MRNAs can turn the tables on their microRNA regulators, according to a study

MicroRNAs are short sequences of RNA that maintain tight control over which genes are expressed and when. They do this by regulating which messenger RNA (mRNA) transcripts – the single-stranded templates for proteins – are actually read by the cell. But what controls these cellular controllers?

In a new study published on November 12 in Science, researchers from David Bartel’s lab at the Whitehead Institute show that mRNAs and other RNAs often turn the tables on their microRNA regulators and show that the path to microRNA degradation is not what scientists expected it to be.

“Many people know that microRNAs repress mRNAs – this is a textbook,” said Charlie Shi, a graduate student in Bartel’s lab and first author of the paper. “But in some cases, this logic is reversed. And I think it’s really interesting and weird, this idea that often the cards on the table are overturned.”

When the transcripts attack

MicroRNAs typically control gene expression by binding to mRNA transcripts and then working together with a protein called Argonaute to “silence” those transcripts by causing them to degrade more rapidly. Since the microRNAs are held comfortably within the Argonaute protein, they are protected from destructive enzymes in the cell and are therefore long-lived enough by cellular standards. They can persist for up to a week, causing many mRNA molecules to be destroyed over time.

Sometimes, however, a microRNA binds to a special target site on an mRNA transcript leading to premature destruction of the microRNA. This phenomenon, called targeted microRNA degradation, or TDMD, occurs naturally in cells and is a way to control how much of certain microRNAs can persist at any given time.

Bartel’s lab began studying this form of degradation after the lab’s researchers discovered that an RNA called CYRANO, which does not code for any protein, leads to the degradation of a specific microRNA called miR-7. This interaction was of interest to the researchers because the mechanism did not appear to be in line with current TDMD theories.

Previous models of TDMD suggested that special target sites, such as the one in CYRANO, cause one end of the microRNA to protrude from Argonaute and become vulnerable to addition and subtraction of nucleotides by cytoplasmic enzymes. This process, called tailing and trimming, was thought to be a key step on the path to microRNA degradation.

“But when you get rid of the enzyme that causes miR-7 tailing, it has no impact on degradation,” Shi said. “So it’s curious, right? So how can we really disrupt this seemingly responsible system and have no impact?”

A new model

In order to further probe the mechanism of TDMD, the researchers focused on this interaction between CYRANO non-coding RNA and miR-7. Shi designed a CRISPR screen to identify genes essential for microRNA degradation when encountered a CYRANO transcript.

The screen produced a gene essential for microRNA degradation, called ZSWIM8. When they looked at the function of the gene, the researchers found that it encodes a component of a ubiquitin ligase. Ubiquitin, so called because it is found in virtually all cell types, acts as a flag to flag proteins for degradation in a cellular waste disposal called a proteasome.

The discovery of the ubiquitin ligase ZSWIM8 implies that CYRANO-mediated degradation of microRNA results in the destruction of the Argonaute protein. In this new molecular model for TDMD, the regulatory RNA, CYRANO, binds to the microRNA, mir-7, encased in its protective Argonaute protein, and then recruits the ubiquitin ligase ZSWIM8. This ligase then attaches certain ubiquitin molecules to the microRNA Argonaute, causing the Argonaute to degrade and thereby exposing its microRNA load to be destroyed by enzymes in the cell. Importantly, this process does not require any microRNA cutting and tailing.

“The discovery of this unexpected path to TDMD illustrates the power of CRISPR screens, which can simultaneously interrogate essentially every protein in the cell, including those you never imagined would be involved,” said Bartel, who is also a Howard Hughes investigator. Medical Institute and professor of biology at the Massachusetts Institute of Technology.

A multitude of micro-horns

When the researchers looked at other known examples of TDMD, they found that ZSWIM8 was essential in all of them. Identifying this key part of the degradation pathway allowed them to search for more microRNAs subject to this regulation.

“When we started this project, there were only about four naturally occurring examples of endogenous RNAs that are encoded by the TDMD-capable cell,” Shi said. “We felt there would be many more, so by finding a required factor for TDMD in a general way – ZSWIM8 – we were then able to ask and answer the question, ‘how widespread is this phenomenon?'”

Apparently, TDMD is quite common in multicellular organisms. The researchers looked for evidence of the microRNA degradation mechanism in different cell types – two from mice and one from fruit flies – and found that in any given cell, up to 20 different microRNAs were regulated by TDMD out of a couple of hundred. of total microRNAs in the cell.

The researchers also observed this mechanism in human cells and nematodes, suggesting that TDMD as a method for regulating microRNAs goes back to the common ancestor of these disparate species. This certainly creates a lot of questions for us, “Shi said.” Each of these microRNAs is a story. ”