Researchers are uncovering new insights into the idea that ‘silent’ or synonymous mutations may play important roles in genetics. Their research indicates that a synonymous mutation in one gene can influence a neighboring gene significantly, leading to an increase in its protein production.
Genetic disorders like cystic fibrosis and Huntington’s disease are often deemed incurable, resulting from gene mutations present in nearly every cell of the body.
Gene mutations happen when one nucleotide in a codon is altered. Non-synonymous mutations disrupt the codon’s ability to produce its corresponding amino acid, while synonymous mutations allow the codon to still produce the correct amino acid. Because of this, synonymous mutations are often regarded as “silent” and thought to have little impact on human health.
Yet, researchers at the University of Notre Dame are shedding new light on the potential importance of these silent mutations. Their findings, published in the Proceedings of the National Academy of Sciences, demonstrate that a synonymous mutation in one gene can significantly increase the protein output of an adjacent gene.
“Currently, the prevailing mindset is that only mutations that change one amino acid to another in the protein-coding section of the genome are significant,” explained Patricia L. Clark, the lead author and O’Hara Professor of Chemistry and Biochemistry at Notre Dame. “This perspective is overly simplistic and could be misleading regarding what’s truly important.”
In this study, supported by Clark’s NIH Director’s Pioneer Award, researchers worked with the genome of the bacteria E. coli due to its simpler genome and cell structure, making it easier to investigate the basic effects of mutations compared to human cells. They created nine different synonymous variants of the CAT (Chloramphenicol acetyltransferase) gene, each utilizing a different synonymous codon to encode the CAT protein.
Upon expressing these variants, the researchers found that four out of the nine synonymous sequences had an impact on the quantity of CAT proteins produced.
“You can think of synonymous mutations as a vast quilt of possible DNA sequences, all leading to the same protein,” Clark remarked. “You can choose any section of the quilt and still produce the same protein, but does it mean you’ll produce the same amount? Will the protein fold correctly? Will the cell remain healthy? These issues were what we aimed to investigate.”
Initially, Clark, an expert in protein folding, hypothesized that these four synonymous mutations might alter the folding of the CAT protein after gene expression. However, the research team, including first author Anabel Rodriguez, who was a doctoral student in Clark’s lab, discovered that the effects of the synonymous mutations occurred during gene expression, particularly influencing the transcription from DNA to RNA.
“What Anabel demonstrated was that the quantity of synthesized CAT protein correlated with the amount of CAT RNA being produced,” Clark noted. “This showed that some synonymous mutations disrupted the RNA synthesis from DNA. That Anabel accomplished this while working in a lab with no prior experience in transcription studies is impressive.”
The study revealed that certain synonymous mutations created hidden transcription sites on the CAT DNA strand. The enzyme RNA polymerase, which is responsible for converting DNA to RNA, was binding to these unexpected sites rather than their anticipated locations.
As a result, these polymerases produced RNA that originated in CAT but also extended to include the entire neighboring upstream gene. In the case of CAT, the upstream gene encodes a repressor protein, and an increase in this protein inhibits the expression of CAT.
In the last decade, the potential for a synonymous mutation to affect its own gene’s processes has gained attention. Therefore, the notion that a synonymous mutation in one gene could also influence the transcription and translation of an adjacent gene is a substantial advancement, and Clark and her lab plan to investigate this further.
“There’s a growing body of important studies revealing how limited our understanding is regarding the effects of synonymous mutations. We should be exploring the impact of these mutations on all diseases and genetic disorders,” Clark stated. “I hope our research contributes to a more thorough understanding of these mechanisms.”
The research team intends to delve into how certain synonymous mutations of the CAT gene effectively attracted RNA polymerase to the hidden binding site. This inquiry is particularly notable as current machine learning algorithms have struggled to predict this phenomenon accurately.
Clark is also the associate vice president of research and director of the Biophysics Instrumentation Core Facility at Notre Dame. Anabel Rodriguez, the lead author and a former graduate student in Clark’s lab, is now an instructor at Coastal Carolina Community College.
Other co-authors include Jacob Diehl, Christopher Bonar, Taylor Lundgren, McKenze Moss, Jun Li, Tijana Milenkovic, Paul Huber, and Matthew Champion from Notre Dame; Gabriel Wright from the Milwaukee School of Engineering; and Scott Emrich from the University of Tennessee.
