RNA sequences involved in regulating gene expression identified

Date:
July 29, 2020
Source:
Massachusetts Institute of Technology
Summary:
By studying RNA-binding proteins, a research consortium has
identified genomic sites that appear to code for RNA molecules
that influence gene expression.



FULL STORY ==========================================================================
The human genome contains about 20,000 protein-coding genes, but the
coding parts of our genes account for only about 2 percent of the entire genome. For the past two decades, scientists have been trying to find
out what the other 98 percent is doing.


==========================================================================
A research consortium known as ENCODE (Encyclopedia of DNA Elements)
has made significant progress toward that goal, identifying many genome locations that bind to regulatory proteins, helping to control which
genes get turned on or off. In a new study that is also part of ENCODE, researchers have now identified many additional sites that code for RNA molecules that are likely to influence gene expression.

These RNA sequences do not get translated into proteins, but act in a
variety of ways to control how much protein is made from protein-coding
genes. The research team, which includes scientists from MIT and several
other institutions, made use of RNA-binding proteins to help them
locate and assign possible functions to tens of thousands of sequences
of the genome.

"This is the first large-scale functional genomic analysis of RNA-binding proteins with multiple different techniques," says Christopher Burge, an
MIT professor of biology. "With the technologies for studying RNA-binding proteins now approaching the level of those that have been available
for studying DNA- binding proteins, we hope to bring RNA function more
fully into the genomic world." Burge is one of the senior authors of
the study, along with Xiang-Dong Fu and Gene Yeo of the University of California at San Diego, Eric Lecuyer of the University of Montreal,
and Brenton Graveley of UConn Health.

The lead authors of the study, which appears today in Nature, are Peter
Freese, a recent MIT PhD recipient in Computational and Systems Biology;
Eric Van Nostrand, Gabriel Pratt, and Rui Xiao of UCSD; Xiaofeng Wang
of the University of Montreal; and Xintao Wei of UConn Health.



==========================================================================
RNA regulation Much of the ENCODE project has thus far relied on detecting regulatory sequences of DNA using a technique called ChIP-seq. This
technique allows researchers to identify DNA sites that are bound to DNA-binding proteins such as transcription factors, helping to determine
the functions of those DNA sequences.

However, Burge points out, this technique won't detect genomic
elements that must be copied into RNA before getting involved in gene regulation. Instead, the RNA team relied on a technique known as eCLIP,
which uses ultraviolet light to cross-link RNA molecules with RNA-binding proteins (RBPs) inside cells.

Researchers then isolate specific RBPs using antibodies and sequence
the RNAs they were bound to.

RBPs have many different functions -- some are splicing factors, which
help to cut out sections of protein-coding messenger RNA, while others terminate transcription, enhance protein translation, break down RNA after translation, or guide RNA to a specific location in the cell. Determining
the RNA sequences that are bound to RBPs can help to reveal information
about the function of those RNA molecules.

"RBP binding sites are candidate functional elements in the
transcriptome," Burge says. "However, not all sites of binding have a
function, so then you need to complement that with other types of assays
to assess function." The researchers performed eCLIP on about 150 RBPs
and integrated those results with data from another set of experiments in
which they knocked down the expression of about 260 RBPs, one at a time,
in human cells. They then measured the effects of this knockdown on the
RNA molecules that interact with the protein.



========================================================================== Using a technique developed by Burge's lab, the researchers were also
able to narrow down more precisely where the RBPs bind to RNA. This
technique, known as RNA Bind-N-Seq, reveals very short sequences,
sometimes containing structural motifs such as bulges or hairpins,
that RBPs bind to.

Overall, the researchers were able to study about 350 of the 1,500
known human RBPs, using one or more of these techniques per protein. RNA splicing factors often have different activity depending on where they
bind in a transcript, for example activating splicing when they bind
at one end of an intron and repressing it when they bind the other
end. Combining the data from these techniques allowed the researchers
to produce an "atlas" of maps describing how each RBP's activity depends
on its binding location.

"Why they activate in one location and repress when they bind to another location is a longstanding puzzle," Burge says. "But having this set
of maps may help researchers to figure out what protein features are
associated with each pattern of activity." Additionally, Lecuyer's
group at the University of Montreal used green fluorescent protein
to tag more than 300 RBPs and pinpoint their locations within cells,
such as the nucleus, the cytoplasm, or the mitochondria. This location information can also help scientists to learn more about the functions
of each RBP and the RNA it binds to.

Linking RNA and disease Many research labs around the world are now
using these data in an effort to uncover links between some of the RNA sequences identified and human diseases.

For many diseases, researchers have identified genetic variants called
single nucleotide polymorphisms (SNPs) that are more common in people
with a particular disease.

"If those occur in a protein-coding region, you can predict the effects on protein structure and function, which is done all the time. But if they
occur in a noncoding region, it's harder to figure out what they may be
doing," Burge says. "If they hit a noncoding region that we identified
as binding to an RBP, and disrupt the RBP's motif, then we could predict
that the SNP may alter the splicing or stability of the gene." Burge and
his colleagues now plan to use their RNA-based techniques to generate
data on additional RNA-binding proteins.

"This work provides a resource that the human genetics community can
use to help identify genetic variants that function at the RNA level,"
he says.

The research was funded by the National Human Genome Research Institute
ENCODE Project, as well as a grant from the Fonds de Recherche de Que'bec-Sante'.


========================================================================== Story Source: Materials provided by
Massachusetts_Institute_of_Technology. Original written by Anne
Trafton. Note: Content may be edited for style and length.


========================================================================== Journal Reference:
1. Abascal, F., Acosta, R., Addleman, N.J. et al. Expanded
encyclopaedias of
DNA elements in the human and mouse genomes. Nature, 2020 DOI:
10.1038/ s41586-020-2493-4 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/07/200729114811.htm

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