Chemical modifications in proteins help control RNA fate within the cell
Study investigated how small chemical modifications in yeast can regulate RNA processing and degradation
By: Thabata Oliveira*
Art by: Livia Bortoletto**
Saccharomyces cerevisiae, brewer’s yeast, was the organism used as a model to investigate RNA processing and regulation – Photo: Rainis Venta/ Wikimedia Commons
A study published in the Journal of Proteome Research addressed one of these gaps and investigated how the cell controls two essential stages in the life of RNA: processing, which prepares the molecule to function, and degradation, which eliminates it when it is no longer needed.
The research was conducted by scientists from USP’s Institute of Chemistry (IQ) and focused on the RNA exosome, a multiprotein complex that acts as a processing and degradation machine. “All RNAs undergo processing, and this is an essential step in regulating gene expression [the process by which a cell converts genetic information into functional molecules]”, explained Carla Columbano de Oliveira, a professor at IQ and coordinator of the study.
One of the most important examples involves ribosomes, the structures responsible for producing the cell’s proteins. Ribosomes themselves are partly composed of RNA, which must be processed before they can function. In the cell nucleus, the exosome participates in ribosomal RNA processing and eliminates molecules that were produced incorrectly.
In the cytoplasm, the region of the cell outside the nucleus, the exosome participates in the degradation of different types of RNA, including messenger RNA, the molecule that carries the instructions needed to produce proteins.
“If a messenger RNA has not been properly processed, it must be eliminated; otherwise, it can produce a defective protein that may harm the cell. In addition, RNA cannot remain in the cell indefinitely: at some point it must be degraded so that it is no longer expressed”, the researcher explained.
The cell and the exosome
To perform such different tasks, the exosome must interact with other proteins that help target it and regulate its activity. The researchers focused precisely on this network of interactions. Their central question was: how does the cell control which proteins associate with the exosome in each situation?
“Our question is this: if the exosome is involved in ribosomal RNA processing and in degrading all types of RNA, both in the nucleus and the cytoplasm, how is it regulated?”, Carla Oliveira summarized.
The study used Saccharomyces cerevisiae, the yeast that has been used for millennia to make bread, beer, and wine. The choice was deliberate: the RNA exosome was discovered in this microorganism, which has become an important model for studying how cells function. Yeast is easy to culture and allows researchers to modify its genes and observe the consequences of those changes. In addition, many cellular mechanisms found in yeast are also present in other organisms, including humans. “In humans, mutations in the genes encoding exosome subunits are associated with diseases affecting the central nervous system”, the professor explained.
Chemical switches
One hypothesis investigated by the researchers was that phosphorylation could help regulate the exosome. Phosphorylation is a small chemical modification that the cell makes to its own proteins by adding a phosphate group. Depending on where it occurs, this modification can act as a kind of switch, changing a protein’s activity or the molecules with which it interacts.
To investigate this hypothesis, the team used two proteins from the exosome itself — Rrp6 and Rrp46 — as “baits” to capture other molecules associated with it. Rrp6 can capture proteins associated with the nuclear exosome, whereas Rrp46 can capture proteins associated with both the nuclear and cytoplasmic exosomes.
Next, the researchers determined which proteins captured by the baits carried phosphate groups. They initially identified 737 phosphorylation sites. Each site corresponds to a specific amino acid residue within a particular protein where a phosphate group was detected.
The researchers then filtered the results for sites related to ribosomal RNA processing and the transport of molecules between the nucleus and cytoplasm. This yielded 114 phosphorylation sites, most of them in exosome-interacting proteins, but some in the exosome’s own subunits. Each site is a candidate — a location where phosphorylation may regulate the exosome and that must now be tested individually.
Phosphorylation patterns differed between proteins associated with Rrp6 and those associated with Rrp46. According to the researchers, this is consistent with the idea that the exosome can assemble with different sets of proteins and therefore act differently depending on the task it performs. Phosphorylation may be one mechanism regulating these interactions. Determining the role of each phosphorylation event, however, will require specific experiments.
RNA degradation
One of these investigations focused on Ski7, a protein that recruits the exosome to degrade RNA in the cytoplasm. Previous work by the group showed that overexpressing Ski7 traps the exosome in the cytoplasm and prevents it from reaching the nucleus, where it performs its essential role in ribosomal RNA processing. The result is impaired cell growth.
The researchers identified two phosphorylation sites in Ski7 and tested one of them, S90. When they prevented phosphorylation at this site, the protein no longer impaired cell growth, even when overexpressed. The result indicates that S90 phosphorylation contributes to regulating Ski7 function.
“Ski7 is part of a protein complex called SKI. In humans, mutations have been identified in genes encoding other proteins in this complex that cause a disease [trichohepatoenteric syndrome, a rare genetic disorder that mainly affects the hair, liver, and intestine]. This is why yeast is useful: it allows us to obtain information that helps us understand what happens in humans”, Oliveira explained.
The next step is to repeat with other proteins what was done with Ski7: prevent each phosphorylation event from occurring and observe whether the protein continues to function and interact normally. This is how the researchers hope to determine which of these modifications actually alter exosome function.
The article Phosphorylation Patterns of Preribosomal Proteins Associated with the RNA Exosome is available online.
More information: ccoliv@iq.usp.br, Carla Columbano de Oliveira
*Written by São Paulo Research Foundation (Fapesp) Media Science Fellow at USP’s Institute of Chemistry (IQ). Adapted for Jornal da USP
English version: Nexus Traduções, edited by Denis Pacheco