5-hydroxymethyl cytosine (5hmC)5-8, 5formyl cytosine (5fC)9 and 5-carboxyl cytosine (5caC)10, 11 in CpG dinucleotides in DNA12; diverse base and 2´ hydroxyl RNA modifications (see Figure 1A) including pseudouridine ()13,

14,

N6-methyl

adenosine (m6A)15-17, N1-methyl adenosine (m1A)18, 19, N6, 2´-O-dimethyladenosine (m6Am)20, 5-methyl cytosine (m5C)21-23, and inosine (I)24; as well as posttranslational modifications of histone proteins25-27. Epigenetic alterations in DNA and histone proteins28,29 dramatically alter gene expression profiles independent of the DNA sequence; can be modulated by environmental stimuli30 and genotoxic stress31-33 (see Figure 2), and are critically important in progression of several diseases, particularly cancer, as summarized in several excellent reviews34-36. Recent advances in high-throughput sequencing (HTS) technologies coupled with development of sensitive new methods for quantitation of modified bases has allowed mapping of numerous diverse RNA modifications on a transcriptome-wide scale37-40.

These RNA base and 2´ hydroxyl modifications

increase the chemical, structural, and biological diversity of all RNAs16, 41 (see Figure 1A). Intriguingly, methylation of m6A39 and m5C42 is ubiquitous in mRNAs and long non-coding RNAs (lncRNAs), particularly in evolutionarily conserved sites in the coding sequence as well as 3´ and 5´ untranslated regions (UTRs) of many genes. In addition, all RNAs can be modified by alkylation damage that occurs on nucleic acids as a result of endogenous compounds or exogenous agents43-45 (see Figures 1B & 2). Repair of this damage on RNA may be critical in some instances (see Figure 1C) because unregulated alkylated RNA is potentially genotoxic and can affect transcription, RNA processing, and mRNA turnover46-48 (see Figure 2). The fact that ten-fold more alkylated RNA is repaired compared to alkylated


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Biochemistry

DNA (due to the abundance of RNA in a cell) highlights the importance of RNA repair49. Chemotherapeutics that induce alkylation damage (see Figure 1B) are one of the most common classes of agents used in cancer treatment50, 51 (see Figure 1B), underscoring the clinical importance of an improved mechanistic foundation for RNA alkylation repair. Families of RNA-specific m6A and m5C S-adenosyl Lmethionine (SAM)-dependent methyltransferases (MTases) such as METTL352-54, METTL1455, METTL1657, and TRDMT158,

59

56,

have been identified as “writers” of RNA methylation. The m6A is

reversible (see Figure 1C), and RNA demethylases or “erasers” belonging to Fe(II)/2-oxoglutarate (2OG)-dependent AlkB (alkylated DNA repair protein B) dioxygenase family45,

60, 61,

such as FTO62-64,

ALKBH165, 66, ALKBH367-70, and ALKBH571 have been shown to be essential for normal reversal of RNA methylation (see Figure 1C) as well as regulation of RNA alkylation. Here we provide a synopsis of recent advances in the area of RNA epitranscriptomics72 that includes normal biological methylation as well as undesired base alkylation due to RNA damage. Some have used the term ‘RNA epigenetics’ to describe this burgeoning field73. However, the implication of generational inheritance by this term can be confusing, as the mechanisms of gene expression changes with these RNA modifications are largely unknown. Thus, we prefer the use of the term ‘RNA epitranscriptomics’38, 74, which we define as functional changes to RNA due to modifications induced physiologically or by damaging agents that do not change the RNA sequence. We furthermore integrate published results on the epitranscriptome with new computational analysis from our laboratories connecting the ALKBH3-ASCC alkylation reversal complexes to cancer outcomes in patients. Based upon this integration, we propose that reversal of undesired RNA alkylation or RNA repair may be important for cancer cell proliferation and survival mechanisms that have promise as targets for advancing anti-cancer therapy75. This is a relatively untapped avenue for future research in RNA research and we expect this area to show substantial growth.


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Figure 1. Nucleotide modifications and reversal mechanisms. (A) Common sites of RNA modification. (B) Commonly used monofunctional chemotherapeutic agents for cancer treatment and types of lesions introduced by these agents on DNA/RNA. (C) AlkB enzymes can reverse methylation on RNA and DNA in a two-step reaction that involves formation of a hydroxymethyl intermediate and release of formaldehyde.


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Biochemistry

NORMAL vs. ABERRANT RNA ALKYLATION Recent transcriptome-wide mapping studies have identified several reversible methylation sites in mRNAs (summarized in Figure 1 and Table 1) and non-coding RNAs: e.g. occupancy of 1-3 sites per mRNA for N6-methyl adenosine (m6A) is reported39, tRNAs76, rRNAs77,

78,

40.

However, alkylation of nucleotide bases in

and snRNAs79 has been recognized for decades80-83. These modifications are

conserved across genomes, becoming more complex and diverse from archaea to humans. They range from addition of simple methyl groups at conserved nucleotide positions, to the addition of complex bulky hydrophobic groups such as N6-dimethylallyl adenosine (i6A37), methylaminomethyl-5-uridine (queuosine),

agmatidine,

N4-acetylcytosine,

wybutosine,

lysidine,

5-methoxycarbonyl-methyl-2-

thiouridine, among many others. A comprehensive list of RNA modifications can be retrieved either from

MODOMICS

(http://modomics.genesilico.pl)

(http://mods.rna.albany.edu/home).

or

The

RNA

Modification

database

Whereas the introduction of a methyl group follows a simple

enzymatic reaction involving specific methyltransferases, in vivo synthesis of the hyper modifications such as queuosine and lysidine involve multiple enzymatic steps. Structure/function studies of modified RNAs reveal that these modifications and hyper modifications occur in specific regions of all RNAs, are not random, and are important for RNA maturation78,

84, 85,

RNA folding86,

87,

for decoding of the tRNA anticodon loop88, for maintaining

translation efficiency63, 89-92, RNA processing93, 94, miRNA biogenesis95, 96, and viral replication97, 98. For example, loss of queuosine (Q) at the wobble position 34 in the E.coli tRNATyr and tRNAHis anticodon arm results in frameshifting and slippage of tRNAs at the P-site due to slow entry of the hypomodified (Q34) tRNATyr and tRNAHis in the A-site99,

100.

Similarly, position 37 in the anticodon loop is almost

always modified in tRNAs: introduction of N6-dimethylallyl adenine (i6A37), (2-thiomethyl, N6dimethylallyl)-adenine (ms2i6A37), N6-(4-hydroxyisopentenyl) adenosine (io6A37) and 2-methylthio-N6(4-hydroxyisopentenyl) adenosine (ms2io6A37) modifications at this position affect the conformation dynamics of the loop88 and is important for the efficiency of peptide bond synthesis101; insertion of wybutosine at position 37 of tRNAPhe causes stacking onto the loop, stabilizes codon-anticodon


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Table 1. Structural and functional consequences of base modifications in RNA Base modification

N1-methyladenosine

Location RNA

in Structural Consequences of modification

Found near start codons and near the first splice site of mRNAs Found in tRNAs, rRNAs, mRNAs, lncRNAs

Disrupts WC base-pairing

Promotes duplex unfolding

Functional consequences

RNAProtein Interactio ns

May promote translation initiation

May bind to several YTH domain family proteins but not YTHDC2

Important for folding of tRNAs

Ten times less abundant than m6A N6-methyladenosine

Most common in 3’ UTRs, near stop codons, and at splice sites in RRACH motifs (R = A/G, H = A/C/U)

Prefers the energeticallyunfavorable syn conformation for a N6Me-A-U pair, resulting in destabilization of RNA secondary structure when found within a helix.

Found in mRNAs, lncRNAs, tRNAs

Inhibits WC basepairing Promotes base stacking when fold at ends of helices, resulting in stabilization of the RNA helix.

Decrease in the half-life of the mRNA in cells

Changes the subcellular localization of the RNA

YTH domain containing proteins

HNRNP family proteins

Regulates RNA processing, splicing, export, translation Important for folding of tRNAs Cell cycle regulation Regulation of circadian rhythms

5-methylcytosine

Identified in tRNAs, rRNAs,

Promotes stability via favorable

May promote mRNA export

Unknown


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Biochemistry

ncRNAs and mRNAs at levels much lower than m6A

base stacking No effect on base-pairing

Important for translational efficiency May be important for RNA mediated epigenetic heredity

Enriched in GCrich regions of mRNA Tissue specific 5-Hydroxy methylcytosine

lncRNAs and mRNAs

Unknown

Increases translation efficiency

Unknown

Involved in brain development Pseudouridine

Most abundant RNA modification. Found in rRNAs, tRNAs, and to a lesser extent in mRNAs

Stabilizes basepairing.

Enhances half-life of the RNA

Reduces flexibility.

May increase or decrease translation efficiency

Unknown


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Figure 2. RNA fate. Cellular RNAs can be modified as part of normal epigenetic pathways or may undergo damage in response to ultraviolet radiation, endogenous and exogenous alkylating agents or oxidative stress. The RNA m6A modification has been shown to regulate the ultraviolet-induced DNA damage response via activation of the METTL3-METTL14 methyltransferase complex by PARP1/21. Normal and aberrant alkylation impacts all aspects of RNA metabolism.


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Biochemistry

interactions88, 102 and is important for translation. Human ribosomal RNAs are also modified by 2´ OMe and at >200 different sites, and a few sites also show base modifications77, 78. These base and sugar modifications are essential for rRNA folding and function. Intriguingly, the nucleolar snoRNAs that are required for rRNA maturation are also modified cotranscriptionally84, 85, 94, illustrating the co-dependence of different pathways and universal importance of RNA modifications for the overall fitness of cells. Internal modifications consisting of base methylation, 2´-OMe, and sites in mRNAs have only recently been discovered37-40 (Figure 1 & Table 1) and have been well-summarized16, 41, 103. As yet, there is no evidence that mRNAs or lncRNAs are modified by larger bulky groups like tRNAs as part of normal cellular pathways, however this has not yet been explored. RNA alkylation can also occur via endogenous and exogenous alkylating agents (Figure 2) that can chemically modify RNA bases at ring nitrogens (N) and extracyclic oxygens (O) by SN1 or SN2 types of nucleophilic reactions, in a manner similar to DNA51, 104-108 (Figure 1B). Unlike regulated biological methylation, the biological consequences of undesired RNA alkylation remain poorly defined and have the potential to disrupt posttranscriptional events such as RNA processing, splicing and translation104. At least two modifications, m1C and m3C, may be induced by chemical methylating agents that are also known products of RNA methyltransferases109,

110.

Besides the fact that these modifications block

canonical Watson-Crick base pairing, the biological function of these modifications is only just starting to be investigated. Unless these two physiological modifications are in some particular sequence context recognized by the cell, there may be no clear distinction between whether such marks are due to damage, or induced physiologically. This may be one mechanism by which alkylating agents induce their cytotoxicity. Such modifications may be induced spontaneously by endogenous sources, such as the cellular methyl donor S-adenosylmethionine (SAM)111 a cofactor in biological methylation reactions. Exogenous sources are abundant and are present in environmental pollutants, including methyl halides and tobacco products112-115 as well as anticancer chemotherapies116, 117 (Figures 1 & 2). In DNA, the most common modifications produced by chemotherapeutics (Figure 1B) that are mono- or bifunctional


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methylating agents, are N7-methylguanine (m7G) and N3-methyladenine (m3A), with 1-methyladenine (m1A), 3-methylcytosine (m3C), O6-methylguanine (O6mG), the backbone phosphoryl oxygens, guanineguanine (G-G) and guanine-adenine (G-A) inter-strand crosslinks being less common. Most alkylating chemotherapeutics (with the exception of Busulfan) currently used in the clinic (Figure 1B) are SN1 type nucleophiles (Figure 1B). In addition, alkylating agents such as methyl methanesulfonate (MMS) and nitrogen mustards perform SN2 type of reactions forming m1A and m3C, which block DNA replication51, 118.

The structural and chemical consequence of each modification in DNA is different: whereas m7G

results in depurination and formation of an abasic site, m3A is highly cytotoxic and blocks DNA synthesis by inhibiting DNA polymerases. Similarly, m1A and m3C can result in fork blocks as well as mismatches and are hence mutagenic, whereas O6mG can result in mismatch with thymine during replication, resulting in a G:C to A:T mutagenic transition. RNA modification at the nucleobases in response to damage due to ultraviolet (UV) radiation119, oxidative damage120, and alkylation70,

121

has been reported and may be linked to diseases such as

neurodegeneration31, 122 and cancer104, 121, 123, 124. Since there is ~4-6 times more RNA than DNA in the cell, RNA damage can have a deleterious effect on gene expression. Undesired RNA modifications in yeast rRNAs125 and mammalian tRNAs107 have been shown to inhibit protein synthesis due to inhibition of rRNA-tRNA base-pairing interactions43. In addition, unwanted RNA modifications can cause RNAs to misfold, can result in mispairing of ncRNA-mRNA interactions leading to altered mRNA decay rates and production of aberrant proteins. As part of quality control mechanisms, eukaryotic cells have developed specialized surveillance mechanisms for either direct reversal of RNA damage or targeting aberrant RNAs for degradation126, 127.

RNA DAMAGE SURVEILLANCE AND REPAIR MECHANISMS It is well established that cells are equipped with a number of DNA repair pathways that facilitate dealkylation/demethylation. In contrast, RNA repair mechanisms have only recently been delineated. Unlike aberrant DNA methylation4, 26, 27 that can have profound effects, ranging from the induction of


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Biochemistry

mutations to replication fork blockage and programmed cell death, the biological consequences of unwanted RNA methylation remain poorly defined. Since new copies of RNA can be made, one relevant solution for the cell is to simply degrade the RNA. Indeed, structure-specific DNA nucleases also act on RNA, which deserves more attention128. However, accumulating evidence suggests that damaged RNAs can be problematic because they can stall ribonucleoprotein complexes (RNPs) and RNA utilizing machines, such as the ribosome. Thus repairing or degrading the RNA via quality control pathways may be critical to free the ribosome and other RNPs for essential functions in protein production105. At least three distinct mechanisms have been shown to repair alkylation damage in DNA: base excision repair (BER), direct reversal by O6-alkylguanine-DNA alkyltransferases (AGT) also known as methylguanine methyltransferase

(MGMT),

and

direct

dealkylation

repair

by

the

AlkB

family

of

demethylases/dealkylases43, 44, 50, 51, 129. An understudied additional mechanism involves a protein in yeast that can link alkylated base damaged and nucleotide-excision repair (NER)130-133, suggesting that novel alkylation damage responses may be found in human cells. Central issues in alkyation and other base repair is damage detection and pathway coordination that have been enlightened by structural results134. These data have cancer relevance as hyperactivation of one or more pathways can be a major factor in alkylation chemotherapy resistance50,

135.

Importantly however, of the known alkylation response

pathways, only direct dealkylation repair by the AlkB family appears to be relevant for repair of alkylated RNA.

AlkB family of oxidative dealkylases A wide array of deleterious alkylated lesions in RNA can be removed through a direct reversal mechanism analogous to DNA damage reversal70 (Figure 1C & Figure 3).

AlkB proteins are

evolutionarily conserved oxidative demethylases present in both prokaryotes and eukaryotes (except S. cerevisiae) and are named after E.coli AlkB45, 60, 123, 136-138. The bacterial AlkB protein can catalyze the direct oxidative demethylation of positively-charged m1A and m3C lesions in DNA and RNA, neutral m3T and m1G adducts, as well as bulky cyclic adducts of adenosine and 3,N4-etheno-cytosine. The activity of


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Figure 3. Reaction mechanism for dealkylation by AlkB family proteins. A two-step mechanism of oxidative demethylation has been proposed from kinetic measurements and X-ray crystallographic studies. The initial reaction involves activation of molecular oxygen by -ketoglutarate and Fe(II) yielding a high-spin Fe(IV) oxo state and release of CO2. The Fe(IV)=O species abstracts a proton from the methyl group to yield a radical intermediate and a Fe(III)-OH. The hydroxyl group is abstracted by the carbon radical to give a hydroxymethyl intermediate, which is released from the AlkB active site in the presence of H2O as the demethylated product.


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AlkB appears indispensable in restoring the biological functions of mRNA and tRNA inactivated by chemical methylation as part of an adaptive response in vivo43, 139-141. AlkB proteins are non-heme Fe(II) and -ketoglutarate-dependent dioxygenases that use molecular oxygen to remove the alkyl groups from modified RNA and DNA bases by oxidative dealkylation45, 60, 142 (Figures 1C & 3). In eukaryotes, the AlkB superfamily consists of nine mammalian homologues (ALKBH1-8, FTO), although not all of them are involved in DNA and RNA repair. ALKBH2 appears to be a dedicated nuclear repair enzyme with significant preference for DNA relative to RNA, as shown both in vitro and in mouse knockout studies143, 70, 144. Whereas ALKBH1 can demethylate m3C in DNA and RNA in vitro145, it likely acts upon m6A in vivo66, 146, 147. On the other hand, strong evidence exists for ALKBH2 and ALKBH3 acting as bona fide DNA repair enzymes68, 70, 143. Similar to E. coli AlkB, purified ALKBH3 has dual specificity towards both RNA and DNA. ALKBH3 (also known as ABH3 or prostate cancer antigen 1, PCA-1) also plays an important role in alkylation damage repair in several tumor cells where it is overexpressed68. In vitro, it has broad substrate specificity, reversing alkylated bases such as m1A in DNA and RNA, m3C in DNA and RNA, 1-ethyl-adenine (e1A) in DNA, m1G in RNA, and m3T in DNA. ALKBH3 prefers ssDNA or ssRNA in contrast to ALKBH2, which strongly prefers dsDNA70,

144.

These two enzymes have distinct subcellular localizations,

suggesting that they may also have different biological roles. ALKBH2 is located exclusively in the nucleus where it mediates DNA dealkylation repair during the phase of DNA replication, via direct association with PCNA148. ALKBH3 is found in the nucleus as well as the cytosol, the latter suggesting a clear function in repairing RNA, although definitive evidence for this is lacking. It is interesting that in many tumor cell lines that overexpress ALKBH3, inhibition of this demethylase is significantly detrimental to cell growth even in the absence of exogenous alkylating agents, suggesting that ALKBH2 cannot adequately compensate for ALKBH3 function in certain contexts68. Beyond these repair-associated AlkB proteins, four other RNA demethylases are ALKBH1, ALKBH5, FTO and ALKBH8. ALKBH5 and FTO are key regulators of the m6A modification in RNA


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Figure 4. Crystal structures of AlkB family proteins and a model for the ALKBH3-ASCC complex. (A) Crystal structure of the demethylase domain of ALKBH3 bound to Fe(II) and ketoglutarate (PDB code 2IUW) showing the overall folding topology of AlkB proteins consisting of 14 -strands and 2 -helices. The active site is capped by a flexible 4-5 hairpin loop, unique to ALKBH3, that also lines the DNA/RNA binding pocket and may act to discriminate between ssRNA/DNA and dsRNA/DNA substrates. (B) Arrangement of active site residues that coordinate the Fe(II) and -ketoglutarate in the E.coli AlkB crystal structure bound to the methylated trinucleotide Tm1A-T (PDB code 2FD8). The coordination at the active site is conserved in AlkB proteins and is required for the chemistry shown in panel (A). (C) Crystal structure of the E.coli AlkB-dsDNA complex (PDB code 2BIE). The AlkB domain is in blue, the damaged DNA strand with the flipped out m1A adduct is in green and the complementary strand is in red. (D) Crystal structure of ALKBH2dsDNA complex (PDB code 3BUC) is shown. The AlkB domain is in blue, the damaged DNA strand with the flipped out m1A adduct is in green and the complementary strand is in red. (E) Crystal structure of the RNA m6A demethylase human FTO (PDB code 3LFM). The AlkB domain is in blue and the C-terminal accessory domain in purple. The protein was crystallized with m3T in the active site. The L1 loop (shown in yellow) flanks the active site, restricting the substrate to a ssRNA. It is unique to FTO. (F) Crystal structure of the RNA m6A demethylase ALKBH5 (PDB code 4NJ4) is shown bound to Mn2+ and the small molecule IOX3. ALKBH5 also has a single-strand nucleic acid recognition loop (shown in red). In panel (G), the domain organization and common structural motifs in the ALKBH3-ASCC complex is depicted. (H) A model for transcription coupled RNA and DNA repair by ALKBH3-ASCC is shown. The helicase domain of ASCC3 could assist in unwinding RNA and DNA, allowing ALKBH3 access to single-stranded RNA/DNA substrates.


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that have been suggested to be important for regulating RNA processing, translation and degradation. Consistent with its role in m6A demethylation of ssRNA, FTO forms discrete foci in the nucleus, partially overlapping with nuclear speckles and mRNA processing factors62. These demethylases are discussed in greater detail in the next section. It is possible that other AlkB homologues also play important roles in that have been suggested to be important for regulating RNA processing, translation and degradation. Consistent with its role in m6A demethylation of ssRNA, FTO forms discrete foci in the nucleus, partially overlapping with nuclear speckles and mRNA processing factors62. These demethylases are discussed in reversing aberrant RNA methylation, since their overall mechanisms of demethylation are similar. Insights into substrate recognition is gained from several crystal structures of AlkB proteins solved with either Fe(II) or Mn(II) ions, -ketoglutarate (-KG) and alkylated nucleic acid substrates67, 149-153.

The catalytic core of the demethylase domain (Figure 4A) has a characteristic -jelly-roll fold of

the Fe(II)/2--KG dependent dioxygenases. The active site is partially buried and the conserved triad residues His-X-Asp-Xn-His and two arginines in an Arg-X5-Arg motif are oriented for optimal coordination of Fe (II) and -KG in all structures (Figures 4A and 4B). The conserved arginines make salt-bridge interactions with the carboxylate groups of -KG and mutation of iron coordinating residues His191 and Asp193 in ALKBH3 completely abrogates demethylase activity67. The crystal structure of AlkB bound to dsDNA via covalent cross-linking (Figure 4C) shows a positively charged binding groove in AlkB that interacts with the phosphodiester backbone of only the damaged strand of the dsDNA substrate. The m1A base is stabilized in the active site via  stacking interactions with Trp69 and His131 residues and hydrogen bonding interactions of Asp135 and Glu136 with the N6 of the methylated adenine. It is assumed that AlkB proteins recognize their targets in a non-sequence specific manner, although this has not been rigorously tested. Notably, DNA base repair enzymes can show increased activity on some sequences compared to others, but this is typically associated with repair from dsDNA with the sequence bias reflecting ease of flipping the damaged base from the duplex, as seen for uracil-DNA glycosylase154.


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Biochemistry

The crystal structure of ALKBH2 bound to dsDNA (Figure 4D) differs from the AlkB-dsDNA complex in that ALKBH2 interacts with both the damaged strand as well as the complementary strand of dsDNA. A unique feature of ALKBH2 is the presence of a hydrophobic -hairpin close to the active site. Phe102 in the -hairpin intercalates into the dsDNA to enable base flipping of the damaged base. The mechanism of substrate recognition for ALKBH3 is unknown and awaits structural studies of the ALKBH3-DNA and ALKBH3-RNA complexes. A positively charged groove lies adjacent to the active site of ALKBH3, and could provide a suitable binding pocket for ssRNA or ssDNA67. The active site of ALKBH3 is capped by a flexible and charged 4-5 hairpin loop that also lines the DNA/RNA binding pocket and may act to discriminate between ssRNA/DNA and dsRNA/DNA substrates67. -hairpin swapping experiments in ALKBH2 and ALKBH3, along with site-directed mutagenesis studies strongly suggest that this hairpin motif may play an important role in conferring substrate specificity towards ssDNA and dsDNA substrates in these enzymes155, 156. An unknown question is how a subset of AlkB proteins can distinguish between RNA and DNA substrates that carry the same base modification. FTO also recognizes m3T in DNA and m3U in ssRNA. The crystal structure of FTO bound to m3T (Figure 4E) shows an N-terminal AlkB domain and a C-terminal -helical domain that adopts a novel fold. In contrast to ALKBH2, FTO has a loop that flanks the active site and occludes binding of the unmethylated strand of dsRNA or DNA, thereby conferring specificity to single-stranded substrates. Similar to FTO, ALKBH5 also has a flexible loop near the active site that limits specificity to ssRNA or ssDNA substrates. ALKBH8 recognizes hypermodifications in tRNAs and has an N-terminal RRM motif important for RNA recognition (Figure 4F). Structures of AlkB demethylases bound to methylated ssRNA and structured RNA substrates should reveal the precise mode of RNA recognition by these proteins. Our understanding of the reaction mechanism has been obtained from kinetics157,

158,

crystallographic studies and molecular dynamics simulations of E.coli AlkB-DNA complexes (PDB codes 3O1M, 3O1O, 3O1P, 3O1R, 3O1S, 3O1T, 3O1U and 3O1V) obtained in the presence of covalently


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linked alkylated substrates, Fe (II) and -ketoglutarate under anaerobic conditions159 as well as NMRbased dynamics measurements160.

When oxidation was initiated in crystallo, the crystal structures

provided evidence for formation of hydroxylated alkyl intermediates. The proposed reaction mechanism (Figure 3) consists of a four-electron reduction of molecular oxygen, with two electrons provided by Fe (II) and two electrons by -ketoglutarate (Figure 3). The substrate is converted into a hydroxylated alkyl intermediate which is unstable, releasing formaldehyde, so as to restore the normal base. NMR and fluorescence-based measurements show that this multistep catalytic cycle is facilitated by conformational transitions in the AlkB protein in which the nucleotide recognition lid (NRL) fluctuates between an open conformation where the NRL is away from the active site and is disordered, to the closed state that is represented by the crystal structures. Binding of -ketoglutarate changes the ratio of the open:closed states from ~5:1 to 0.5:1, increasing the affinity for the DNA/RNA substrate160. Exceptions to this reaction mechanism include the AlkB domain of FTO, which is capable of converting m6A to N6hydroxymethyladenosine as well as N6-formyladenosine161 and the AlkB domain of ALKBH8, that does not

demethylate

tRNA

substrates

but

generates

the

stable

hydroxylated

product

methoxycarbonyl(hydroxymethyl)uridine from 5-methoxycarbonylmethyluridine162, 163. One might expect that the direct alkylation reversal enzyme ALKBH3 functions alone in demethylation reactions, as compared to multi-step DNA break and nucleotide excision repair processes that require ATPases or helicases and other macromolecular assemblies to function164, 165; XPB and XPD helicases in TFIIH orchestrate DNA duplex opening and damage verification to coordinate repair with transcription and cell cycle via CAK kinase166. Surprisingly this is not the case. ALKBH3, which has been shown to dealkylate DNA and RNA, uniquely partners with the Activating Signal Cointegrator Complex (also known as ASCC, ASC1 or TRIP4) heterotrimer to demethylate DNA targets in the cell50, 167

(Figure 4G). The largest subunit, ASCC368 binds directly to ALKBH3 and contains two tandem

superfamily 2 helicase cassettes, conserved with DEAD-box RNA helicases (Figure 4G)68. Although some Fe2+ and 2-oxoglutarate dependent oxygenases form functional dimers (Pro-metastatic collagen


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lysyl hydroxylase dimer assemblies stabilized by Fe2+-binding168, there is no evidence for ALKBH3 selfassembly suggesting that an ALKBH3 monomer binds to ASCC3. The ASCC2169 subunit may bind to TBP, TFIIA, and p300170, linking it to transcription. Recruitment of the ALKBH3-ASCC repair complex to nuclear speckle bodies occurs specifically during alkylation damage and requires K63-linked polyubiquitin recognition of the CUE domain of ASCC2169. The ASCC1 subunit has a K-homology (KH) domain, a sequence-specific ssRNA binding motif167, 171, 172 plus an RNA ligase-like domain consistent with the putative role of this complex in RNA repair. The ASCC1 C-terminal RNA ligase-like domain is homologous to bacterial 2´-5´ RNA ligases and belongs to a larger family of 2H phosphoesterase domaincontaining proteins. Its closest human homologue is AKAP18, which specifically binds mononucleotides 5ÁMP and 5ĆMP over 5′GMP/TMP via two of its HxT motifs173. This domain also serves as a 2´-5´ oligoadenylate phosphodiesterase, although the function of this activity is not clear173. ASCC1 knock-out in mammalian cells shows increased ASCC3 foci formation during alkylation damage, suggesting ASCC1 negatively regulates ASCC recruitment169. Another unique feature of ASCC is that in response to the alkylation agent MMS, the ALKBH3ASCC complex forms distinct ASCC foci that are not induced with DNA damaging agents such as irradiation, UV, topoisomerase inhibitor camptothecin (CPT), or the DNA break-inducing agent bleomycin169. Intriguingly, alkylation-induced ASCC foci partially co-localize with other alkylation repair factors like DNA pol and the demethylase ALKBH2169.

Furthermore, pre-incubation of

permeabilized cells with RNaseA reduced these foci. These data suggest that a distinct alkylationspecific response pathway exists in human cells, which was previously unknown169. Proteomic analysis with or without alkylation damage169 has revealed that the ASCC complex interacts with the RNA Pol II transcription machinery, transcription termination factors, polyadenylation factors, splicing factors and DNA repair proteins. These collective results suggest that ALKBH3-ASCC is recruited to active transcription sites during alkylation damage (Figure 4H). These data, combined with ALKBH3 specificity towards methylated RNAs, strongly support a role of ALKBH3-ASCC in RNA repair and regulation.


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Figure 5. Emerging implications of abnormal RNA modifications reversal in cancer. Modifications in RNA and their functional implications in cellular physiology (shown in green boxes). This involves a cascade of molecular reactions and interactions mediated by writers, readers and eraser proteins that assure fine-tuning of appropriate levels of modified RNA. Overexpression of erasers (ALKBH5/FTO and ALKBH3-ASCC) result in an abnormal reversal of methylated RNA and may contribute to cancer progression. Damaged RNA due to endogenous and exogenous alkylating agents or oxidative stress (orange boxes) may lead to RNA degradation or defects in protein synthesis. These damaged RNAs may be repaired/demethylated by ALKBH3 and possibly by ALKBH5 and FTO.


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Repair of RNA breaks by RNA ligases RNA repair of breaks by RNA ligases is another well-characterized RNA repair mechanism more commonly observed in phage, plants, prokaryotes and lower eukaryotes such as yeast174, 175. RNA breaks can occur due to the action of endogenous endonucleases during RNA processing or due to radiation and stress such as viral infections or the unfolded protein response. There is evidence that such breaks can occur during tRNA splicing, or due to damage to ribosomes. Resealing the break is a repair process that involves modification of the RNA ends followed by resealing the ends by RNA ligases176. RNA breaks could leave either (1) a 2´, 3´ cyclic-PO4 and a 5ÓH (due to a transesterification reaction) or (2) 3ÓH and 5´PO4 ends (due to a hydrolytic reaction). Similar to DNA ligases, several ATP-dependent RNA ligases have been characterized. Although noteworthy, these do not appear to act in a major RNA damage reversal mechanism in humans177.

CONNECTIONS BETWEEN RNA METHYLATION AND RNA PROCESSING Among the approximately 150 chemical modifications that can occur in eukaryotic cellular RNA, the N6‐methyladenosine (m6A), is the most prevalent methylation mark in mRNAs40 and lncRNAs39, and to a lesser extent in tRNAs178 and rRNA39, 179. Besides m6A, which accounts for about 80% of all RNA base methylation, the spectrum of major physiological mRNA methylation marks comprises m1A, m3C, m5C and hm5C, which is derived from the hydroxylation of m5C. As mentioned earlier, some of these physiological modifications, such as m1A and m3C, overlap with those induced by alkylating agents. The discovery of an interplay between the introduction and removal of methyl marks by “writer’’ and “eraser” proteins16,

180,

as well as their recognition by “readers” has the potential to vastly expand the splicing

code181 and has revealed a wide range of implications and functional consequences for chemically modified RNA16 (Figure 5). Insights into the function of the m6A modification come from mapping studies, which show enrichment around stop codons, in 3ʹ untranslated regions (3ʹUTRs), and within internal long exons41, 180. The structural consequences of the m6A modification in RNA are position-dependent: in general, m6A


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alters Watson-Crick base-pairing and results in helix unfolding by switching the N6Me-A-U pair to a syn conformation182. However, when found at the ends of RNA helices, m6A can stabilize the duplex via favorable stacking interactions (Table 1). At least two mammalian m6A methyltransferases have been characterized, namely the METTL3/METT1455, 183 complex and the METTL1657 methyltransferase. The METTL3/METLL14 complex includes additional adaptor proteins critical for proper function, including the Wilms tumor 1-associated protein (WTAP) and KIAA142952,

55, 184-186.

At least two mRNA

demethylases can reverse the m6A modification in vitro, namely FTO (Fat mass and obesity-associated) and ALKBH5 (AlkB homolog 5)71. Similar to other AlkB proteins, the catalytic activities of FTO and ALKBH5 depend on the presence of Fe (II) and α-ketoglutarate as co-factors to oxidize the N-methyl moiety of m6A to a hydroxymethyl group, which is then hydrolyzed to the unmodified base45, as elaborated earlier (Figures 1C & 3). We should note, however, that whether m6A is the major substrate for these enzymes in vivo is not yet clear. Deposition of the m6A mark in RNA has diverse effects on RNA metabolism that includes RNA processing, RNA splicing, mRNA export, mRNA translation and decay187. Functional and structural characterization of the YTH domain-containing protein family, which can directly or indirectly recognize the m6A modification in RNA, has been crucial in understanding the role/s of m6A modification188-191. These proteins include YTHDF1-3, as well as YTHDC1-2, and exhibit distinct functions. The YTHDF1 selectively recognizes m6A-containing RNAs, recruits translation initiation factors and expedite access to the ribosome decoding center, thus promoting translation efficiency90. Remarkably, on the other hand, YTHDF2 accelerates mRNA decay of cytoplasmic m6A-modified mRNAs by shuttling them into processing bodies (P-bodies)192. The specific function of YTHDF3 remains undefined. However, it can regulate both translation and decay of methylated mRNAs by forming complexes with either YTHDF1 or YTHDF2193 respectively193, 194. The YTHDC1 protein is predominantly located in the nucleus, while YTHDC2 is present in the nucleus and the cytoplasm195, 196. In mammalian cells YTHDC1 was found to interact with the serine and arginine-rich splicing factors SRSF1, SRSF3, SRSF7, SRSF9-10, and its depletion by small interfering


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Biochemistry

RNA results in splicing abnormalities, which can be rescued by re-expression of YTHDC1 protein, thus strongly implicating a role in regulating alternative splicing of pre-mRNA189, 197. In addition, several other factors including HNRNPA2B1 and ELAVL1/HUR may act as m6A readers95, 96. m6A may also control gene expression by modulating mRNA export from the nucleus to the cytoplasm198,

199

and well as

modulating stability of small non-coding RNAs. Intriguingly, knockdown of the m6A demethylase ALKBH5 enhances mRNA export to the cytoplasm71 and in vivo studies in ALKBH5 knockout mice show that m6A-containing mRNA levels are abnormally increased in the cytoplasm leading to defects in spermatogenesis in mice testes71. Notably, these mRNA export phenotypes may be due to defective RNA processing in the nucleus as opposed to effects on the rates of mRNA export per se, and thus specific molecular mechanisms of these phenotypes remain to be defined. The m1A modification is found primarily on rRNAs and tRNAs, and can alter RNA structure by disrupting Watson-Crick base-pairing and promoting duplex unfolding87 (Table 1). Similar to m6A, m1A has mostly been detected in the 5ÚTRs of mRNAs, where it appears to promote translation initiation through the specific interactions with YTHDF1 and YTHDF319,

200,

although the mechanisms are

unknown. The methyltransferases TRMT6/TRM61A are primarily responsible for formation of m1A201, 202,

and m1A may be enzymatically demethylated from tRNA and mRNA in vitro and in vivo by the

ALKBH3 demethylase43,

67, 70, 203.

However, other demethylases may also perform this function. The

methylation modifications m3C and m5C have also been detected in many RNA species in organisms from all three kingdoms of life. However, their exact positions and roles in RNA metabolism is poorly understood21,

42, 109, 204-206.

METTL2 and METTL6 catalyze m3C deposition in tRNAs,109 whereas

METTL8 specifically directs mRNA methylation109. Intriguingly some of these modifications, specifically m1A and m3C, inhibit Watson-Crick base pairing207, which would be detrimental for any canonical use of such RNA templates. Certainly, additional biochemical and cell biological characterization of these enzymes are appropriate to understand their physiological roles.


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Figure 6. High gene expression of RNA demethylation enzymes correlates with poor prognosis in cancer. (A), box plot of ALKBH3 normalized gene expression levels in tumor (red) and matched normal controls (blue) from TCGA2. Only datasets with at least 10 matched controls were plotted. BLCA,

bladder

urothelial

carcinoma;

BRCA,

breast

invasive

carcinoma;

COAD,

colon

adenocarcinoma; ESCA, esophageal carcinoma; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; PRAD, prostate adenocarcinoma; STAD, stomach adenocarcinoma; THCA, thyroid carcinoma; UCEC, uterine corpus endometrial carcinoma. Gene expression data were downloaded

with

the

TCGA-Assembler2

and

correspond

to

the

Illumina

HiSeq

.rsem.genes.normalized_results files, which were processed using custom scripts. (B), bar graph of – log10 P-values obtained from Wilcoxon tests for tumor-matched control gene expression pair data, as exemplified in panel A. Only –log10 values >3 (significant) are shown. Tumor > Normal, P-values for cases in which gene expression was higher in the tumor than in controls; Tumor < Normal, P-values for cases in which gene expression was higher in controls than in the tumor. (C), gene expression data for each of the 33 TCGA tumor types was separated into two groups: group 1 contained patients with a signal above or equal to the mean in the tumor sample, and group 2 contained patients with a signal below the mean in the tumor sample. Patient time of death and censored information was then used to construct Kaplan-Meier survival plots (KM estimator) for groups 1 and 2 using the survminer R package. Red square, significant (< 0.05) P-value for the KM estimator in which group 1 performed worse than group 2. Green square, significant (< 0.05) P-value for the KM estimator in which group 1 performed better than group 2. (D), example of KM estimator for ASCC2 gene expression in KIRC, with confidence intervals.


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RNA MODIFICATION ENZYMES IN CANCER DNA damaging drugs used in cancer therapy also modify RNA, inducing a wave of aberrant modifications44, 49, 51, 106. Understanding how cancer cells differentially cope with such modified RNAs and repair proteins can help improve therapeutic strategies in cancer. Although the correlation between defects in RNA damage repair and human disease has not been firmly established, a growing body of evidence suggests that dysregulation of the RNA methylation/demethylation is associated with neurodegeneration and cancer, particularly leukemias, glioblastoma (GBM), breast and colon cancers208. The m6A “eraser” proteins in particular, which are so far limited to AlkB family members, have been suggested to promote the RNA stability and abundance of many oncoproteins209-213. By affecting the self-renewal and proliferation of cancer stem cells, ALKBH5 is reported to play an oncogenic role in the development of glioblastoma (GBM) and breast cancer209,

212, 214.

Indeed, abnormal upregulation of

ALKBH5 correlates with poor outcome in patients with GBM209. Increased ALKBH5 expression and demethylase activities lead to m6A “cleansing” in transcripts of specific genes such as FOXM1, hypoxic environment, ALKBH5 expression is reported to be elevated and promotes NANOG correlating with increased expression, which subsequently promotes tumorigenesis. Similarly, in a tumor mRNA stability and expression by reversing m6A formation212. The dealkylase FTO gene is highly expressed in acute amyloid leukemia (AML) and cervical cancers, although mechanistic implications of this are poorly understood210. A recent report suggested that patients diagnosed with cervical cancer exhibit a higher expression of FTO, in turn correlating with beta-catenin mRNA expression and a poor prognosis215. Similarly, overexpression of ALKBH3 in several forms of cancer has been linked to an aggressive tumor phenotype or poor prognosis216-218, in large part due to increased DNA alkylation repair by the ALKBH3-ASCC complex68. A potential new mechanism by which ALKBH3 may promote tumor progression is associated tRNA m6A demethylation219, although the activities of ALKBH3 that are most relevant in cancer are as yet unclear. Nevertheless, inhibiting ALKBH3 demethylase activity with small molecule inhibitors has significant promise in preclinical cancer models220-223.


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Given the roles of human AlkB family members in RNA demethylation with impacts on various types of cancer, we reasoned that we could further test this concept with cancer patient data on ALKBH3 and the ASCC complex. To examine and possibly relate the knowledge accumulated from biochemical and biophysical studies on ALKBH3 and the ASCC complex with clinical outcomes in cancer, we used TCGA data to assess the difference in the average gene expression levels between tumor and matched normal tissues (controls) for those tumor types for which at least ten control samples were available. A total of 15 tumor types were thus analyzed in which ALKBH3, ASCC2, and ASCC3 gene expression was compared using statistical tests between tumor and controls. For all three genes the range in gene expression in control tissues (~3 logs or an ~8-fold variation from the lowest to the highest levels) was overall smaller than in the tumors, in which up to ~7 logs or >100-fold variation was observed. Consistent with published studies, ALKBH3 and ASCC gene expression is widely deregulated in cancer as compared to normal tissues (Figure 6A). For each of the 15 tumor types, we assessed the statistical significance in gene expression between tumor and normal samples for the 3 genes by using the unpaired two-samples Wilcoxon test and considered to be significant those with a p-value below 0.001 (i.e. –log10 p-value > 3). A total of 23 tumor-normal pairs were identified in which ALKBH3 and/or ASCC2-3 were differentially expressed (Figure 6B), 17 (74%) of which displayed higher mRNA levels in the tumor than in controls and 6 (26%) of which displayed higher mRNA levels in controls than in the tumor (p-value between the two proportions from z-test, 0.001). Therefore, although ALKBH3 and ASCC2-3 overexpression and underexpression are both observed in cancer, overexpression occurs most frequently, approximately in 1/3 of tumor types (17/45 = 0.38). To address if deregulation of ALKBH3 and ASCC2-3 gene expression may provide a readout for clinical outcome, we evaluated survival probabilities using the Kaplan-Meier estimator for all 33 TCGA cancer datasets, in which patients were stratified into two groups: group 1 with gene expression in the tumor tissue above average and group 2 with gene expression in the tumor tissue below average. Five cases displayed a significant clinical outcome between patients with high and low gene expression levels,


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two for ASCC2 and three for ASCC3, with 3/5 cases incurring worse survival with high gene expression (Figure 6C and D). Thus, in total, high gene expression of dealkylating enzymes is a sensitive readout for poor survival in 4 different cancer types, with ASCC2 in kidney renal clear cell carcinoma providing a particularly sensitive prognostic indication.

SUMMARY AND PERSPECTIVE The recent identification of reversible RNA modifications, particularly methylation in the area of RNA epitranscriptomics, is providing important new insight into the “nature vs. nurture” debate of how genetic pre-wiring of the genome can be changed by external factors. It is clear that the external environment can control signaling pathways, which lead to altered gene expression profiles. New epigenetic regulators and adaptor proteins are being identified, and we are beginning to understand how these modifications influence gene expression patterns and impact human disease and behavior at the molecular level. Furthermore, advances in the applications of solution X-ray structural methods for RNA as well as proteins224, 225 and their integration with cellular imaging226, 227 promise to enable structural data to inform enigmatic molecular and cellular mechanisms for RNA modifications, their reversal by repair and erasers and their impact on cancer. In fact, epigenetic-based medicines are already under development for treatment of several cancers and neurodegeneration. With the development of new next-generation DNA sequencing technologies, we expect to see an elaboration of the RNA epigenetic code to include additional base and sugar modifications including alkylations more complex than simple methylation. Yet, to grasp the mechanisms by which the epigenome is regulated, the macromolecular assemblies involved will need to be identified and defined by molecular and structural biological tools. In these efforts we view RNA repair as an underexplored area that intersects with epitranscriptomics as well as epigenetics due to RNA and DNA damage by oxidation, alkylation, and UV-radiation. Whereas some types of RNA damage may be resolved by degradation48, 126, 228, in other cases, repair may offer selective advantages. With this in mind, the interplay of RNA repair pathways with RNA modifications and damage to RNA will need to be


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Biochemistry

defined at the molecular and cellular levels. We anticipate that investigations in these areas will be relevant to cancer etiology, prognosis, and treatment. The knowledge of basic RNA modification and repair mechanisms may therefore not only provide a foundation for a greater understanding of RNA biology but also of cancer cell sensitivity and resistance to alkylating chemotherapy.

ACKNOWLEDGEMENTS N.M. acknowledges grant support from the NIH (R01 CA193318 and R01 CA227001), the Alvin Siteman Cancer Research Fund, the Siteman Investment Program and an American Cancer Society Research Scholar Award. J.A.T. is supported by a Robert A. Welch Chemistry Chair, the Cancer Prevention and Research Institute of Texas, and the University of Texas System Science and Technology Acquisition and Retention. R.T. acknowledges support from the Knowledge GAP award, MD Anderson Cancer Center.

The authors acknowledge the Texas Advanced Computing Center (TACC) at The

University of Texas at Austin for providing HPC resources that have contributed to the research results reported within this paper. URL: http://www.tacc.utexas.edu


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Table 1. Structural and functional consequences of base modifications in RNA Base modification Location in RNA Structural Functional Consequences of consequences modification N1-methyladenosine

Found near start codons and near the first splice site of mRNAs

Disrupts WC base-pairing

May promote translation initiation

Promotes duplex unfolding Important for folding of tRNAs

RNA-Protein Interactions May bind to several YTH domain family proteins but not YTHDC2

Found in tRNAs, rRNAs, mRNAs, lncRNAs Ten times less abundant than m6A N6-methyladenosine

Most common in 3’ UTRs, near stop codons, and at splice sites in RRACH motifs (R = A/G, H = A/C/U) Found in mRNAs, lncRNAs, tRNAs

Prefers the energeticallyunfavorable syn conformation for a N6Me-A-U pair, resulting in destabilization of RNA secondary structure when found within a helix.

Decrease in the half-life of the mRNA in cells Changes the subcellular localization of the RNA

Inhibits WC base-pairing

Regulates RNA processing, splicing, export, translation

Promotes base stacking when fold at ends of helices, resulting in stabilization of the RNA helix.

Important for folding of tRNAs Cell cycle regulation Regulation of circadian rhythms


YTH domain containing proteins HNRNP family proteins

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Biochemistry

5-methylcytosine

Identified in tRNAs, rRNAs, ncRNAs and mRNAs at levels much lower than m6A

Promotes stability via favorable base stacking No effect on base-pairing

Enriched in GC-rich regions of mRNA

May promote mRNA export

Unknown

Important for translational efficiency May be important for RNA mediated epigenetic heredity

Tissue specific 5-Hydroxy methylcytosine

lncRNAs and mRNAs

Unknown

Increases translation efficiency

Unknown

Involved in brain development Pseudouridine

Most abundant RNA modification.

Stabilizes base-pairing.

Enhances half-life of the RNA

Reduces flexibility. Found in rRNAs, tRNAs, and to a lesser extent in mRNAs

May increase or decrease translation efficiency


Unknown

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1 177x193mm (300 x 300 DPI)


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Figure 2 82x101mm (300 x 300 DPI)


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Figure 3 177x173mm (300 x 300 DPI)


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Biochemistry

Figure 4 177x201mm (300 x 300 DPI)


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Figure 5 145x115mm (300 x 300 DPI)


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Biochemistry

Figure 6 166x194mm (300 x 300 DPI)


RNA Modifications: Reversal Mechanisms And Cancer - Biochemistry

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