exons being correspondingly underenriched
(3). How exactly the depletion of m6A could
alter splicing patterns is currently unknown.
A provocative study recently reported that
inhibition of methylation by either a drug or
knockdown of the m6A methylase perturbed
the circadian clock (5). When methylation
was reduced by either treatment, the period of
the clock was lengthened. By contrast, overexpression of the methylase caused a shorter
period. Reduction of internal methylation
resulted in a pronounced delay in export of
mRNAs from the nucleus and, consequently,
marked nuclear retention of those mRNAs. A
second, perhaps related, phenomenon linked
to inhibition of methylation was the widespread stabilization of mRNAs, in particular mRNAs encoding proteins known to be
involved in the circadian clock. This observation may explain how the clock is perturbed
when methylation is inhibited, but how specificity is achieved remains unknown. It is also
unclear why overexpression of the methylase
speeds up the clock; the methylation status of
specific mRNAs was not examined.
Whereas these studies focused on the
effects of inhibiting RNA methylation,
another report examined the effect of a
genetic null in an RNA demethylase. FTO,
the first demethylase discovered, belongs
to a family of enzymes that catalyze a wide
range of oxidation reactions (2). Systematic
study of these enzymes revealed that another
family member, ALKBH5, was also a m6A
demethylase (6). Both FTO and ALKBH5
are localized to nuclei, and both colocalize with nuclear speckles, sites of concentrations of splicing factors. It is tempting to
speculate that this localization is tied to the
effects of methylation on splicing. Indeed,
more than 3000 altered mRNA isoforms were
observed when ALKBH5 was knocked down
in tissue culture cells, indicating that splicing was affected. Despite this, mice null for
ALKBH5 were anatomically normal and
grew to adulthood. However, male mice lacking the demethylase were sterile, with gene
expression and splicing massively altered in
their testes. The sterility phenotype was manifested as a metaphase arrest during spermatogenesis. The tissue specificity of the phenomenon might be explained by the fact that
ALKBH5 is more highly expressed in testes
relative to other tissues, but perhaps the most
puzzling observation is that deletion of the
demethylase resulted in only a very modest
increase in overall levels of m6A in the testis.
The existence of a methylase and at least
two demethylases coupled with phenotypes
observed when either activity was altered
strongly suggested that one or more factors
recognize m6A. A first step in identifying such
factors has come from the demonstration that
an RNA binding protein, YTHDF2, specifi-
cally recognizes m6A-modified RNAs (3, 7).
In vivo cross-linking and immunoprecipita-
tion showed that YTHDF2-bound RNAs were
highly enriched for mRNAs known to be meth-
ylated (7). Moreover, a variety of approaches,
including ribosome profiling and immunoflu-
orescence microscopy, provided evidence that
the binding of YTHDF2 to mRNA resulted
in relocalization of the mRNA from translat-
ing ribosomes to cytoplasmic foci (P bodies)
known to be enriched in RNA degradative
activities. As a consequence, targeted mRNAs
were destabilized, and the magnitude of desta-
bilization correlated with the number of meth-
ylation sites in the mRNA. Knockdown of
YTHDF2 led to an increase in mRNA stabil-
ity for targeted transcripts.
Although these studies have provided new
insight into the distribution and functional
importance of the m6A modification, several
fundamental questions remain. Prime among
these is how specificity of modification is
achieved. Clearly, the sequence that consti-
tutes the consensus site is not sufficient on
its own, nor does secondary structure appear
to play a role. If methylation is cotranscrip-
tional, it may be possible that chromatin sta-
tus could play a role in site selection. The
function(s) of m6A in nuclear RNA metabo-
lism are also unclear. Although it is possible
that nuclear factors recognize the modifica-
tion, it seems equally plausible that modifica-
tions could function by preventing or altering
the binding of some proteins. The importance
or function of modifications in the vicinity
of stop codons remains to be established, as
does the importance of the FTO demethyl-
ase. Perhaps the most challenging question—
and most difficult to answer—is how such a
widespread modification has apparently quite
specific effects. Are all modified sites equally
important, or is only a small subset of them
important? Hopefully, answers to at least
some of these questions will emerge soon in
this now fast-moving field.
1. J. A. Bokar, M. E. Shambaugh, D. Polayes, A. G. Matera,
F. M. Rottman, RNA 3, 1233 (1997).
2. G. Jia et al., Nat. Chem. Biol. 7, 885 (2011).
3. D. Dominissini et al., Nature 485, 201 (2012).
4. K. D. Meyer et al., Cell 149, 1635 (2012).
5. G. Zheng et al., Mol. Cell 49, 18 (2013).
6. J.-M. Fustin et al., Cell 155, 793 (2013).
7. X. Wang et al., Nature 505, 117 (2014).
Stirring the Simmering
“Designer Baby” Pot
Thomas H. Murray1, 2
How much discretion should parents be granted in determining what sort of child they have?
In February 2014, the U.S. Food and Drug Administration’s (FDA’s) Cellular, Tis- sue, and Gene Therapies Advisory Com-
mittee met to consider the possibility of future
clinical trials that would test mitochondrial
manipulation technologies for two purposes:
to treat infertility and to prevent the transmis-
sion of mitochondrial disease from women to
their future children. This meeting focused on
scientific, technological, and clinical issues.
The FDA acknowledged “ethical and social
policy issues related to genetic modification
of eggs and embryos” but chose not to engage
with them, at least not yet (1). Good ethics
begins with good facts, but the effort by the
FDA to get the facts straight is just the begin-
ning, not the end, of the conversation we must
have on the wisdom of mitochondrial manip-
ulation and other reproductive technologies
that potentially provide parents with more of a
say about the children they have. Preventing a
lethal disease is one thing; choosing the traits
we desire is quite another.
The replacement of defective mitochon-dria involves placing nuclear DNA from the
egg of a woman with a mitochondrial defect
into a donated egg that has had its nuclear
DNA removed, but contains healthy mitochondrial DNA. The egg can then be fertilized by artificial insemination. Daughters produced by this procedure could pass down the
mitochondrial DNA to future offspring. Up to
now, the United States has not allowed such
genetic changes across generations.
1The Hastings Center, Garrison, New York, USA. 2The Centre for Biomedical Ethics, National University of Singapore,
Singapore 117597. E-mail: email@example.com