lncRNA structure affects its function
Several years ago, biologists discovered a new type of genetic material known as long noncoding RNA or lcnRNA. This RNA does not code for proteins and is copied from sections of the genome. In the 1970's it was considered "junk DNA."
Since then, scientists have found evidence that long noncoding RNA, or lncRNA, plays a role in many cell processes — even guiding cell fate during embryo development. However, exactly how was uncertain.
But scientists were inspired to keep looking by work revealing structure and function in other RNA classes — such as with transfer RNA which transports amino acids from the cytoplasm of a cell to a ribosome — reflecting some kind of interactive support.
MIT biologists now have deciphered how the structure of one lncRNA acts on a cellular protein to control the development of heart muscle cells — one of the first studies to connect lncRNA structure to a functional outcome.
"Emerging data, points to fundamental roles for many of these molecules in development and disease. So, we believe that determining the structure of lncRNAs is critical for understanding how they function," says Laurie Boyer, the Irwin and Helen Sizer Career Development Associate Professor of Biology and Biological Engineering at the Massachusetts Institute of Technology (MIT) and the senior author of the study, which appears in the journal Molecular Cell on Sept. 8, 2016.
Learning more about how lncRNAs control cell differentiation could offer a new approach to developing drugs for patients whose hearts have been damaged by cardiovascular disease, aging, or cancer.
The paper's lead author is MIT postdoc Zhihong Xue. Other MIT authors are undergraduate Boryana Doyle and Sarnoff Fellow Arune Gulati. Scott Hennelly, Irina Novikova, and Karissa Sanbonmatsu of Los Alamos National Laboratory are also authors of the paper.
Boyer's lab previously identified a mouse lncRNA known as Braveheart, found at higher levels in the heart when compared to other tissues. In 2013, Boyer found this lncRNA molecule is needed for normal heart muscle development. Researchers decided to investigate which regions of Braveheart's 600-nucleotide RNA molecule were crucial to its function.
"We knew Braveheart was critical for heart muscle cell development, but we didn't know the detailed molecular mechanism of how this lncRNA functioned, so we hypothesized that determining its structure could reveal new clues," said Xue.
To determine Braveheart's structure, they used a chemical probing technique using a reagent - a substance added to cause a chemical reaction. Analyzing which nucleotides bound to the reagent, researchers identified single-stranded regions, double-stranded helices, loops, and other Braveheart structures.
This analysis revealed that Braveheart has several distinct structures or motifs. Researchers then tested which motif was the most important to the molecule's ability to function.
To their surprise, by removing 11 nucleotides (a loop that represents just 2 percent of the entire molecule) they stopped normal heart cell development.
They then searched for proteins that the Braveheart loop interacted with. Screening about 10,000 proteins, they discovered cellular nucleic acid binding protein (CNBP) bound strongly to that particular lncRNA loop.
Previous studies had already shown that mutations in CNBP can lead to heart defects in mice and humans. More research revealed CNBP acts as a potential roadblock to cardiac development, and that Braveheart supresses CNBP, allowing cells to continue to become heart muscle.
Scientists have not yet identified a human counterpart to the mouse Braveheart lncRNA, partly because human and mouse lncRNA sequences are poorly conserved across our two species, even though protein-coding genes for both are typically very similar.
However, now that researchers know the structure of the mouse Braveheart lncRNA, they plan to analyze human lncRNA molecules and identify similar structures that may also have similar functions.
"We're taking this motif and using it to build a fingerprint, so that we can potentially find motifs resembling this lncRNA across species," Boyer adds. "We also hope to extend this work to identify modes of action of a catalog of motifs so we can better predict lncRNAs with important functions."
The researchers also plan to apply what they have learned about lncRNA toward engineering new therapeutics. "We fully expect that unraveling lncRNA structure-to-function relationships will open up exciting new therapeutic modalities in the near future," Boyer says.
•Long non-coding RNA Braveheart (Bvht) adopts a modular secondary structure
•An internal G-rich RNA motif (AGIL) of Bvht is required for cardiac specification
•AGIL interacts with nucleic acid binding proteins, including CNBP
•AGIL functionally antagonizes the zinc-finger protein CNBP to promote cardiac fate
Long non-coding RNAs (lncRNAs) are an emerging class of transcripts that can modulate gene expression; however, their mechanisms of action remain poorly understood. Here, we experimentally determine the secondary structure of Braveheart (Bvht) using chemical probing methods and show that this ∼590 nt transcript has a modular fold. Using CRISPR/Cas9-mediated editing of mouse embryonic stem cells, we find that deletion of 11 nt in a 5′ asymmetric G-rich internal loop (AGIL) of Bvht (bvhtdAGIL) dramatically impairs cardiomyocyte differentiation. We demonstrate a specific interaction between AGIL and cellular nucleic acid binding protein (CNBP/ZNF9), a zinc-finger protein known to bind single-stranded G-rich sequences. We further show that CNBP deletion partially rescues the bvhtdAGIL mutant phenotype by restoring differentiation capacity. Together, our work shows that Bvht functions with CNBP through a well-defined RNA motif to regulate cardiovascular lineage commitment, opening the door for exploring broader roles of RNA structure in development and disease.
Braveheart, cardiac, CNBP, long non-coding RNA, SHAPE
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