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PERSPECTIVES

R N A P R O C E S S I N G A N D M O D I F I C AT I O N
to uridine14 are not discussed here),
OPINION
andwe provide an updated view of gene
expression that incorporates the dynamics
RNA modifications and structures and physiological relevance of these RNA
features and how they shape binding
cooperate to guide RNAprotein byRBPs.

interactions RNA modifications


The epitranscriptome is shaped through
the activity of evolutionarily conserved
Cole J.T.Lewis, Tao Pan and Auinash Kalsotra factors that encode (writers), decode
(readers) and remove (erasers) various
Abstract | An emerging body of evidence indicates that post-transcriptional gene chemical modifications of RNA bases
regulation relies not only on the sequence of mRNAs but also on their folding into (FIG.1). More than 100 distinct types
intricate secondary structures and on the chemical modifications of the RNA bases. of RNA modifications have been
These features, which are highly dynamic and interdependent, exert direct control found to date15, and more will likely be
identified. Although most modifications
over the transcriptome and thereby influence many aspects of cell function. which include N6methyladenosine
Here,we consider how the coupling of RNA modifications and structures shapes (m6A), 5methylcytosine (m5C),
RNAprotein interactions at different steps of the gene expression process. N 1methyladenosine (m1A) and
pseudouridine () were originally
identified in the highly abundant ribosomal
Gene expression relies on the intricate the different processing steps is achieved. RNAs (rRNAs), transfer RNAs (tRNAs)
coordination of precursor mRNA ThemRNA sequence is often insufficient and small nuclear RNA (snRNAs)16,17,
(pre-mRNA) processing stages, to drive protein binding, as RBP consensus recent advances in sequencing and mass
which include capping, splicing and motifs are highly overrepresented compared spectrometry enabled their detection and
polyadenylation, as well as the export with the incidence of actual binding 68, characterization in the relatively lowly
of mature transcripts to the cytoplasm which indicates that additional features are expressed mRNAs. Genome-wide analyses,
for translation. How do cells exert responsible for determining the specificity coupled with gain- and lossoffunction
tight spatiotemporal control over these of proteinRNA interactions in cells. Recent studies of readers, writers and erasers,
multistepprocesses? Coupling of nuclear studies have revealed that post-transcriptional have expanded the number of mRNA
and cytosolic processes can be accomplished modifications of mRNA through the addition modifications in eukaryotic transcriptomes,
through two distinct mechanisms. First, or removal of chemical groups (together annotated their abundance and distribution
RNA-binding proteins (RBPs) that shuttle comprising the e pitranscriptome) can along transcripts, mapped the modification
between the nucleus and the cytoplasm can expand the information encoded by RNA9. sites that are conserved between species,
bind their target transcripts in the nucleus As we discuss below, RNA modifications, unravelled changes in modification
to influence their processing. Anexcellent which are dynamic and reversible, can abundance and position in response to
example of this is the serine/arginine-rich modulate proteinRNA interactions and environmental changes, demonstrated their
(SR) proteins, which are RBPs that were mediate rapid responses to environmental impact on mRNA processing, stability and
initially found to have diverse roles in changes. In addition to the panoply of translation, and revealed the physiological
splicing and were later found to be important chemical modifications, mRNAs also consequences of modification dynamics.
for transcription elongation, polyadenylation, fold into intricate secondary structures.
mRNA export and translation15. Second, Until recently, the structural aspect of the RNA modifications expand the splicing
nuclear processing, which includes splicing transcriptome was largely unexplored owing code. The splicing code represents the full
and polyadenylation as well as dynamic to technical limitations. The development set of RNA features that directly influence
changes in RNA chemical modifications and of new transcriptome-wide approaches to pre-mRNA splicing 18. Although sequence
secondary structures (discussed below), can study RNA structure invivo has unravelled elements such as the binding motifs of RBPs
facilitate downstream associations with RBPs the plasticity, complexity and functionality have been studied extensively, evidence
in the cytoplasm and thereby affect mRNA ofmRNA structures1012. now indicates that RNA modifications also
translation anddecay. In this Opinion article, we discuss recent have a role in determining the splicing code.
As RBPs link multiple steps of gene findings that demonstrate the functional Ofthe internal mRNA modifications (that is,
expression, factors governing their binding coupling and interdependence of mRNA excluding the 5 7methylguanosine cap and
to target RNAs must be considered in modifications and structures (the editing 3 poly(A) tail), m6A is the most prevalent,
order to understand how coupling between of adenosine to inosine13 and cytidine accounting for approximately 80% of all

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a b
NH 2 Modication Writer Eraser Reader
N
N N6-Methyladenosine Methyltransferase- AlkB homologue 5 YTH domain family
Adenosine like 3 (METTL3) (ALKBH5) proteins 13 (YTHDF13)
N N and METTL14 Fat mass and YTH domain-containing
Wilms tumour 1- obesity- protein 1 (YTHDC1)
associated protein associated Eukaryotic translation
(WTAP) protein (FTO) initiation factor 3 (eIF3)
CH 3 RNA-binding motif complex
HN NH 2 protein 15 (RBM15) Heterogeneous nuclear
N N CH 3 and RBM15B ribonucleoproteins
N N Protein virilizer A2/B1 (HNRNPA2B1)
homologue and HNRNPC
N N N N (KIAA1429) Serine/arginine-rich
splicing factor 2 (SRSF2)
N6-Methyladenosine N1-Methyladenosine
(m6A) (m1A) N1-Methyladenosine Unknown ALKBH3 Unknown
5-Methylcytosine NOL1/NOP2/Sun Unknown Unknown
NH 2 domain family
member 2 (NSUN2)
N DNA methyltransferase-
Cytosine like protein 2
N O (DNMT2)

5-Hydroxymethylcytosine* Ten-eleven Unknown Unknown


NH 2 NH 2 translocation
protein 13
H 3C
N HO N (TET1TET3)

N O N O Pseudouridine Pseudouridine Unknown Unknown


synthase 14
(PUS1PUS4),
5-Methylcytosine 5-Hydroxymethylcytosine PUS6, PUS7, PUS9
(m5C) (hm5C) Dyskeratosis
congenita
protein 1 (DKC1)
O O
*5-Hydroxymethylcytosine is formed by the hydroxylation of 5-methylcytosine.
NH HN NH
Uridine Pseudouridine
N O C O () Figure 1 | RNA modifications and their writers, erasers and readers. a | Chemical structures
of unmodified and modified RNA bases. b | The writers, readers and erasers of RNAmodifications.

Nature Reviews | Molecular Cell Biology


RNA base methylations in eukaryotes19,20. A sizeable proportion (approximately that m6A potentiates SRSF2 binding and
Emerging evidence demonstrates that 30%) of m6A is found in introns, which splicing regulation. However, it is unclear
m6A deposition catalysed by a writer indicates that these modifications are whether SRSF2 binds m6A directly or
complex comprising methyltransferase-like probably deposited cotranscriptionally through m6A-binding proteins. The m6A
protein3 (METTL3; also known as MTA70) and before splicing 24,31. Similarly, the reader YTH domain-containing protein1
andMETTL14, Wilms tumour 1associated colocalization of m6A writers and erasers (YTHDC1) is an example that supports
protein (WTAP), protein virilizer with pre-mRNA splicing factors in the the second scenario. By binding to m6A,
homologue (KIAA1429), RNA-binding splicing-associated nuclear bodies known YTHDC1 modulates thousands of splicing
motif protein 15 (RBM15) and RBM15B1927 as speckles suggests an intrinsic connection events through the promotion or inhibition
(FIG.1b) has important roles in regulating between m6A and splicing 24,32. Furthermore, of transcript binding by SRSF3 or SRSF10,
pre-mRNA processing. In mammals, m6A a deficiency of the m6A demethylase alkB respectively 35. The Drosophila melanogaster
is enriched in 3 untranslated regions homologue 5 (ALKBH5) disrupts the homologue of YTHDC1, YT521-B, is also
(3UTRs), introns and alternatively spliced recruitment of the splicing factors serine/ involved in splicing regulation. More than
exons19,20,28. Depletion of METTL3, WTAP arginine-rich splicing factor 1 (SRSF1), 100 splicing events are differentially regulated
or fat mass and obesity-associated protein SRSF2 and SRSF protein kinase 1 (SRPK1) in the fly following knockdown of subunits
(FTO), which is an m6A eraser 29, results in to nuclear speckles32. As the localization ofthe m6A writer complex36,37. Approximately
splicing defects in hundreds of genes19,26,30. of SR proteins and their phosphorylation 70% of these events are also misregulated
A portion of these events occurs in 3 UTRs by SRPKs are intimately linked with upon depletion of YT521-B, indicating
and final exons, which suggests that m6A splicing 33,34, these results may explain how that this protein is the primary mediator
could also affect polyadenylation. Indeed, changes in m6A could affect global splice of m6A-dependent splicing regulation
ahigher m6A density near proximal poly(A) site choices. In another study, 6080% of in D.melanogaster37. Notably, through
sites correlates with a decreased use of exonic FTO-regulated m6A sites were found binding to m6A in the Sex lethal mRNA,
those sites for polyadenylation, whereas to overlap with exonic splicing-enhancer YT521-B regulates sex determination by
thepresence of m6A near (and upstreamto) sequences that were bound by SRSF1 and repressing the inclusion of the male-specific
distal poly(A) sites correlates with an SRSF2 (REF.30). Strikingly, SRSF2 binding exon ofthe transcript36,37. Splicing factors
increased use for polyadenylation in a subset and exon inclusion are inversely correlated can also bind directly to m6A-containing
of mRNAs28. with FTO expression32, which indicates sequences. A member of the heterogeneous

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PERSPECTIVES

nuclear ribonucleoprotein (HNRNP) family binding to m6A recruits the CCR4NOT HNRNPA2B1, which recruits DGCR8
of splicing factors, HNRNPA2B1, binds deadenylase complex 46 and facilitates mRNA to pri-miRNAs51. In addition, although
directly to METTL3deposited m6A-bearing transport to processing bodies47, which are the abundance of m5C in mRNAs is low,
regions near alternative exons and thereby sites of mRNA decay 48. However, YTHDF2 transcriptome-wide profiling revealed
affects their splicing 38. Thus, m6A serves was recently reported to stabilize HIV substantial overlap between m5C sites
as a docking site for proteins that can mRNAs and YTHDF2tethered reporter and AGO1AGO4 binding sites52, which
directly or indirectly influence pre-mRNA constructs49, which raises questions about suggests that m5C might have a role in
processing(FIG.2). the sole role of YTHDF2 in destabilizing miRNA-mediated silencing. Recently,
Modifications and microRNAs cooperate mRNAs. By contrast, HUR stabilizes another modified nucleotide N6,2-O-
to control mRNA decay. The development of these mRNAs by binding to their 3 UTRs dimethyladenosine (m6Am) was found
chemical labelling techniques has led to the and thereby blocks microRNA (miRNA) to be highly abundant adjacent to the 5
transcriptome-wide mapping of in yeast binding 31. The decreased methylation that cap of mRNAs in human cells53. m6Am
and human mRNAs3942. Pseudouridylation follows METTL3 depletion promotes HUR increases mRNA stability by conferring
of mRNAs is catalysed by the pseudouridine binding and stabilizes target transcripts by resistance to mRNA-decapping enzyme 2
synthase (PUS) family of enzymes. blocking miRNA-dependent mRNA decay. (DCP2). As miRNA-mediated mRNA decay
Following heat shock in yeast, Pus7 catalyses The spatial proximity of m6A and involves decapping, m6Am-modified mRNAs
formation at over 200 distinct sites in miRNA-binding sites in 3 UTRs raises an are more resistant to this mechanism of
mRNAs42. The levels of Pus7 mRNA targets interesting question: do miRNAs guide m6A degradation53. Collectively, these studies
are higher than in Pus7deficient cells, writers to specific adenosines? Recently, highlight the dynamic cooperation between
whichsuggests that actively regulates miRNAs were shown to modulate the the epitranscriptome and miRNAs in
mRNA stability (FIG.2). Conversely, depletion binding of METTL3 to transcripts through modulating mRNA decay.
of the m6A writer METTL3 results in a a sequence-dependent pairing mechanism
global increase in the half-life of METTL3 and to confer methylation specificity The epitranscriptome regulates
target mRNAs43,44. In mouse and human independently of the miRNA-interacting translation under stress conditions.
embryonic stem cells, this stabilization Argonaute (AGO) proteins50. Although Translation regulation is a highly dynamic
occurs in pluripotency genes, which intriguing, this result is yet to be validated process that facilitates rapid responses to
results in a failure to exit the naive state of by others. METTL3 also methylates environmental stimuli, owing to the ample
pluripotency 44,45. These effects are regulated many primary miRNAs (pri-miRNAs), availability of translationally competent
by the m6A reader YTH domain family which is obligatory for their processing mRNA pools. Translation initiation can
protein 2 (YTHDF2) and by the RBP human by the Microprocessor complex subunit be cap-dependent or cap-independent 54,
antigen R (HUR; also known as ELAVL1), DiGeorge syndrome critical region 8 andthe epitranscriptome has profound
with opposing consequences. YTHDF2 protein (DGCR8)51. This is mediated by effects onboth mechanisms.

m1A m6A AUG hm5C m6A STOP m6A m5C A(n)


m7G
YTHDC1 YTHDF1 YTHDF2
eIF3 HNRNPC
YTHDF2 HNRNPA2B1
(nuclear) SRSF2
Translation Translation Cap- RNA Translation Increased
eciency Cap- eciency Alternative dependent degradation eciency stability
independent splicing and translation
translation polyadenylation

Heat shock and UV exposure


nutrient deprivation and heat shock Brain Sex Stem cell dierentiation; Heat shock
responses (?) responses development Adipogenesis determination circadian clock control Senescence response

Figure 2 | Biological functions of mRNA modifications. The levels of protein1(YTHDC1), heterogeneous nuclear ribonucleoprotein C (HNRNPC),
N 1methyladenosine (m1A) and N 6methyladenosine (m6A) in 5 untranslated HNRNPA2B1 and serine/arginine-rich Nature Reviews
splicing | Molecular
factor 2 (SRSF2).Cell Biology
Removal of
regions (5 UTRs) are dynamically altered in response to stress. m1A promotes m6A by fat mass and obesity-associated protein (FTO) (not shown) antago-
cap-dependent translation through an unknown reader. m1A levels are nizes SRSF2 binding and is necessary for adipogenesis. A homologue of
altered by nutrient deprivation or heat shock, which indicates a possible role YTHDC1, YT521-B, binds to m6A in the Sex lethal mRNA and regulates its
in directing cellular responses during these conditions (the question mark splicing, which in turn controls sex determination in D. melanogaster. m6A in
indicates that no direct link has been found). m6A facilitates cap-independent 3 UTRs also affects RNA processing via the aforementioned readers, as well
translation through interactions with the eukaryotic translation initiation as cap-dependent translation via YTHDF1 and RNA degradation via YTHDF2.
factor 3 (eIF3) complex in response to heat shock or UV exposure. YTH Regulation of mRNA stability by m6A is crucial for stem cell differentiation
domain-containing family protein 2 (YTHDF2) protects 5 UTR m6A from and circadian clock control. 5Methylcytosine (m5C) is linked to the trans
demethylation. In coding sequences, 5hydroxymethylcytosine (hm5C) lational control of senescence-related genes. Pseudouridine () in 3 UTRs
increases translation efficiency via an unknown mechanism; hm5C levels are has been shown to increase the stability of modified transcripts during
highest in the brain, and ablation of Teneleven translocation (Tet) genes, heat shock. Blue arrows indicate modifications with no known readers.
which encode proteins that catalyse hm5C formation, affects brain develop- Red lines connect readers to biological processes that they are directly
ment in Drosophila melanogaster. m6A in coding regions affects RNA associated with. A(n), polyadenylation; AUG, translation start codon;
processing by modulating the binding of YTH domain-containing m7G,7methylguanosine; STOP, translation stop codon.

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a Normal conditions b Heat shock conditions


m7G FTO
FTO A(n) YTHDF2 A(n)
m6A A(n) m6A A(n)
A(n) FTO YTHDF2 A(n)
FTO
m6A A(n) m6A A(n)
A(n) A(n)
A(n) A(n)
F2 Nucleus YTHDF
YTHD 2

Non-stress-responsive Cytoplasm Stress-responsive Non-stress-responsive Stress-responsive


gene transcripts gene transcripts gene transcripts gene transcripts
m7G
eIF4G eIF3
A(n) A(n) A(n) A(n)
eIF3
eIF4E eIF4A Ribosome Rapid m6A-mediated cap-
Ecient cap-dependent translation Minimal cap-dependent translation Cap-dependent translation inhibited independent translation

Figure 3 | Stress induces N6methyladenosine-dependent translation. translocates from the cytoplasm to the nucleus, binds to m6A in the 5 UTR
Nature Reviews | Molecular Cell Biology
Under normal conditions (part a), N6methyladenosine (m6A) in the 5 UTRs of stress-responsive mRNAs and prevents their FTO-mediated demethyl
of stress-responsive gene transcripts is removed by the eraser fat mass and ation. Stress conditions globally inhibit cap-dependent translation of
obesity-associated protein (FTO). Unmodified transcripts undergo efficient unmodified transcripts. However, the m6A residues in the 5 UTRs of stress
cap-dependent translation, whereas the demethylated transcripts of responsive genes are directly bound by the eukaryotic translation initiation
stress-responsive genes are minimally translated. Following heat shock factor 3 (eIF3) complex, and this enables their rapid cap-independent
(partb), the m6A reader YTH domain-containing family protein 2 (YTHDF2) translation. A(n), polyadenylation; m7G, 7methylguanosine.

In human cells, the m6A reader YTHDF1 m5C and its derivative 5hydroxy Although somewhat preliminary, these
promotes cap-dependent translation methylcytosine (hm5C) are also implicated studies demonstrate the effects of RNA
initiation through its associations with in translation regulation. Expression of modifications on different steps of gene
theeukaryotic translation initiation factor 3 the m5C writer NOL1/NOP2/Sun domain expression, which raises the question of
(eIF3) complex and other RBPs55. However, family member 2 (NSUN2) is tightly how the modifications link various nuclear
YTHDF1 has also been shown to regulate regulated during the cell cycle, and the and cytosolic processes. For example,
mRNA decay 46,49,56, which suggests that it overexpression of NSUN2 delays replicative in the case of m6A, the discrete cellular
has a role outside of translation. Although senescence through the methylation of localization of readers and their distinct
m6A levels are low in 5 UTRs, heat cyclin-dependent kinase 1 (CDK1) and protein interaction domains seem to allow
shock significantly increases m6A levels CDK inhibitor1B (CDKN1B; which encodes their interaction with different effectors to
specifically in the 5 UTRs of heat shock- p27) transcripts. Deposition of m5C in the regulate specific processes in the nucleus
responsive mRNAs in mammalian cells in CDK1 3 UTR enhances its translation, andcytoplasm30,35,38,47,55,58.
a YTHDF2dependent manner 57,58. Heat whereas m5C in the 5 UTR of CDKN1B
shock inhibits cap-dependent translation represses its translation, which together RNA structure
globally 59 but, remarkably, asingle result in increased cellular proliferation60,61. Unlike DNA, which universally adopts
m6A residue in a 5 UTR is sufficient to The formation of hm5C from m5C by a double helical conformation, RNA has
facilitate cap-independent translation of methylcytosine dioxygenases62 in mRNA extensive intramolecular interactions that
heat shock-responsive mRNAs, which correlates with an enhanced association cause it to fold into an array of complex
includes those encoding heat shock protein withpolyribosomes in D.melanogaster 63. structures67. RNA structure is highly
70kDa (HSP70)57 (FIG.3). Under normal Recently, another modification, m1A, dynamic and is governed by factors such
conditions, FTO removes m6A from the wasdiscovered64,65. Interestingly, m1A as temperature, cellular energy state,
5 UTRs of HSP70 and other heat shock- displays plasticity in response to nitrogen ATP-dependent RNA helicases, chaperone
responsive mRNAs, which prevents their deprivation in yeast 64, as well as tissue proteins and other RBPs68. RNA structures
cap-independent translation. During specificity and a rapid response to multiple enable a myriad of functions, which include
heat shock, YTHDF2 translocates from stimuli in mammalian cells64,65. Most m1A- encoding genetic information and catalysing
the cytoplasm to the nucleus and binds modified transcripts harbour a single chemical reactions69,70. The dynamic nature
m6A residues in the 5 UTRs of heat m1A in their 5 UTRs, and m1A-bearing of RNA structure is illustrated by splicing,
shock-responsive mRNAs, which prevents transcripts have higher translation during which structural rearrangements
demethylation by FTO and promotes rapid efficiency and protein levels compared of snRNAs permit the recognition of
cap-independent translation58. In this to unmodified transcripts. Finally, splice sites and branch point sequences
way, 5 UTR m6A deposition ensures the pseudouridylation can affect the translation in pre-mRNAs and facilitate the stepwise
production of proteins that are essential for of some transcripts by alternative assembly of spliceosomes71. Numerous
survival during heat shock, even as general decodingofpseudouridylated codons, studies have confirmed that mRNAs also
translation ishalted. whichmay improvestresstolerance66. contain structured regions, although the

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extent of folding is significantly lower than Third, RNA modifications may act to these stems are often N6methylated, which
that of non-coding RNAs. The advent of stabilize or disrupt structural elements destabilizes the stem structure to expose
chemical modification-based procedures and and influence RBP accessibility. This the Utract and allow binding by HNRNPC
RBPRNA crosslinking methods11,12,72,73 have mechanism has been shown to regulate (FIG.4b). Thousands of such m6A structural
enabled transcriptome-wide mapping of RNA the binding of HNRNPC84, an RBP switches have been identified, and the
structures in different species. These studies that is involved in mRNA stability and expression of nearly 2,000 genes and more
have unveiled a previously unappreciated processing 7,85,86. The Utract sequences than 100 splicing events are regulated
andcomplex layer of generegulation. that are recognized by HNRNPC are often by HNRNPC and METTL3METTL14
buried within stem structures, which (REF.84). Taken together, these studies
RNA structures shape protein binding and prevent HNRNPC binding. Adenosines in indicate that the structural landscape of
splicing. RNA folding begins as the transcript
is synthesized by RNA polymeraseII a Distal RBFOX2-binding site (minimal activity)
(PolII)7476. Transcript elongation by Pol II is Alternative exon
not continuous, but instead entails periods m7G A(n)
RBFOX2
of active elongation that are interrupted by >500 nt
pauses and backtracking. The formation
of RNA structures such as hairpins on the Bridged RBFOX2-binding site (high activity)
nascent transcript can create physical barriers
that prevent backtracking and promote
>500 nt
forward elongation by PolII74,75. As splicing
and polyadenylation are dependent on PolII
A(n)
elongation kinetics77,78, the effect of RNA
structure on transcript elongation probably
affects downstream RNA processing.
RNA structures may also regulateRNA b
processing directly by three distinct Annealed Exposed
mechanisms. First, local RNA structure U-tract m6A U-tract m6A HNRNPC
motif motif
variations can promote or inhibit the
binding of RBPs8. For example, secondary m6A deposition, RBP binding
structures surrounding the consensus structure destabilization
motifs for the splicing factors RBP
FOX-1 homologue 2 (RBFOX2) and
muscleblind-like protein 1 (MBNL1) are c RNA H
key determinants of whether these sites will N N N
H
N N RNA
be bound invivo. Furthermore, motifs near N N
H
evolutionarily conserved alternative exons H O O N
tend to be more single-stranded and to H N H N
N H N H
exhibit stronger RBP binding than species- N O O H
specific alternative exons or constitutive H
N N
exons8. Distinct structural features also RNA N N
H
N N N
occur at 5 and 3 splice sites, at strong H RNA
andweak splice sites and at polyadenylation
signals, which indicates that local structures Translation miRNA
regulate RNA processing globally 72,79,80. initiation binding
Second, larger RNA structures can miRNA-
binding site
regulate splicing by bringing distal
regulatory elements in close proximity to
m7G AUG STOP A(n)
target exons. For example, more than half
of the RBFOX2binding sites are found over Figure 4 | The effect of RNA structures on mRNA modification and
Nature gene expression.
Reviews a | A majority
| Molecular Cell Biology
500 nucleotides away from any annotated of RNA-binding protein FOX1 homologue 2 (RBFOX2) binding sites are over 500 nucleotides (nt) away
exon81. Regulation from these deep intronic from the nearest exon. To enable splicing regulation by RBFOX2 from these distal binding sites,
sites occurs by the formation of long-range base-pairing interactions occur within the intron, which bring RBFOX2 into close proximity to its
intronic structures that deliver RBFOX2 target exons. b | N6methyladenosine (m6A) deposition destabilizes base-pairing interactions and dis-
close to its target exons (FIG.4a). Moreover, rupts local secondary structures. In doing so, m6A can expose previously buried RNA-binding motifs
to RNA-binding proteins (RBPs), such as the Utracts that are bound by heterogeneous nuclear
recent studies utilizing methods that detect
ribonucleoprotein C (HNRNPC). c | RNA Gquadruplexes (RGQs) form by the stacking of three or more
large RNA duplexes have revealed that up guanine quartets (top panel). In 5 untranslated regions (5 UTRs), RGQs impede the initiation and
to 40% of transcript structures span more scanning steps of translation (bottom panel). In 3 UTRs, RGQs can block access to microRNA (miRNA)-
than 300 nucleotides10,82,83. Thus, the RNA binding sites and thus prevent miRNA-mediated decay and translation repression. RGQs are dynamic
structure-based proximity system observed structures and can be unwound by RNA helicases. A(n), polyadenylation; AUG, translation start codon;
for RBFOX2 may represent a mechanism m7G, 7methylguanosine; STOP, translation stop codon. Part b is adapted with permission from REF.84,
that is common to otherRBPs. Macmillan Publishers Ltd.

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PERSPECTIVES

Gene Intron mRNAs, which potentially enables RNA


Exon structures to affect transcript stability.
Transcription
Indeed, 3 UTR structures that involve the
poly(A) tail are the primary determinants
Unmodied transcripts Modied transcripts of transcript stability in yeast and result in
alternative polyadenylation isoforms with
drastically different stabilities95,96, although
RBP stability differences between such 3 UTR
Modied
mG7 base isoforms are subtler in mammalian cells97.
A(n) A(n) Interestingly, a sizeable portion (1525%)
Reader RBP of annotated mRNAs and long non-coding
RBP
RNAs (lncRNAs) are not polyadenylated
Splicing Splicing or are bimorphic, so that some transcripts
contain and others lack a poly(A) tail98,
Fate 1 Translation Fate 2 Translation
which raises the question of how these
Decay Decay transcripts are stabilized and, in the case
Cell state 1 90% 10% of mRNAs, translated. The intronless,
Cell state 2 30% 70% replication-dependent histones are one such
group of non-polyadenylated mRNAs. These
Figure 5 | The dynamics of mRNA modification stoichiometries. Within the pool of transcripts from
Nature Reviews | Molecular Cell Biology mRNAs are instead stabilized by highly
a given gene, each potential modification site is modified in only a fraction of transcripts, which is
conserved stem-loop structures that form
known as the modification stoichiometry. The modification stoichiometry may be very low under
certain cellular conditions (cell state 1). By causing RNA structural rearrangements and directly in their 3 UTRs, which are also required for
recruiting modification reader proteins, the modified mRNAs acquire a different fate, which could their translation99. Furthermore, additional
result, for example, in increased turnover (decay). As cellular conditions change, modification 3 structures (such as triple helices) can also
stoichiometry may change as well, which can result in a higher (cell state 2) or lower percentage of stabilize mRNAs and lncRNAs.
modified transcripts owing to alterations in reader, writer and/or eraser activity or expression. RNA triple helices are structures
Thedashed arrows indicate an indirect effect. A(n), polyadenylation; m7G, 7methylguanosine; that result when an RNA strand forms
RBP,RNAbinding protein. Hoogsteen hydrogen bonds withthe
major groove of an RNA duplex.
Thetriple helix structure that forms
thetranscriptome is an essential mediator regulation. Although they are prevalent atthe expression and nuclear retention
of RNA processing and is yet another factor invitro especially in 5 and 3 UTRs element (ENE) in the 3 end of the Kaposi
that must be fully understood to complete recent invivo genome-wide analysis has sarcoma-associated herpesvirus lncRNA,
the splicingcode. revealed that RGQs are globally unfolded polyadenylated nuclear RNA (PAN RNA),
in mammalian cells90. Nonetheless, many protects the transcript from rapid nuclear
RNA structures tune translation and mRNA individual examples of functionally deadenylation-dependent decay 100102.
stability. RNA structure has also been important RGQs exist 89,91. In T cell acute Recently, a pair of studies discovered
linked to translation regulation. Inplants, lymphoblastic leukaemia cells, inhibition similar ENEs in two mammalian non-
animals and fungi, the approximately of the RGQ-helicase eukaryotic translation polyadenylated lncRNAs, metastasis-
five-nucleotide region surrounding the initiation factor 4A1 (eIF4A1) induces associated lung adenocarcinoma
start codon displays a remarkable lack of apoptosis and delays tumour growth owing transcript1 (MALAT1) and multiple
structure11,72,80,87,88. This feature is enriched in to decreased translation of oncogenes endocrine neoplasia (MEN; also known
genes with high translation efficiency and is such as myelocytomatosis proto-oncogene as NEAT1)103,104. Both MALAT1 and MEN
absent in those with low efficiency 11,72,80,87,88, (MYC), Notch, and BCL2 (REF.92). are not processed by the canonical cleavage
which indicates that start codon accessibility Transcripts affected by eIF4A1 inhibition and polyadenylation machinery, and instead
promotes translation. RNA secondary have longer 5 UTRs on average and they contain tRNA-like structures in their
structure is also highly dynamic in response display a strong enrichment for the twelve 3 ends that are cleaved by RNaseP105,106.
to external stimuli. In yeast, following the nucleotide RGQ-forming motif (CGG)4. Following cleavage, genomically encoded
transition from 30C to 37C, base-pairing Thus, the tumorigenic properties of eIF4A1 Arich tracts at the 3 ends of both lncRNAs
of over 25,000 bases at approximately may be based on its ability to unwind form triple helices, which are nearly
2,000structured sites in mRNAs is translation-inhibiting RGQs in the 5 UTRs identical to those formed in the PAN
disrupted88. These heat-sensitive bases of oncogenes. In addition to 5 structures, RNA103,104. These structures are essential
are enriched in 5UTRs, regions in which RGQs in the 3 UTRs of mRNAs can also for the stability of MALAT1 and MEN,
structure is known to influence translation89. affect mRNA translation and stability as shown by deletions of or destabilizing
Another structural element that has through the occlusion of miRNA-binding mutations in these structures. Strikingly,
gained attention in the past decade is the sites93 (FIG.4c). placement of the MALAT1 and MEN 3
RNA Gquadruplex (RGQ), which is a The degradation of mRNAs is structures into polyadenylation-deficient
four-stranded structure that results from primarily controlled by the exosome, reporter genes not only significantly
the stacking of multiple planar guanine which is a multiprotein exonuclease94. stabilizes mRNAs but also facilitates
quartets (FIG.4c). RGQs are implicated in Exosome-mediated degradation requires efficient translation104, suggesting that
splicing, transcript stability and translation a single-stranded region at the 3 end of such RNA structures may have a wider

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role in the translation and/or stabilization complex through the occlusion, exposure Finally, we also need to further explore
of other poly(A)-lacking transcripts. orformation of RBP-binding sites. Thus,the how structural and chemical alterations
Recently, bioinformatics analysis identified tracking of individual transcript structures cooperate to allow the functional coupling
similar structures that stabilize hundreds of throughout their life cycle will be necessary of the different steps of the mRNA life cycle.
transposon transcripts in fungi, providing to fully understand the impact of mRNA In addition to the functions provided by
the first evidence of the existence of triple structure on gene expression. Future studies direct readers, RNA modifications may
helices in mRNAs107. should discern how external cues fine-tune be one of the primary drivers of structural
During heat shock in yeast, RNA the structural and chemical variations plasticity within the transcriptome by
structures with low melting temperatures within transcript pools to produce a stabilizing or disrupting base-pairing
are rapidly degraded by the exosome as their coherent biological response. Forexample, interactions. As such, the phenotypes
3 ends become accessible88. As discussed do different signalling pathways recruit observed following perturbation of the
above, deposition increases following heat discrete epitranscriptome readers, writers activity of writers or erasers may partially
shock, which increases mRNA stability 42. or erasers as effectors to influence the be the result of global changes in the RNA
As A base pairs are more stable than UA fates of individual mRNAs? Efforts to map structural topography.
base pairs66, it is possible that increases the epitranscriptome at single-nucleotide Cole J.T.Lewis and Auinash Kalsotra are at the
mRNA stability by preventing the melting resolution must also continue in order Department of Biochemistry, University of Illinois;
of RNA structures that protect the 3 end to identify the exact nucleotides that are A.K.is also at the Institute of Genomic Biology,
University of Illinois, Urbana-Champaign,
at increased temperatures. In contrast to , modified in each transcript, define how
Illinois61801, USA.
m6A acts as a spring-loaded modification, these sites are selected and determine
Tao Pan is at the Department of Biochemistry and
which disrupts RNA duplexes owing to the whether these dynamics are achieved Molecular Biology, University of Chicago,
adoption of an unfavourable, high-energy through the active control of writers Chicago, Illinois 60637, USA.
conformation108,109. Indeed, transcriptome- versuserasers. Correspondence to T.P.and A.K.
wide structure analysis revealed a distinct Another important aspect that is yet taopan@uchicago.edu;
structural profile at m6A sites, which is to be addressed is the dynamics of RNA kalsotra@illinois.edu
consistent with unpaired RNA11. Besides its modification stoichiometry. Current doi:10.1038/nrm.2016.163
role in miRNA-mediated decay, the strong epitranscriptome studies deal mostly with Published online 1 Feb 2017

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