Rei1 and Reh1 facilitate the loading of eL24

This study demonstrates that the yeast proteins Rei1 and Reh1 facilitate the cytoplasmic loading of the ribosomal protein eL24 onto pre-60S subunits, a function revealed through the analysis of double mutants and the identification of bypass suppressors that restore eL24 incorporation.

The Gist

Imagine your cell is a bustling factory, and the most important machines it runs are called ribosomes. These machines are responsible for reading your genetic instructions (DNA) and building the proteins that keep you alive.

To build a ribosome, the factory doesn't just assemble it in one go. It's like building a complex car: you build the chassis in the "design office" (the nucleus), ship it out to the "assembly floor" (the cytoplasm), and then add the final touches, like the wheels and the radio, before it's ready to drive.

This paper is about a specific step in that final assembly process. Here is the story in simple terms:

The Old Story vs. The New Discovery

The Old Story (The Misunderstanding):
Scientists used to think the assembly line worked like this:

1. A specific part, called eL24 (think of it as a "key"), gets installed on the machine.
2. Once the key is in, it acts like a magnet, calling in a helper protein named Rei1 (and its twin, Reh1).
3. Rei1's job was thought to be just to help remove a temporary "placeholder" part (Arx1) so the machine could finish up.

The New Discovery (The Plot Twist):
The researchers in this paper found out the story was actually backwards! They discovered that:

1. Rei1 and Reh1 are the foremen. They arrive first.
2. Their main job isn't just to remove the placeholder; it's to install the key (eL24).
3. Without these foremen, the key (eL24) never gets installed, and the machine (ribosome) is broken and can't work.

The Detective Work: How They Figured It Out

The scientists were puzzled because when they removed both foremen (Rei1 and Reh1) from the yeast factory, the machines stopped working badly. But they didn't know why.

Clue 1: The Missing Part
They looked at the broken machines and realized the eL24 key was missing.

• Analogy: It's like finding a car on the assembly line that has no steering wheel. You can't drive it.

Clue 2: The "More is Better" Test
They wondered, "If we just force more keys (eL24) onto the assembly line, will the machines work again?"

• Result: Yes! When they added extra copies of the eL24 gene, the broken machines started working much better. This proved that the problem wasn't that the key was broken, but that the factory couldn't install it without the foremen.

Clue 3: The "Supervisor" Hunt
To understand how the foremen (Rei1/Reh1) helped install the key, the scientists played a game of "find the bug." They let the broken factory run for a while and waited for random mutations (accidental changes) that made the factory work again.

They found three lucky accidents that fixed the problem:

1. A change in the GTPase (Lsg1): This is another helper protein. The mutation made it let go of the machine slightly earlier.
2. A change in a structural part (uL3): This is a piece of the machine itself. The mutation made a little "pocket" on the machine bigger, making it easier to shove the key in.
3. A change in a chemical switch (Ppq1): This is an enzyme that usually adds "chemical tags" (phosphorylation) to proteins. The mutation stopped it from working, which somehow helped the key get installed.

The Big Picture of the Clues:
All three fixes helped the factory install the missing key (eL24). This confirmed that the foremen (Rei1/Reh1) act as a bridge. They hold the machine in the right position so the key can snap into place. Without them, the key just bounces off.

Why Does This Matter?

1. It changes the rulebook: For years, textbooks said "Key first, then Foreman." This paper says "Foreman first, then Key." Science is all about updating our understanding when new evidence appears.
2. Human Health: Yeast is a simple model for humans. Humans have a version of Rei1 called ZNF622. If our human "foremen" are broken, our ribosomes might not assemble correctly. This could lead to diseases related to protein production.
3. Quality Control: The paper also hints that while Rei1 is a construction worker, its twin Reh1 might also be a "quality control inspector" that checks if the machine is built correctly before letting it leave the factory.

The Takeaway

Think of building a ribosome like assembling a high-tech drone. You might think you need to attach the propeller (eL24) first to attract the technician (Rei1). But this study shows that the technician actually needs to arrive first to hold the drone steady and guide the propeller into place. Without the technician, the propeller just falls off, and the drone never flies.

Technical Summary

1. Problem Statement

Ribosome biogenesis is a highly orchestrated process involving over 200 trans-acting factors. In eukaryotes, the large (60S) ribosomal subunit is largely assembled in the nucleolus but undergoes final maturation in the cytoplasm. While most ribosomal proteins (rproteins) are loaded in the nucleus, a specific subset, including eL24, is loaded in the cytoplasm.

The prevailing model suggested that eL24 loads onto the pre-60S subunit first and subsequently recruits the zinc-finger protein Rei1 (ZNF622 in humans). Rei1 is known to facilitate the release of the assembly factor Arx1. In Saccharomyces cerevisiae, Rei1 has a paralog, Reh1. While the single deletion of REI1 causes a mild growth defect (partially suppressible by ARX1 deletion), the simultaneous deletion of REI1 and REH1 results in a severe growth defect that cannot be suppressed by ARX1 deletion. This suggests that Rei1 and Reh1 possess an additional, redundant function beyond Arx1 release, the nature of which was previously unknown.

2. Methodology

The authors employed a multi-faceted approach combining genetics, biochemistry, and structural biology:

• Mass Spectrometry (MS) of Affinity-Purified Pre-60S: The authors affinity-purified pre-60S subunits from wild-type (WT) and rei1∆ reh1∆ arx1-S347P mutant strains using a TAP-tagged Lsg1 (a GTPase that remains bound during cytoplasmic maturation). They analyzed the protein composition via LC-MS/MS to identify missing components.
• Genetic Suppression Screens:
  • Overexpression: Tested if high-copy expression of RPL24A (encoding eL24) could suppress the growth defect of rei1∆ reh1∆ cells.
  • Spontaneous Suppressor Screen: Screened for spontaneous mutations that restored growth in rei1∆ reh1∆ arx1-S347P cells. Identified suppressors were sequenced via Whole Genome Sequencing (WGS).
• Biochemical Assays:
  • Sucrose Cushion Sedimentation: Used to separate ribosome-bound fractions (pellet) from soluble fractions (supernatant) to assess the loading status of eL24, Rei1, and Lsg1.
  • Polysome Profiling: Analyzed translation efficiency and subunit joining defects.
  • Western Blotting: Used specific antibodies (anti-FLAG, anti-HA, anti-Rpl8, etc.) to quantify protein levels in different fractions.
• Structural Modeling: Utilized AlphaFold3 predictions and existing cryo-EM data to map suppressor mutations (in Lsg1, Rpl3, and Ppq1) onto the 3D structure of the pre-60S particle.
• Mutagenesis: Generated specific point mutations and deletions (e.g., lsg1-∆34, rei1-∆270-342, phosphomimetic/null mutations in Lsg1) to validate the function of identified residues.

3. Key Contributions and Results

A. Redefining the Order of Assembly Events

Contrary to the established model that eL24 recruits Rei1, the authors found that:

• eL24 is deficient in rei1∆ reh1∆ mutants: MS analysis revealed that pre-60S particles from rei1∆ reh1∆ cells were specifically depleted of eL24 (and Ybl028c), while other factors like Nmd3 and Tif6 remained unaffected.
• Rei1 binding is independent of eL24: In rpl24a∆ rpl24b∆ (eL24 null) cells, Rei1 still bound efficiently to pre-60S particles. This proves that eL24 is not required for Rei1 recruitment.
• Rei1/Reh1 are required for eL24 loading: Complementation of rei1∆ reh1∆ with either REI1 or REH1 restored eL24 levels on the ribosome. Furthermore, overexpression of RPL24A partially suppressed the growth defect and restored eL24 loading, confirming that the primary defect in the double mutant is the failure to load eL24.

B. Identification of Extragenic Suppressors

To understand the mechanism of eL24 loading and the additional role of Rei1/Reh1, the authors identified spontaneous suppressors of the rei1∆ reh1∆ growth defect:

1. LSG1 Mutations: Two point mutations (K619Q, H621L) and a C-terminal deletion (∆607-640) in the GTPase Lsg1 suppressed the defect.
   • Mechanism: The C-terminus of Lsg1 is highly basic and interacts with the ribosome. Deletion of this region reduced Lsg1's affinity for the pre-60S. Structural modeling suggests the Lsg1 C-terminus passes beneath Rei1/Reh1 and near the eL24 binding site. The authors propose that Rei1/Reh1 normally position the Lsg1 C-terminus to facilitate eL24 incorporation; removing this interaction (via deletion) bypasses the need for Rei1/Reh1.
2. RPL3 Mutation: A mutation in ribosomal protein uL3 (V57G) suppressed the defect.
   • Mechanism: uL3 V57 is located in a cleft that embraces the N-terminus of eL24. This mutation likely alters the local structure to favor eL24 loading even in the absence of Rei1/Reh1.
3. PPQ1 Mutation: A mutation in the phosphatase Ppq1 (G491C) acted as a suppressor.
   • Mechanism: Ppq1 is a PP1-family phosphatase. The mutation likely reduces its activity. Since Lsg1 is phosphorylated at its C-terminus (by Rim15), the authors hypothesize that Ppq1 may dephosphorylate Lsg1. Loss of Ppq1 function might lead to hyperphosphorylation of Lsg1, weakening its ribosome binding (similar to the lsg1-∆34 deletion), thereby suppressing the defect. However, direct phosphomimetic mutations in Lsg1 did not fully recapitulate the suppression, suggesting a complex regulatory mechanism.

C. Functional Divergence of Rei1 and Reh1

The study clarifies the distinct roles of these paralogs:

• Rei1: Primarily responsible for the release of Arx1.
• Reh1: Acts as a quality control factor for defective ribosomes (specifically those with uL16 mutations) and is the last factor released during early translation.
• Redundant Function: Both Rei1 and Reh1 are required for the efficient loading of eL24.

4. Significance

• Revised Assembly Pathway: The paper fundamentally revises the cytoplasmic maturation pathway of the 60S subunit, establishing that Rei1 and Reh1 facilitate the recruitment of eL24, rather than eL24 recruiting Rei1.
• Mechanistic Insight: It provides a structural and genetic framework for how assembly factors (Rei1/Reh1) coordinate with GTPases (Lsg1) and ribosomal proteins (uL3) to ensure the correct incorporation of late-loading rproteins.
• Human Relevance: Since humans possess only a single homolog, ZNF622 (Rei1), understanding its dual role in assembly (eL24 loading) and potential quality control (analogous to Reh1) is crucial for understanding ribosomopathies and diseases linked to ribosome biogenesis defects.
• Phosphoregulation: The identification of Ppq1 as a potential regulator of Lsg1 ribosome association introduces a new layer of post-translational control in ribosome assembly.

In conclusion, this study demonstrates that Rei1 and Reh1 act as chaperones that position the Lsg1 C-terminus and/or the ribosomal surface to enable the stable loading of eL24, a critical step for the final maturation and functional licensing of the 60S ribosomal subunit.