Whitehead Institute
scientists have identified conserved, long intervening non-coding RNAs
(lincRNAs) that play key roles during embryonic brain development in
zebrafish. They also show that the human versions of the lincRNAs can
substitute for the zebrafish versions, which implies that the functions
of these non-coding RNAs have been retained in humans as well as fish.
Until now, lincRNAs have been studied primarily in cell lines rather
than at the organismal level, which has precluded research into how
lincRNAs affect growth and development.
"These studies show that zebrafish, an animal that is frequently
used to study the genetics of animal development, can also serve as a
tool to uncover in systematic fashion the functions of lincRNAs," says
Whitehead Member David Bartel, who is also a Howard Hughes Medical
Institute investigator and a professor of biology at MIT. "This is
another case in which a phenomenon in zebrafish provides insight into
what's probably happening in humans, as has been established in many
studies of protein-coding genes."
Only a minority of RNAs transcribed in a human cell goes on to
template protein production, according to a 2007 assessment of the human
genome by the Encyclopedia of DNA Elements (ENCODE) Project Consortium,
which was funded by the National Human Genome Research Institute. The
rest of the RNAs are dubbed non-coding RNAs (ncRNAs), with those located
between protein-coding genes and with lengths of 200 base pairs or
longer referred to as lincRNAs.
Despite their prevalence in the cell, lincRNAs have been referred to
as the "dark matter" of all the transcribed RNAs because little is
known of their functions or mechanisms. One limitation to studying this
class of RNAs is their low sequence similarity between species. Unlike
protein-coding genes, which are frequently well-conserved between
species, lincRNA genes typically have a very small bit of conserved DNA
between species, if any. This lack of conservation makes identification
of related lincRNAs difficult in closely related species and nearly
impossible in distantly related species.
For example, Bartel lab scientists Igor Ulitsky and Alena Shkumatava
identified more than 500 lincRNAs in zebrafish but found that only 29
of these have homologs in both humans and mice.
Ulitsky and Shkumatava, who report their findings in this week's issue of the journal Cell,
tested the function of two of the 29 lincRNAs by knocking them down in
zebrafish embryos. Both knockdowns had striking effects on the
zebrafish's brain development. Reduction of one of the lincRNAs, which
they called cyrano, caused the zebrafish to have enlarged snouts, small
heads and eyes, and short, curly tails, while the zebrafish lacking the
lincRNA they called megamind had abnormally shaped heads and enlarged
brain ventricles.
To test if the human homologs of the cyrano and megamind lincRNAs
are functionally equivalent, Shkumatava injected the human versions into
the knocked-down zebrafish. Remarkably, the human lincRNAs rescued the
zebrafish and restored brain development and head size for both
lincRNAs, indicating that the human lincRNAs may have the same role in
embryonic development as their zebrafish analogs.
"This work represents a major advance because it provides a
framework for studying lincRNAs, a poorly understood, but abundant class
of molecules," says Michael Bender, who oversees RNA processing and
function grants at the National Institutes of Health's National
Institute of General Medical Sciences, which partially funded the work.
"The discovery that human lincRNAs appear to function much like their
zebrafish counterparts in embryonic development suggests that the
framework will prove valuable in bringing new insights on the roles
played by lincRNAs in mammalian organisms."
The zebrafish is already a powerful tool for studying genetics.
Whitehead Member Hazel Sive, who collaborated with Bartel and his lab
members on the Cell paper, uses zebrafish to study brain development and genetic mutations linked to autism.
Says Sive, "The zebrafish is a fantastic, facile system for discovering the mechanisms by which genes work."
"We humans share with zebrafish this subset of ancient, peculiar
genes, and the functionality has been retained in them," says Ulitsky.
"We can perturb them in zebrafish and then replace them with the human
ones and, at least in the lincRNAs we look at, the human ones function
to restore proper development."
"Because of this functional conservation of lincRNAs between
zebrafish and humans, we're introducing the zebrafish as a new
vertebrate tool that could be used basically to uncover the functions of
other lincRNAs," says Shkumatava.
This work was supported by the National Institutes of Health's
(NIH's) National Institute of General Medical Sciences (NIGMS), European
Molecular Biology Organization (EMBO), Human Frontiers Science Program,
and the National Science Foundation (NSF).
