The nascent peptide cypher: a novel controller of mRNA stability

From the Subramaniam Lab, Cancer Basic Biology Program, Cancer Consortium.

For a protein-coding mRNA, “life” should be pretty simple: get assembled through transcription, produce a protein through translation, then get recycled by cellular machinery so you can do it all again. Unfortunately, what I just wrote leaves out a colossal orchestra of quality-control machinery. Oh, you think I mean splicing? Try again. In an article published last month in Nature Communications, Dr. Phillip Burke, a recent doctoral graduate in the Subramaniam Lab, showed there is a translational control cypher encoded in nascent peptides that has potent effects on the stability of the encoding mRNA. Decoding this translational cypher took years of painstakingly precise work, and the creation of an innovative screening approach.

“In the past few years, several groups have implicated coding sequence metrics such as codon [use], amino acid [sequence], and GC content as regulators of mRNA stability in human cells,” shared Dr. Burke. “These studies relied on a correlation between these metrics and measured stability of endogenous human mRNAs. However, since the sequences of endogenous mRNAs are the result of complex evolutionary pressures, we have little mechanistic insight into how the implicated metrics influence mRNA stability. We felt there was a critical need in the translation field for a bottom-up high throughput approach for systematically mapping the effects of increasingly complex coding sequence motifs on mRNA stability.” Effectively, Dr. Burke and his advisor, Dr. Arvind “Rasi” Subramaniam, wanted to define the “individual contribution[s] of mRNA features implicated in previous studies.” However, no one had a method to do this. So, they built one. “Developing the initial high-throughput assay took a lot of optimization,” confessed Dr. Burke. “We tried without success for a while, then completely dropped the project for about a year…We ended up coming back to this approach while trying to study mRNA quality control pathways in human cells, when we realized we still had no idea what human coding sequence motifs could reliably trigger mRNA instability.”

A graphic showing the correlation between dipeptides and mRNA stability.
In this study, Dr. Burke used a reporter screen (left) to tease out combinations of amino acids in newly translated proteins (nascent peptides) that destabilized (red) or stabilized (blue) their template mRNAs. Figure provided by Dr. Phillip Burke

What they found was a complex network of interactions between pairs of amino acids, called dipeptides, in the nascently translated proteins encoded by the mRNAs. “[mRNAs that encode] nascent peptides with a combination of beta-strand (β-strand) structures and bulky and positively charged sequences trigger acute instability,” explained Dr. Burke. That is to say, their screen for mRNA features accidentally uncovered the importance of features of the nascent protein encoded by said mRNAs. “This discovery transforms our understanding of the ribosome from being a passive translator of the genetic code to an active filter against aggregation-prone peptides such as bulky β-strands. Our findings also imply that the universal interpretation of missense mutations as altering protein activity needs to be revised to account for their effects on mRNA stability.”

As for what’s next, “we would love to do some Cryo-EM studies to dissect the atomic level interactions mediating the peptide code we report here,” said Dr. Burke. “We think these studies will expand our mechanistic understanding of how ribosome arrest peptides work. We are also working on using the high-throughput assay we developed here to profile effects of viral coding sequence motifs on mRNA stability!”

This big of a project relied heavily on the resources established by the Fred Hutch/University of Washington Cancer Consortium. Said Mr. Burke, “The Fred Hutch sequencing core was absolutely essential; we never would have completed this project without their expertise. We also got a lot of great feedback on the project from many members of the Basic Sciences Division and the Computational Biology program here at the Fred Hutch.”

This work was funded by the National Institutes of Health, the National Science Foundation, the Sidney Kimmel Scholarship, the Genomics Shared Resource and Fred Hutch Scientific Computing.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Dr. Rasi Subramaniam contributed to this work.

P Burke, H Park, and AR Subramaniam. 2022. A nascent peptide code for translational control of mRNA stability in human cells. Nature Communications. 13: 6829.