When RNA rules
A newly discovered class of molecules plays an astonishingly
powerful role in biology
What do newly discovered molecules called microRNAs
and the Internet have in common? Both reshaped entire
fields in the past decade, says Whitehead postdoctoral
fellow Andrew Grimson.
“That’s a fairly grandiose claim for microRNAs,”
acknowledges Grimson, who studies them. “But the
discovery of the widespread role of these molecules
changed the landscape of biology very quickly.”
“Labs across the world, working on a variety
of biological questions, are now integrating microRNAs
into their research,” says David
Bartel, Whitehead Member and Howard Hughes Medical
Institute investigator.
Bartel and his colleagues have helped to fuel the frenzy
by identifying hundreds of the small RNA molecules and
providing compelling evidence that they regulate the
production of thousands of proteins in plants and animals.
| “Computational work has produced a very
big picture of what microRNAs are likely to be doing
in a very short time,” says Nobel laureate
and MIT Institute Professor Phillip Sharp. |
Until the early 1990s, no one had a clue about microRNAs,
which flew under the radar because of their tiny size.
Each one contains only 21 to 24 nucleotides, or letters
of the genetic alphabet, so scientists simply missed
them. Victor Ambros’s group found the first microRNA—lin-4—in
1993 at Harvard Medical School while studying a mutation
in the worm Caenorhabditis elegans.
Another Harvard researcher detected a second microRNA
in 2000. One year later, the floodgates opened with
the discovery of nearly a hundred in worms, insects
and humans. At this point researchers began calling
these tiny regulatory molecules “microRNAs.”
The discoveries changed conceptions of RNA. Scientists
have known for decades that RNA molecules serve as messengers
and translators, building proteins from DNA sequences.
But microRNAs determine which DNA sequences get translated
in a given cell, a responsibility once considered the
purview of proteins known as transcription factors.
MicroRNAs essentially choreograph biological ballets,
helping to determine where and when proteins can appear
to perform. Thus RNA can add “regulator”
to the roles listed on its résumé.
MicroRNAs bind to messenger RNAs that code for proteins
involved in activities ranging from development to cancer,
and disrupt the production of these proteins. In humans,
microRNAs regulate roughly one-third of protein-coding
genes, and that’s a conservative estimate.
Going through the genome
“This is the first discovery of a broad biological
mechanism that’s been made since genomics,”
says Nobel laureate Phillip Sharp, who is investigating
how microRNAs work at MIT, where he is an Institute
Professor.
Scientists determined the scope of microRNA activity
in a matter of years by mining recently published DNA
sequences. Bartel, an RNA biochemist, and computational
biologist Christopher Burge of MIT played a leading
role. They collaborated to develop computer programs
that scanned genomes to identify microRNAs and their
messenger RNA targets. Their work helped to ignite interest
in microRNAs as biologists in labs around the world
realized the tiny molecules regulate a large portion
of the protein-coding genes in plant and animal cells.
“Computational work has produced a very big picture
of what microRNAs are likely to be doing in a very short
time,” says Sharp. “ “It feels like
the field is moving at warp speed,” agrees Burge,
a Whitehead Career Development Associate Professor of
Biology. “Genomic approaches have provided a number
of important insights, and there has been nice synergy
with molecular and biochemical studies.”
Finding the first microRNAs
Rosalind Lee and Rhonda Feinbaum, researchers in the
Ambros lab, were conducting painstaking experiments
on C. elegans when they bumped into the first
microRNA.
They knew that early development of worm larvae required
proper levels of the novel protein lin-14. They also
knew that something was regulating those levels and
assumed it was another protein, so they set out to isolate
the gene for that protein. The result amazed them.
The gene fell on a stretch of DNA once termed “junk”
by some, a stretch outside the protein-coding region
of the chromosome. It appeared to code for a small RNA
molecule— lin-4—that somehow regulated lin-14
levels.
The researchers wondered if lin-4 was an esoteric molecule
or a harbinger of a new class of RNAs. “We had
no basis for saying that lin-4 was part of something
much broader,” says Ambros, who now works at Dartmouth
Medical School.
His lab had no luck searching for additional RNAs in
the next few years. He was thrilled when researchers
in the lab of Harvard Medical School’s Gary Ruvkun
discovered another gene in C. elegans that
coded for a small RNA called let-7 in 2000. In addition
to cloning let-7, Ruvkun’s group examined the
genomes of a number of other animals and found the gene
for let-7 in most of them. The study foreshadowed the
role of genomics in later research.
In 2001, Rockefeller University associate professor
Thomas Tuschl (formerly a postdoctoral fellow in the
Bartel lab), Ambros and Bartel independently found dozens
of additional small RNA genes in worms, flies and humans
and decided to call them microRNAs.
Leveraging genomics
Bartel realized he needed to look outside the toolbox
of classical biology. In 2001, he approached Burge—who
had previously developed algorithms to identify protein-coding
genes in the human genome—and Lee Lim, who had
just completed his PhD training with Burge. The researchers
jumped at the chance to explore a new class of genes.
Lim worked jointly with the two labs to write a computer
program that could scan DNA sequences and predict microRNA
genes.
He started by examining known microRNAs. Each microRNA
is generated from a piece of RNA that folds back on
itself to form a structure that resembles a hairpin.
Lim scanned the genome of C. elegans for DNA
sequences that would give rise to hairpins after being
transcribed into RNA. He then looked for ways to further
refine the search.
The double-stranded RNA of a hairpin is chopped and
processed into a single-stranded microRNA by proteins
called Drosha and Dicer. But apparently these proteins
don’t recognize every hairpin. Lim whittled down
the list of potential microRNAs by eliminating DNA templates
for hairpins that lacked Dicer-friendly characteristics.
Lim then screened the remaining microRNA candidates
by comparing the genomic sequence of C. elegans
with that of the related worm C. briggsae.
He reasoned that most of the genuine microRNAs, those
performing critical biological functions, would be conserved
across species.
Eventually, the team showed that the human genome contains
more than 200 microRNA genes. “We were excited
to find new microRNAs,” says Burge. “But
then the big question was—what do they do?”
This question had been largely answered in plants. Matthew
Jones-Rhoades, a graduate student in the Bartel lab,
had discovered that plant microRNAs have extensive and
highly conserved pairing to plant messenger RNAs, so
he could easily identify many targets of the plant microRNAs.
“At a time when we had about 50 plant targets,
we were still in the dark regarding which genes were
targeted in animals,” says Bartel.
Benjamin Lewis, a graduate student in both the Bartel
and Burge labs, developed a second computer program
to bridge this gap. He took the sequences of known micro-RNAs,
scanned animal genomes for corresponding messenger RNA
targets and, like Lim, used conservation across species
to screen the results. The goal was to find many more
conserved microRNA-mRNA pairings than would result by
chance. But the initial program failed to deliver.
The researchers then tried another twist. Previous work
showed that some microRNAs pair only partially with
their mRNA targets, so the team hypothesized that one
part of each microRNA sequence might be particularly
important. They were right. Lewis hit the jackpot when
he required perfect pairing near one end of the microRNAs.
He found tiny sequences, matching short stretches of
microRNAs, conserved much more frequently than chance
would dictate in the mRNAs of mice, rats and humans.
Lewis named the critical stretch that matches targeted
mRNAs the “seed” of the microRNA. The discovery
of the seed gave scientists working on the biochemical
interaction between microRNAs and mRNAs a big boost.
It also allowed Bartel, Burge and Lewis to move forward
with predicting targets.
They showed that many animal microRNAs have hundreds
of conserved targets involved in a variety of processes,
and in January 2005, they conservatively estimated that
micro-RNAs regulate one-third of protein-coding genes
in humans. This was a shock, as each plant microRNA
appears to have just a few targets linked to development.
By the end of 2005, Kyle Kai-How Farh, another graduate
student in Bartel’s lab, together with Andrew
Grimson, showed there is also a large potential for
species-specific targeting, and that in many cases protein-coding
genes are evolving to avoid pairing with microRNAs.
Thus micro-RNAs are affecting the majority of human
protein-coding genes, at either a functional level or
an evolutionary level.
Springboard for new studies
While the human genome is clearly full of potential
microRNA targets, scientists in the lab have confirmed
only a handful of interactions between mammalian microRNAs
and mRNAs in living cells. Investigators are just beginning
to use classical tools to probe the functions of the
interactions identified computationally by Bartel and
Burge, who are refining their computer programs and
designing experiments to test past predictions.
“We’re improving the prediction programs
to make them more inclusive and more accurate, and we’re
sequencing millions of small RNAs in plants and animals
to get a clearer picture of what’s really in the
cell environment,” says Bartel.
Graduate student Graham Ruby, for example, is overhauling
Lim’s microRNA prediction program. The original
application missed many real microRNAs, and Ruby hopes
to catch some of the molecules that fell through the
cracks. Lim narrowed the list of microRNA precursors
by scoring each hairpin according to its microRNA-like
characteristics. Ruby adds a new twist. His program
includes more than one round of scoring, like the American
Idol show. After each round, he eliminates the
lowest-scoring hairpins from the pool of candidates.
He examines the rejects and uses their characteristics
to fine-tune the scoring criteria for the next round,
which should make predictions more accurate.
Other researchers in Bartel’s lab are working
to determine the mechanism by which microRNAs lower
protein levels, as much of it remains a mystery. The
picture is clearer in plants, where microRNAs pair fully
with and direct the cleavage of messenger RNAs.
But most of the new studies on microRNAs deal with their
specific functions. Cancer researchers are particularly
interested in the tiny RNAs, as many of them appear
to regulate cell proliferation. Several papers last
year confirmed this link. Gregory Hannon of Cold Spring
Harbor and Scott Hammond of the University of North
Carolina, for example, showed that overabundance of
a specific group of micro-RNAs probably contributes
to human B cell lymphomas.
“Studies are beginning to show the relevance of
micro-RNAs to human disease,” says postdoctoral
fellow Michael Lam, who is working with mice in Bartel’s
lab to probe some of the other microRNAs connected to
cancer.
“It’s exciting to watch the parallel currents
in microRNA research,” says Ambros. “As
a classical geneticist, I find it interesting to know
how particular micro-RNAs work in particular situations.
But I’m also intrigued by the work of people such
as Dave Bartel, who are taking a more genomic view and
discerning general patterns of microRNA function and
evolution.”
“This will occupy thousands of people for years,”
Sharp says. “It will take decades to work out
the specifics of many different microRNA-regulated processes
and integrate those into whole-organism biology.”
***************
Running interference
RNA interference (RNAi) advanced genomic exploration
a few years before the abundance of microRNAs was recognized.
Scientists found they could knock down the output of
genes by introducing double-stranded RNA into an animal.
They soon found that this double-stranded RNA was processed
into short strands of RNA, and that these small interfering
RNAs (siRNAs) pair to and direct the cleavage of messenger
RNA molecules that control protein production.
Sound familiar?
It turns out that microRNAs and siRNAs use similar cellular
machinery to achieve their goals. Both rely on a protein
called Dicer for processing, and a “silencing
complex” facilitates their interaction with mRNAs.
Within this silencing complex, the two types of RNA
molecules both can direct messenger RNA cleavage when
they have extensive pairing to the messengers, or dampen
protein output by other means when pairing is not as
tight.
However, a microRNA comes from a single strand of RNA
coded in the genome that folds back on itself to form
a structure resembling a hairpin, while an siRNA comes
from a long piece of double-stranded RNA.
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