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Nucleotides - not amino acids - change gene function
Actin is so ubiquitious and essential, it's called the "housekeeping protein." It's the most abundant protein found in our cells, and in its different forms it affects cell migration, the contraction of muscles, and is critical in early fetal development. For a time, scientists thought its different forms existed as back-up in case another verion of actin was defective.
Now, researchers believe these various forms of actin are not redundant. Some forms of actin are located in specific parts of a cell, while others are incorporated into different parts of the cytoskeleton. And when actin proteins are tampered with, outcomes become different too.
Humans possess six versions of the actin protein. Two in particular, ß-actin and y-actin, are nearly identical, only different by four amino acids. Yet these near twin proteins carry out distinctly different roles. A long standing question for biologists: how is this possible?
When ß-actin is diminished, fetal mice die in early stages of embryonic development. But mice lacking y-actin, though typically deaf and smaller than normal, can survive to adulthood.
"It's a mystery that's been debated in the field for the past 40 years," explains Anna Kashina, professor of biochemistry in the University of Pennsylvania School of Veterinary Medicine. New findings by Kashina and colleagues have pointed them to a surprising conclusion. The different functions of these proteins are not determined by their amino acid sequences — but by their genetic code.
"We like to call it the 'silent code. Our findings show that the parts of genes that we think of as being silent actually encode very key functional information."
The researchers think "silent" differences in nucleotide sequences influence the density of ribosomes, those molecular machines that translate RNA into proteins. Such "silent" differences might be enabling individual forms of actin to play a different role in the cell.
In a 2010 paper published in Science, Kashina's group took a step toward determining differences between actin types. Looking at a protein modification that normally only exists in ß-actin, they found that the reason it was not also present on y-actin was due to variations in the coding sequence between the two actin genes. Kashina: "We wanted to build on this, and decided to test the hypothesis, 'What if their functional differences had nothing to do with their amino acid sequence; what if it's all in the genes?'"
So, they took advantage of the precision gene editing made possible by the CRISPR/Cas-9 system. While the two actin isoforms differ by only four amino acids, their mRNA coding sequences differ by almost 13 percent. Their "silent" nucleotide differences encode the same amino acids. Making changes to only five nucleotides in the ß-actin gene, the researchers were able to transform it to produce the exact same amino acid output as the y-actin protein. All that distinguishes it are the silent nucleotide substitutions.
CRISPR/Cas-9 gene editing worked. Mice with these edits had no ß-actin protein. But unlike true ß-actin knockout mice, they were completely healthy and viable, just as if they possessed the proper proporitions of ß-actin and y-actin proteins. They survived to reproduce and averaged the same litter sizes as normal animals.
The researchers performed the same experiment, editing the y-actin gene to encode the ß-actin protein but were only able to change the coding sequence for three of the four amino acides. Still, mice subject to this partial replacement also appeared normal and healthy, despite lacking y-actin protein. In follow-up experiments, Kashina's team found that the y-actin proteins made from the edited ß-actin gene formed a normal cytoskeleton and enabled cells to migrate in a normal fashion. "If only the nucleotide sequence is important to protein function, then the mice shouldn't care what protein they have," Kashina added. "And the mice didn't care."
Getting at a mechanism for how DNA sequence could influence protein function, the researchers found that ribosome density on ß-actin RNA is more than a thousand times higher than on y-actin RNA, and indeed all six actin genes had differences in ribosome density.
And curious as to how widespread this phenomenon might be, the researchers looked for protein families with nearly identical members that are encoded by different genes and had significant variations in ribosome density across the family. They found many groups that were shared across mice, zebrafish and human genomes.
"We think this form of functional regulation is a global phenomenon," explains Kashina, and it is one her lab will continue to investigate. Kashina coauthored the group's latest paper now published in eLife.
ß- and y- cytoplasmic actin are nearly indistinguishable in their amino acid sequence, but are encoded by different genes that play non-redundant biological roles. The key determinants that drive their functional distinction are unknown. Here we tested the hypothesis that ß- and y-actin functions are defined by their nucleotide, rather than their amino acid sequence, using targeted editing of the mouse genome. Although previous studies have shown that disruption of ß-actin gene critically impacts cell migration and mouse embryogenesis, we demonstrate here that generation of a mouse lacking ß-actin protein by editing ß-actin gene to encode y-actin protein, and vice versa, does not affect cell migration and/or organism survival. Our data suggest that the essential in vivo function of ß-actin is provided by the gene sequence independent of the encoded protein isoform. We propose that this regulation constitutes a global 'silent code' mechanism that controls the functional diversity of protein isoforms.
Authors: Nicolae Adrian Leu, Yuri I Wolf, Svetlana A Shabalina, Junling Wang, Stephanie Sterling, Dawei Dong, Anna Kashina
Funding: The study was supported by the National Institutes of Health (grants GM104003 and GM117984).
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Actin is an abundant protein, present in every cell. Research shows that differences in the order of nucleotides - not amino acids - governs distinct functions in two forms of the actin protein.
Image credit: University of Pennsylvania.