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SUNDAY SCIENCE: REVOLUTIONARY GENETICS RESEARCH SHOWS RNA MAY RULE
OUR GENOME  
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Phillip Ball
May 14, 2024
Scientific American
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_ Scientists have recently discovered thousands of active RNA
molecules that can control the human body _

, James Yang

 

Thomas Gingeras did not intend to upend basic ideas about how the
human body works. In 2012 the geneticist, now at Cold Spring Harbor
Laboratory in New York State, was one of a few hundred colleagues who
were simply trying to put together a compendium of human DNA
functions. Their ­project was called ENCODE, for the Encyclopedia of
DNA Elements. About a decade earlier almost all of the three billion
DNA building blocks that make up the human genome had been identified.
Gingeras and the other ENCODE scientists were trying to figure out
what all that DNA did.

The assumption made by most biologists at that time was that most of
it didn’t do much. The early genome mappers estimated that perhaps 1
to 2 percent of our DNA consisted of genes as classically defined:
stretches of the genome that coded for proteins, the workhorses of the
human body that carry oxygen to different organs, build heart muscles
and brain cells, and do just about everything else people need to stay
alive. Making proteins was thought to be the genome’s primary job.
Genes do this by putting manufacturing instructions into messenger
molecules called mRNAs, which in turn travel to a cell’s
protein-making machinery. As for the rest of the genome’s DNA? The
“protein-­cod­ing regions,” Gingeras says, were supposedly
“surrounded by oceans of biologically functionless se­­quences.”
In other words, it was mostly junk DNA.

So it came as rather a shock when, in several 2012 papers
in _Nature_, he and the rest of the ENCODE team reported that at one
time or another, at least 75 percent of the genome gets transcribed
into RNAs. The ENCODE work, using techniques that could map RNA
activity happening along genome sections, had begun in 2003 and came
up with preliminary results in 2007. But not until five years later
did the extent of all this transcription become clear. If only 1 to 2
percent of this RNA was encoding proteins, what was the rest for? Some
of it, scientists knew, carried out crucial tasks such as turning
genes on or off; a lot of the other functions had yet to be pinned
down. Still, no one had imagined that three quarters of our DNA turns
into RNA, let alone that so much of it could do anything useful.

Some biologists greeted this announcement with skepticism bordering
on outrage
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The ENCODE team was accused of hyping its findings; some critics
argued that most of this RNA was made accidentally because the
RNA-making enzyme that travels along the genome is rather
indiscriminate about which bits of DNA it reads.

Now it looks like ENCODE was basically right. Dozens of other research
groups, scoping out acti­­vity along the human genome, also have
found that much of our DNA is churning out “noncoding” RNA. It
doesn’t encode proteins, as mRNA does, but en­­gages with other
molecules to conduct some biochemical task. By 2020 the ENCODE project
said it had identified around 37,600 noncoding genes—that is, DNA
stretches with instructions for RNA molecules that do not code for
proteins. That is almost twice as many as there are protein-coding
genes. Other tallies vary widely, from around 18,000 to close to
96,000. There are still doubters, but there are also enthusiastic
biologists such as Jeanne Lawrence and Lisa Hall of the University of
Massachusetts Chan Medical School. In a 2024 commentary for the
journal _Science_, the duo described these findings as part of an
“RNA revolution.”

What makes these discoveries revolutionary is what all this noncoding
RNA—abbreviated as ncRNA—does. Much of it indeed seems involved in
gene regulation: not simply turning them off or on but also
fine-­tuning their activity. So although some genes hold the
blueprint for proteins, ncRNA can control the activity of those genes
and thus ultimately determine whether their proteins are made. This is
a far cry from the basic narrative of biology that has held sway since
the discovery of the DNA double helix some 70 years ago, which was all
about DNA leading to proteins. “It appears that we may have
fundamentally misunderstood the nature of genetic programming,”
wrote molecular biologists Kevin Morris of Queensland University of
Technology and John Mattick of the University of New South Wales in
Australia in a 2014 article.

Another important discovery is that some ncRNAs appear to play a role
in disease, for example, by regulating the cell processes involved in
some forms of cancer. So researchers are investigating whether it is
possible to develop drugs that target such ncRNAs or, conversely, to
use ncRNAs themselves as drugs. If a gene codes for a protein that
helps a cancer cell grow, for example, an ncRNA that shuts down the
gene might help treat the cancer.

A few noncoding RNAs had been known for many decades, but those seemed
to have some role in protein manufacture. For instance, only a few
years after Francis Crick, James Watson and several of their
colleagues deduced the structure of DNA, researchers found that some
RNA, called transfer RNA, grabs onto amino acids that eventually get
strung together into proteins.

In the 1990s, however, scientists realized ncRNA could do things quite
unrelated to protein construction. These new roles came to light from
efforts to understand the process of X-inactivation, wherein one of
the two X chromosomes carried by females is silenced, all 1,000 or so
of its genes (in humans) being turned off. This process seemed to be
controlled by a gene called _XIST_. But attempts to find the
corresponding XIST protein consistently failed.

The reason, it turned out, was that the gene did not work through a
protein but instead did so by producing a long noncoding (lnc) RNA
molecule. Such RNAs are typically longer than about 200 nucleotides,
which are the chemical building blocks of DNA and RNA. Using a
microscopy technique called fluorescence in situ hybridization,
Lawrence and her colleagues showed that this RNA wraps itself around
one X chromosome (selected at random in each cell) to induce
persistent changes that silence the genes. “This was the first
evidence of a lncRNA that does something,” Lawrence says, “and it
was totally surprising.”

If noncoding RNAs power the way a cell processes genetic information,
it is possible they can be used in medicine.

_XIST_ isn’t that unusual in generating an ncRNA, though. In the
early 2000s it became clear that transcription of noncoding DNA
sequences is widespread. For example, in 2002 a team at biotech
company Affymetrix in Santa Clara, Calif., led by Gingeras, who was
working there at the time, re­­port­ed that much more of human
chromosomes 21 and 22 gets transcribed than just the protein-coding
regions.

It was only after ENCODE published its results in 2012, however, that
ncRNA became impossible to ignore. Part of the antipathy toward those
findings, says Peter Stadler, a bioinformatics expert at Leipzig
University in Germany, is that they seemed like an unwanted and
unneeded complication. “The biological community figured we already
knew how the cell works, and so the discovery of [ncRNAs] was more of
an annoyance,” he says. What’s more, it showed that simpler
organisms were not always a reliable guide to human biology: there is
far less ncRNA in bacteria, studies of which had long shaped thinking
about how genes are regulated.

But now there is no turning back the tide: many thousands of human
lncRNAs have been reported, and Mattick suspects the real number is
greater than 500,000. Yet only a few of these have been shown to have
specific functions, and how many of them really do remains an open
question. “I personally don’t think all of those RNAs have an
individual role,” Lawrence says. Some, though, may act in groups to
regulate other molecules.

How lncRNAs perform such regulation is also still a matter of debate.
One idea is that they help to form so-called condensates: dense fluid
blobs containing a range of different regulatory molecules.
Condensates are thought to hold all the relevant players in one place
long enough for them to do their job collectively. Another idea is
that lncRNAs affect the structure of chromatin—the combination of
DNA and proteins that makes up chromosome fibers in the cell nucleus.
How chromatin is structured determines which of its genes are
accessible and can be transcribed; if parts of chromatin are too
tightly packed, the enzyme machinery of transcription can’t reach
it. “Some lncRNAs appear to be involved with chromatin-modifying
complexes,” says Marcel Dinger, a genomics researcher at the
University of Sydney.

If only 1 to 2 percent of the RNA from our genome was encoding
proteins, what was the rest for? Some, scientists knew, carried out
crucial tasks such as turning genes on or off.

Lawrence and Hall suspect that lncRNAs could supply scaffolds for
organizing other molecules, for example, by holding some of the many
hundreds of RNA-binding proteins in functional assemblies. One lncRNA
called NEAT1, which is involved in the formation of small compartments
in the nucleus called paraspeckles, has been shown capable of binding
up to 60 of these proteins. Or such RNA scaffolding could arrange
chromatin itself into particular structures and thereby affect gene
regulation. Such RNA scaffolding could have regularly repeating
modules and thus repetitive sequences—a feature that has long been
regarded as a hallmark of junk DNA but lately is appearing to be not
so junky after all. This view of lncRNA as scaffolding is supported by
a 2024 report of repeat-rich ncRNAs in mouse brain cells that persist
for at least two years. The research, by Sara Zocher of the German
Center for Neurodegenerative Diseases in Dresden and her co-workers.
found these ncRNAs seem to be needed to keep parts of chromatin in a
compact and silent state.

These lncRNAs are just one branch of the noncoding RNA family, and
biologists keep discovering others that appear to have different
functions and different ways of affecting what happens to a cell—and
thus the entire human body.

Some of these RNAs are not long at all but surprisingly short. Their
story began in the 1980s, when Victor Ambros, working as a
postdoctoral researcher in the laboratory of biologist Robert Horvitz
at the Massachusetts Institute of Technology, was studying a gene
denoted _lin-4_ in the worm _Caenorhabditis elegans_. Mutations
of _lin-4_ caused developmental de­­fects in which “the cells
repeated whole developmental programs that they should have
transitioned beyond,” says Ambros, now at the University of
Massachusetts Medical School. It seemed that _lin-4_ might be a kind
of “master regulator” controlling the timing of different stages
of development.

“We thought _lin-4_ would be a protein-coding gene,” Ambros
says. To figure out what role this putative protein plays, Ambros and
his colleagues cloned the _C. elegans_ gene and looked at its
product—and found that the effects of the gene may not be mediated
by any protein but by the gene’s RNA product alone. This molecule
looked ridiculously short: just 22 nucleotides long, a mere scrap of a
molecule for such big developmental effects.

This was the first known microRNA (miRNA). At first “we thought this
might be a peculiar characteristic of _C. elegans_,” Ambros says.
But in 2000 Gary Ruvkun, another former postdoc in the Horvitz lab,
and his co-­work­ers found that another of these miRNA genes in _C.
elegans_, called _let-7_, appears in essentially identical form in
many other organisms, including vertebrates, mollusks and insects.
This implies that it is a very ancient gene and “must have been
around for 600 million to 700 million years” before these diverse
lineages went their separate ways, Ambros says. If miRNAs are so
ancient, “there had to be others out there.”

Indeed, there are. Today more than 2,000 ­miRNAs have been identified
in the human genome, generally with regulatory roles. One of the main
ways miRNAs work is by interfering with the translation of a gene’s
mRNA transcript into its corresponding protein. Typically the miRNA
comes from a longer molecule, perhaps around 70 nucleotides long,
known as pre-­miRNA. This molecule is seized by an enzyme called
Dicer, which chops it into smaller fragments. These pieces, now
miRNAs, move to a class of proteins called Argonautes, components of a
protein assembly called the RNA-­induced silencing complex (RISC).
The miRNAs guide the RISC to an mRNA, and this either stops the mRNA
from being translated into a protein or leads to its degradation,
which has the same effect. This regulatory action of miRNAs guides
processes ranging from the determination of cell “fate” (the
specialized cell types they become) to cell death and management of
the cell cycle.

Key insights into how such small RNAs can regulate other RNA emerged
from studies in _C. elegans_ in 1998 by molecular biologists Andrew
Fire, Craig Mello and their co-workers, for which Fire and Mello were
awarded the 2006 Nobel Prize in Physiology or Medicine. They learned
that RISC is guided by slightly different RNA strands named small
interfering (si) RNA. The process ends with the mRNA being snipped in
half, a process called RNA interference.

MiRNAs do pose a puzzle, however. A given miRNA typically has a
sequence that matches up with lots of mRNAs. How, then, is there any
selectivity about which genes they silence? One possibility is that
miRNAs work in gangs, with several miRNAs joining forces to regulate a
given gene. The different combinations, rather than individual
snippets, are what match specific genes and their miRNAs.

Why would miRNA gene regulation work in this complicated way? Ambros
suspects it might allow for “evolutionary fluidity”: the many ways
in which different miRNAs can work together, and the number of
possible targets each of them can have, offer a lot of flexibility in
how genes are regulated and thus in what traits might result. That
gives an organism many evolutionary options, so that it is more able
to adapt to changing circumstances.

One class of small RNAs regulates gene expression by directly
interfering with transcription in the cell nucleus, triggering mRNA
degradation. These PIWI-interacting (pi) RNAs work in conjunction with
a class of proteins called PIWI Argonautes. PiRNAs operate in germline
cells (gametes), where they combat “selfish” DNA sequences called
transposons or “jumping genes”: sequences that can insert copies
of themselves throughout the genome in a disruptive way. Thus, piRNAs
are “a part of the genome’s immune system,” says Julius
Brennecke of the Institute of Molecular Biotechnology of the Austrian
Academy of Sciences. If the piRNA system is artificially shut down,
“the gametes’ genomes are completely shredded, and the organism is
completely sterile,” he says.

Still other types of ncRNAs, called small nucleolar RNAs, work within
cell compartments called nucleoli to help modify the RNA in
ribosomes—a cell’s protein-making factories—as well as transfer
RNA and mRNA. These are all ways to regulate gene expression. Then
there are circular RNAs: mRNA molecules (particularly in neurons) that
get stitched into a circular form before they are moved beyond the
nucleus into the cytoplasm. It’s not clear how many circular RNAs
are important—some might just be transcriptional “noise”—but
there is some evidence that at least some of them have regulatory
functions.

In addition, there are vault RNAs that help to transport other
molecules within and between cells, “small Cajal-body-specific
RNAs” that modify other ncRNAs involved in RNA processing, and more.
The proliferation of ncRNA varieties lends strength to Mattick’s
claim that RNA, not DNA, is “the computational engine of the
cell.”

If ncRNAs indeed power the way a cell processes genetic information,
it is possible they can be used in medicine. Disease is often the
result of a cell doing the wrong thing because it gets the wrong
regulatory instructions: cells that lose proper control of their cycle
of growth and division can become tumors, for example. Currently
medical efforts to target ncRNAs and alter their regulatory effects
often use RNA strings called antisense 
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(ASOs). These strands of nucleic acid have sequences that are
complementary to the target RNA, so they will pair up with and disable
it. ASOs have been around since the late 1970s. But it has been hard
to make them clinically useful because they get degraded quickly in
cells and have a tendency to bind to the wrong targets, with
potentially drastic consequences.

Some ASOs, however, are being developed to disable lncRNAs that are
associated with cancers such as lung cancer and acute myeloid
leukemia. Other lncRNAs might act as drugs themselves. One known as
MEG3 has been found, preliminarily, to act as a tumor suppressor.
Small synthetic molecules, which are easier than ASOs to fine-tune and
deliver into the body as pharmaceuticals, are also being explored for
binding to lncRNAs or otherwise inhibiting their interactions with
proteins. Getting these approaches to work, however, has not been
easy. “As far as I am aware, no lncRNA target or therapeutic has
entered clinical development,” Gingeras says.

James Yang

Targeting the smaller regulatory RNAs such as miRNAs might prove more
clinically amenable. Because miRNAs typically hit many targets, they
can do many things at once. For example, miRNAs in families denoted
miR-15a and miR-16-1 act as tumor suppressors by targeting several
genes that themselves suppress cell death (apoptosis, a defense
against cancer) and are being explored for cancer therapies.

Yet a problem with using small RNAs as drugs is that they elicit an
immune re­­sponse. Precisely be­­cause the immune system aims to
protect against viral RNA, it usually recognizes and attacks any
“nonself” RNA. One strategy for protecting therapeutic RNA from
immune assault and degradation is to chemically modify its backbone so
that it forms a nonnatural “locked” ring structure that the
degrading en­­zymes can’t easily recognize.

Some short ASOs that target RNAs are already approved for clinical
use, such as the drugs inotersen to treat amyloidosis and golodirsen
for Duchenne muscular dystrophy. Researchers are also exploring
antisense RNAs fewer than 21 nucleotides long that target natural
regulatory miRNAs because it is only beyond that length that an RNA
tends to trigger an immune reaction.

These are early days for RNA-­based medicine, precisely because the
significance of ncRNA itself in human biology is still relatively new
and imperfectly understood. The more we appreciate its pervasive
nature, the more we can expect to see RNA being used to control and
improve our well-being. Nils Walter of the Center for RNA Biomedicine
at the University of Michigan wrote in an article early in 2024 that
the burgeoning promise of RNA therapeutics “only makes the need for
deciphering ncRNA function more urgent.” Succeeding in this goal, he
adds, “would finally fulfill the promise of the Human Genome
Project.”

Despite this potential of noncoding RNA in medicine, the debate
continues about how much of it truly matters for our cells.
Geneticists Chris Ponting of the University of Edinburgh and Wilfried
Haerty of the Earlham Institute in Norwich, England, are among the
skeptics. In a 2022 article they argued that most lncRNAs are just
“transcriptional noise,” accidentally transcribed from random bits
of DNA. “Relatively few human lncRNAs ... contribute centrally to
human development, physiology, or behavior,” they wrote.

Brennecke advises caution about current high estimates of the number
of noncoding genes. Al­­though he agrees that such genes “have
been underappreciated for a long time,” he says we should not leap
to assuming that all lncRNAs have functions. Many of them are
transcribed only at low levels, which is what one would expect if
indeed they were just random noise. Geneticist Adrian Bird of the
University of Edinburgh points out that the abundance of the vast
majority of ncRNAs seems to be well below one molecule per cell. “It
is difficult to see how essential functions can be exerted by an ncRNA
if it is absent in most cells,” he says.

But Gingeras counters that this low expression rate might reflect the
very tissue-specific roles of ncRNAs. Some, he says, are expressed
more in one part of a tissue than in another, suggesting that
ex­­pres­sion levels in each cell are sensitive to signals coming
from surrounding tissues. Lawrence points out that, de­­spite the
low expression levels, there are often shared patterns of expression
across cells of a particular type, making it harder to argue that the
transcription is simply random. And Hall doubts that cells are really
so prone to “bad housekeeping” that they will habitually churn out
lots of useless RNA. Lawrence and Hall’s suggestion that some
lncRNAs have collective effects on chromatin structure would mean that
no individual one of them is needed at high expression levels and that
their precise sequence doesn’t matter too much.

That lack of specificity in sequence and binding targets, Dinger says,
means that a mutation of a nucleotide in an ncRNA typically won’t
have the same negative impact on its function as it tends to in a
pro­tein-­cod­ing DNA sequence. So it would not be surprising to
see quite a lot of sequence variation. Dinger argues that it makes
more sense to assume that “genetically encoded molecules are
potentially functional until shown otherwise, rather than junk unless
proven functional.” Some in the ENCODE team now agree that not all
of the 75 percent or so of human genome transcription might be
functionally significant. But many researchers make the point that
surely many more of the noncoding molecules do meaningful things than
was suspected before.

Demonstrating functional roles for lncRNAs is often tricky. In part,
Gingeras says, this may be be­­cause lncRNA might not be the
biochemically active molecule in a given process: it might be snipped
up into short RNAs that actually do the work. But be­­cause long and
short RNAs tend to be characterized via different techniques,
researchers may end up searching for the wrong thing. What’s more,
long RNAs are often cut up into fragments and then spliced back
together again in various combinations, the exact order often
de­­pend­ing on the condition of the host cell.

At its roots, the controversy over noncoding RNA is partly about what
qualifies a molecule as “functional.” Should the criterion be
based on whether the sequence is maintained between different species?
Or whether deleting the molecule from an organism’s repertoire leads
to some observable change in a trait? Or simply whether it can be
shown to be involved in some biochemical process in the cell? If
repetitive RNA acts collectively as a chromosome “scaffold” or if
miRNAs act in a kind of regulatory swarm, can any individual one of
them really be considered to have a “function”?

Gingeras says he is perplexed by ongoing claims that ncRNAs are merely
noise or junk, as evidence is mounting that they do many things. “It
is puzzling why there is such an effort to persuade colleagues to move
from a sense of interest and curiosity in the ncRNA field to a more
dubious and critical one,” he says.

Perhaps the arguments are so intense because they undercut the way we
think our biology works. Ever since the epochal discovery about
DNA’s double helix and how it encodes information, the bedrock idea
of molecular biology has been that there are precisely encoded
instructions that program specific molecules for particular tasks. But
ncRNAs seem to point to a fuzzier, more collective, logic to life. It
is a logic that is harder to discern and harder to understand. But if
scientists can learn to live with the fuzziness, this view of life may
turn out to be more complete.

_PHILIP BALL is a science writer and former Nature editor based in
London. His most recent book is How Life Works (University of
Chicago Press, 2023)._

_SCIENTIFIC AMERICAN covers the most important and exciting research,
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Spots, Stripes and More: Working out the Logic of Animal Patterns
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By Amber Dance
Knowable Magazine
More than 70 years ago, mathematician Alan Turing proposed a mechanism
that explained how patterns could emerge from bland uniformity.
Scientists are still using his model — and adding new twists — to
gain a deeper understanding of animal markings.
May 23, 2024

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