ThreePharingos
This image of three worms illustrates functional mosaicism in action. The worms have been genetically modified to express a fluorescent protein that appears as colorful glowing dots. The worm on the left shows fluorescence in every cell. In the middle worm, intestinal cells do not fluoresce because a protein that blocks the fluorescent gene has caused RNA interference (RNAi). In the worm on the right, scientists have removed the enzyme previously believed to be responsible for RNAi in intestinal cells, but fluorescence remains blocked in some of those cells, suggesting that RNAi is being carried out by another enzyme. [Snusha Ravikumar/University of Maryland}

Scientists at the University of Maryland (UMD) have discovered that what may seem to be identical cells can use different proteins to carry out the same function. This “functional mosaicism,” which the researchers identified through their studies in the roundworm Caenorhabditis elegans, could have important implications for drug development, as drugs are commonly designed to function by targeting a specific protein or gene. It may be that some cells in a tissue, for example, a tumor, may use functional mosaicism to resist drug therapy by utilizing alternative molecules for the same process. This ability may explain disease persistence and relapse, or resistance to drug therapy.

“Functional mosaicism introduces a cautionary message,” said Antony Jose, PhD, associate professor of cell biology and molecular genetics at UMD. “It means that if you’re developing a drug therapy that targets a certain process within cells, you can no longer assume that all cells of the same type use the same molecule or molecules to carry out that process. If you do, you may miss a few cells where things work differently.” Jose is senior author of the team’s published paper in Nucleic Acids Research, alongside co-authors Sindhuja Devanapally, PhD, and first author Snusha Ravikumar, PhD. Their paper is titled, “Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference.”

Scientists have long recognized biological redundancy in cells, effectively the ability of cells to carry out important functions in multiple ways. But the general tenet has been that the same cell types typically will demonstrate consistency. In effect, all cells of the same type will exhibit the same redundancies and use the same—and possibly multiple—processes and molecules for the same functions. Different cell types may use alternative pathways to carry out the same common function, but again, they will also demonstrate cell type-specific redundancies. “Animals have diverse cell types that perform specialized functions while retaining the ability to perform common functions,” the authors explained. “Such common functions could rely on the same molecular machinery in all cells or on different machinery in different cells.”

Functional mosaicism, on the other hand, means that some individual cells within a population of seemingly identical cells can also use an alternate pathway for certain functions. “With biological redundancy, if you’re developing a drug to target a particular function and the cells have an alternative way to accomplish the function, your drug will fail because all cells will use the alternative,” Jose explained. “Functional mosaicism is different, and it can fool you. Your drug may appear to work because it works in some cells—even most cells, but there may be a few cells that persist because they can use an alternative you’re not aware of.”

Jose and colleagues discovered functional mosaicism through their study of RNA interference (RNAi) in the C. elegans model. RNAi is a key biological process for gene silencing that helps cells to fight infection. In the study, Jose and his team were investigating how RNA moves from one tissue type to another to turn off genes. RNA usually occurs in single strands, except in some viruses, which introduce double-stranded RNA (dsRNA) into the cells they are infecting. However, cells can recognize this double-stranded RNA and turn off genes with matching DNA sequences, through the RNAi interference process.

RNAi utilizes enzymes called RNA-dependent RNA polymerases (RdRp), which replicate strands of RNA that can find and silence their matching genes. Many different RdRp enzymes exist, but researchers assumed the different types were tissue specific, and that all cells from the same tissue used the same RdRp enzyme to carry out RNAi. In C. elegans, for example, the authors explained, “amplification occurs through the production of small RNAs by two RNA-dependent RNA polymerases (dRPs) that are thought to be tissue-specific—EGO-1 in the germline, and RRF-1 in somatic cells.”

For their studies Jose and colleagues first engineered nematode worms to fluoresce green. They then genetically modified the worms to make dsRNA in neurons with the sequence matching the gene for green fluorescence. The dsRNA moved throughout the worms’ bodies and turned off the gene for green fluorescence in many cell types, including intestinal cells.

The researchers then deleted the RRF-1 RdRp enzyme that they thought was responsible for enabling RNAi gene silencing in intestinal cells, and switching off the green fluorescence. What they found was that even after RRF-1 deletion only some of the cells fluoresced green. This indicated that something was continuing to silence the fluorescent gene in some of the intestinal cells. The scientists then eliminated, in turn, each of three other RdRp enzymes present in the nematode, to determine which of the enzymes, other than RRF-1, was turning off the green fluorescent gene in the worms’ intestines. Their results found the culprit to be EGO-1, which has traditionally been thought active only in germline cells. Further tests in multiple worms showed that some cells required the RRF-1 RdRp for gene silencing, but others could use EGO-1 as an alternative. Which intestinal cells used EGO-1 appeared to be random. “ … the lineal origins of cells that can use EGO-1 varies,” the authors commented. “This variability could be because random sets of cells can either receive different amounts of dsRNA from the same source or use different RdRPs to perform the same function.”

The team concluded, “Here we reveal that silencing by neuronal dsRNA can differ from silencing by other sources of dsRNA in its requirement for EGO-1 in the absence of RRF-1 … we demonstrate that EGO-1 can compensate for the lack of RRF-1 when dsRNA from neurons is used to silence genes in intestinal cells … We provide a single-cell resolution view of silencing by neuronal dsRNA and find that each animal has a different set of intestinal cells that can rely on EGO-1 for gene silencing.”

” … which enzyme gets used varies from cell to cell, and which cells use which enzyme varies from worm to worm,” Jose said. “You can’t tell from looking at them which cells use the alternative molecule, and from the cell’s perspective, it doesn’t matter which molecule it uses.” He suggests that functional mosaicism could explain why current drugs may not be able to completely eradicate bacterial infections or cancer cells. Some cancer cells or bacteria might persist by using a different mechanism to the one targeted by therapy, and so lead to relapse or antibiotic resistance. “We speculate that functional mosaicism could contribute to the escape from targeted therapies …” the authors wrote.

“It may not be that these diseases are developing some new mechanism to survive,” Jose noted. “It may be that they already had more than one way of doing the same thing, and the alternates step in to replace whatever the therapeutic treatments knock out.”

Cellular processes are commonly examined at the level of body tissues, with the assumption that a process will be carried out in the same way in all cells of the same type. According to Jose, scientists should now consider the possibility of functional mosaicism, and so may need to carry out far more detailed examination of processes at the single-cell level both before and after treatment.

Although the investigators have not yet looked for functional mosaicism in other cellular processes, the new findings represent a previously unknown and unexpected level of variability that, according to Jose, is likely to exist in many other cellular processes. The researchers suggest that functional mosaicism may also provide new insights into the evolution of processes that develop using different mechanisms. It could, effectively, “… allow developmental systems to drift over evolutionary time,” the investigators stated.

“Imagine starting off with two equally good ways of doing one thing, and as populations separate and begin to speciate, a mutation could cause one population to get stuck doing that thing one way, and the other population could get stuck doing it the alternative way,” Jose said. “You could end up with two completely different ways of doing the same thing.”

For example, vision exists throughout the animal kingdom, but eyes have developed differently among animal groups. Functional mosaicism in a biological process responsible for sensing light in an early ancestor could have led to the divergence in visual systems that exist among the different species today. “Now that we know about functional mosaicism, we can go looking for it,” Jose said. “It doesn’t exist in every process to be sure, but it is something we have to be aware of.”

 

 

 

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