Ribosome Assembly: Key to Growth and Health

Ribosomes are vital machines found in all living organisms. They play a key role in reading the genetic instructions contained in DNA, which ultimately helps in building cells that have unique identities and functions. Because of this, ribosomes need to be assembled correctly. If there are mistakes in how they are made, it can lead to problems in the cells and eventually cause diseases in the organism. In humans, issues with ribosome assembly can lead to a group of diseases known as ribosomopathies. These can cause various health problems, such as reduced brain size, learning disabilities, neurodegeneration, seizures, different types of cancers, and several other syndromes.

#Early Discoveries in Drosophila

More than a hundred years ago, scientists studying the fruit fly Drosophila discovered how important proper ribosome assembly is. They identified Mutants called 'Minute' mutants, which develop with thin and small bristles. These mutants delay their Development, have irregularly shaped eyes, and often struggle with survival and reproduction. Mutations in these mutants mostly affect ribosomal protein units.

Further studies revealed a fascinating phenomenon called cell competition. This occurs when slow-growing cells, known as "loser" cells, are surrounded by healthy, fast-growing cells, known as "winner" cells. The loser cells undergo programmed cell death (apoptosis) and get eliminated. This process is observed not just in Minute mutants but also in other instances where neighboring cells are genetically different.

Cell competition has been found in mice, zebrafish, and cells in culture, suggesting it could be a common way for tissues to detect and remove cells that are not growing properly.

#Mechanisms Behind Cell Competition

Scientists are still figuring out the exact mechanisms behind cell competition. In Minute mutants, it has been discovered that a new stress response pathway is activated, which involves a protein called RpS12. This protein helps activate a transcription factor known as Xrp1. Xrp1 can work with another protein, Irbp18, to turn on genes related to cell death, DNA repair, and protection against damage.

Xrp1 also encourages the production of a protein called PKR-like endoplasmic reticulum kinase (PERK), which then leads to decreased protein translation in the loser cells, resulting in their eventual death.

#The Role of Dilp8

One gene that is heavily influenced by Xrp1 in Minute cells is called dilp8. This gene produces a factor related to insulin that is sent out from the mutant cells and inhibits another signal that is important for timely development. When there is a problem with either Xrp1 or dilp8, the developmental timing can return to normal, indicating that dilp8 plays a major role in delaying the development in these mutants.

In addition to mutations affecting ribosomal proteins, similar developmental issues are seen in mutations affecting other parts of ribosome production.

#Other Factors Affecting Ribosome Production

In Drosophila, various components within the nucleolus can also lead to reduced growth and developmental issues when they are compromised. For instance, proteins like Nop60b, Nop140, and Noc1 can impact growth negatively. When Noc1 is reduced, it causes an increase in dilp8 levels, similar to what happens in Minute mutants.

Other proteins, such as vertebrate ribosomal protein uS5/RPS2 and its partners, PDCD2 and PDCD2L, are well-studied regulators of ribosome production. In Drosophila, the equivalent gene is string of pearls (sop)/RpS2. Mutations in these proteins lead to the same Minute-like symptoms, suggesting they are critical for proper ribosome biogenesis.

#Ribosome Assembly and Its Consequences

When proteins like uS5/RPS2 are lost, this impacts the formation and movement of ribosomal subunits, which can lead to various developmental defects. Biochemical studies have shown that uS5 can bind to PDCD2 and PDCD2L, which are proteins that seem to have evolved through gene duplication before different life forms diverged.

In mice, losing PDCD2 results in issues with the early development of embryos, while PDCD2L mutants also develop but have lethal outcomes at certain stages. Both PDCD2 and PDCD2L are crucial for creating ribosomal subunits.

In Drosophila, the loss of Zfrp8, which corresponds to PDCD2, leads to several developmental problems, including issues with growth and cellular proliferation. Studies have shown that Zfrp8 interacts with many different proteins, indicating that it has multiple roles beyond just being a chaperone for uS5.

#Investigating Trus Function

In this study, we wanted to better understand the function of Trus, the Drosophila counterpart of PDCD2L. We compared the characteristics of trus mutants with other mutants like Minute and Zfrp8 to see commonalities and differences, which might hint at the specific role of Trus in ribosome assembly or other biological processes.

#Creating trus Mutants

To explore the function of Trus, we created trus mutants using a gene-editing technique known as CRISPR/Cas9. One of the mutations we found, trus1, causes a significant delay in development, which makes affected larvae take up to 10 days to complete their lifespan before reaching the pupal stage. These larvae often do not survive to adulthood.

Another mutation we observed led to different developmental delays and structural defects. When we looked at tissues from the mutants, we found that the brain and wing tissue were significantly smaller compared to normal flies. This suggests that Trus is necessary for proper tissue growth during larval development.

#Investigating the Effects of trus Mutations

To analyze the effects of trus mutations in depth, we dissected the brains and imaginal discs of mutant larvae. We used specific antibodies to mark cells that were actively dividing. The brains of trus mutants appeared smaller and less organized than those of normal larvae. The wing discs were also not properly developed, showing reduced size and abnormal shapes.

As we further studied other similar mutants, we noted that the differences between them might be due to how Trus interacts with various proteins during ribosome assembly.

#Understanding Trus Structure

The structure of Trus and its related proteins is highly conserved across different organisms. By analyzing its protein domains, we found that Trus shares many structural features with PDCD2L and PDCD2.

A predicted 3D model of Trus showed that it has a core structure, which supports the idea that it performs similar functions in ribosome assembly like its vertebrate counterparts. The structure allows Trus to interact with other important proteins involved in ribosome production.

#Expression of Trus in Larval Stages

We investigated where Trus is expressed in Drosophila larvae. High levels of Trus were found in tissues where cells were dividing, such as the brain and imaginal discs, indicating it is crucial in these areas during growth. It was also present in non-dividing tissues, but at much lower levels.

#Localization of Trus Protein

Using antibodies specific for Trus, we found that the protein is mainly located in the cytoplasm of cells. It can move between the nucleus and cytoplasm, which suggests it may have various roles depending on where it is functioning within the cell.

Blocking the movement of Trus from the nucleus resulted in its accumulation there, indicating that the export of Trus from the nucleus is important for its function.

#Developmental Delay Rescue Experiments

To see if we could correct the developmental delays caused by trus mutations, we treated the larvae with ecdysone, the hormone responsible for shedding and metamorphosis in insects. Feeding either the hormone or its precursor resulted in some improvement in development timing, but unfortunately, all of the treated trus mutants still died before reaching adulthood.

#Exploring Tissue Growth and Proliferation Defects

When we looked at tissue growth in wing discs where Trus levels were reduced using specific drivers, we observed that there was a significant decrease in the number of cells actively dividing. This implies that Trus plays a key role in promoting growth during development.

In contrast, there was no increase in cell death (apoptosis) in the trus mutant brains or wings, which suggests that Trus is essential for normal growth but does not prompt excessive cell death when it is missing.

#Identifying Key Pathways Involved

We also studied whether the Xrp1-Dilp8 pathway was affected by the absence of Trus. We knocked down Xrp1 or Dilp8 in trus mutants and found that this significantly reduced the developmental delays, confirming that this specific pathway impacts the growth and timing of development.

#Trus Function in Oogenesis

In addition to its role in general growth and development, we discovered that Trus is also crucial for the formation of ovarian structures in female flies. Even when we managed to rescue some aspects of the trus mutant's lethality through specific expression of Trus, females still ended up being sterile.

Examining the ovaries of rescued females showed that egg formation was severely impaired, leading to a complete absence of mature eggs.

#Conclusion

Through a detailed examination of trus mutants, we have gained important insights into the role of Trus in growth, ribosome biogenesis, and oogenesis in Drosophila. The findings highlight how alterations in the function of ribosome assembly factors can have profound effects on development and tissue growth. The similarities observed between Trus and its vertebrate counterparts suggest that studying these proteins could lead to a better understanding of their functions and implications in various diseases.