The RNA splicing factor Prp45 plays an evolutionarily conserved role in histone H2B ubiquitination

This study reveals that the C-terminus of the RNA splicing factor Prp45 (SKIP) plays an evolutionarily conserved, extra-spliceosomal role in stabilizing the scaffold protein Lge1 to facilitate histone H2B ubiquitination and maintain proper cellular morphology.

The Gist

The Big Picture: A "Swiss Army Knife" Protein with a Hidden Tool

Imagine the cell as a bustling, high-tech factory. Inside this factory, there are millions of instructions (DNA) that need to be turned into working products (proteins). To do this, the factory has a massive assembly line.

One of the most important workers on this line is a protein called Prp45. For a long time, scientists thought Prp45 was like a specialized mechanic whose only job was to fix a specific part of the instruction manual called "splicing" (cutting out the junk and stitching the good parts together).

However, this new study discovered that Prp45 has a secret second job. It's not just a mechanic; it's also a construction foreman that helps manage the factory's "security badges" (histone modifications) to make sure the assembly line runs smoothly.

The Mystery of the "Fluffy Tail"

Prp45 is a long protein. The front part is rigid and structured (like a solid metal tool), but the back part is a long, floppy, disordered tail. Scientists have known about this tail for years, but they didn't know what it did. It was like having a car with a giant, floppy antenna that everyone ignored because it didn't seem to connect to the radio.

The researchers asked: What happens if we cut off this floppy tail?

The Discovery: The Tail Holds the Factory Together

When the scientists cut off the tail (the C-terminus) of Prp45, two major things happened:

1. The Factory Slowed Down: The cells grew very slowly and became huge and misshapen (like a balloon that was over-inflated).
2. The Security System Broke: The factory lost its ability to put "Active Work" badges on its instruction manuals.

In scientific terms, the "badges" are called H2B ubiquitination. Think of these badges as a green sticker that says, "This section is open for business! Start building!" Without these stickers, the factory gets confused, stops working efficiently, and the workers (cells) get stressed and grow too big.

The Mechanism: The Tail is a "Velcro Strap"

How does a floppy tail put stickers on a factory manual? The study found the answer: Stability.

The floppy tail of Prp45 acts like a Velcro strap or a safety tether.

• There is another protein in the factory called Lge1. Lge1 is the "glue" that holds the sticker-painting machine (Bre1) in place so it can do its job.
• Lge1 is naturally wobbly and unstable. It needs to be held tight.
• Prp45's tail grabs onto Lge1 and holds it steady.

The Analogy: Imagine Lge1 is a wobbly ladder. The sticker-painting machine (Bre1) is a painter trying to climb it. If the ladder isn't held steady, the painter falls off, and the wall never gets painted. Prp45's tail is the person holding the ladder steady. When the researchers cut off the tail, the ladder (Lge1) became unstable, the painter (Bre1) fell off, and the wall (the DNA) never got its "Active Work" stickers.

The "Universal" Tail

Here is the most fascinating part: Evolution.

The researchers tested if this "Velcro tail" works in other species. They took the tail from Humans and Plants (which are very different from yeast) and attached them to the yeast's broken protein.

• Result: It worked! The human and plant tails could hold the yeast ladder steady, put the stickers back on, and fix the cell's shape.

This means that for hundreds of millions of years, from plants to humans, this specific part of the protein has been doing the exact same job: holding the "sticker machine" steady to keep our genes working correctly.

Why Does This Matter?

This discovery changes how we see the cell's operations:

1. Two Jobs, One Protein: It proves that a protein known for "splicing" (fixing instructions) also directly controls "chromatin modification" (managing the factory floor). These two processes are more connected than we thought.
2. The "Tail" is Vital: We often think of floppy, disordered parts of proteins as useless junk. This study shows they are actually critical structural supports that keep the whole system from falling apart.
3. Cell Size Control: The study explains why cells get huge when this system breaks. Without the "Active Work" stickers, the cell cycle gets confused, and the cell keeps growing without dividing.

Summary in a Nutshell

Think of Prp45 as a construction worker.

• The Head: Fixes the blueprints (Splicing).
• The Tail: Holds the ladder steady for the painter (Lge1/Bre1).

If you cut off the tail, the ladder falls, the painter can't paint the "Open for Business" signs, and the whole construction site (the cell) gets chaotic, grows too big, and stops working efficiently. And the best part? This specific tail design has been the same in plants, humans, and yeast for eons because it works perfectly.

Technical Summary

1. Problem Statement

The RNA splicing factor Prp45 (known as SKIP in mammals) is a well-characterized component of the Prp19 complex (NTC) within the spliceosome, essential for pre-mRNA splicing. While Prp45/SKIP has been implicated in transcription elongation in metazoans, the mechanism by which the yeast protein functions outside of splicing remains unclear. Specifically, the C-terminal region of Prp45 is intrinsically disordered (IDR) and contains a conserved helical domain, yet its specific biological function beyond splicing fidelity has been enigmatic. The authors sought to determine if this C-terminal region mediates an extra-spliceosomal role, potentially linking splicing machinery to chromatin modification.

2. Methodology

The study employed a multidisciplinary approach combining genetics, proteomics, biochemistry, and cell biology in Saccharomyces cerevisiae:

• Genetic Truncation & Phenotyping: Generation of various C-terminal truncation mutants of Prp45 (e.g., Prp45ΔCTR, Prp45(1-169), Prp45(1-260)) to assess temperature-sensitive growth defects and cell morphology.
• Proximity Labeling (TurboID): Fusion of biotin ligase (BirA/TurboID) to the C-terminus of full-length Prp45 and the truncation mutant (Prp45ΔCTR) to identify C-terminus-dependent protein interactors via mass spectrometry.
• Genetic Interaction Analysis: Construction of double mutants combining prp45ΔCTR with deletions of H2B ubiquitination machinery (BRE1, RAD6, LGE1) and deubiquitinases (UBP8, SGF73) to assess synthetic lethality and suppression.
• Biochemical Assays:
  • Co-Immunoprecipitation (Co-IP): To verify physical interactions between Prp45 and H2B ubiquitination components.
  • Immunoblotting: To quantify levels of H2B monoubiquitination (H2BK123ub), protein stability (Prp45, Lge1), and RNA levels.
  • Auxin-Inducible Degron (AID) System: Used to acutely deplete Prp45 or Lge1 to observe immediate effects on protein stability and H2B ubiquitination.
• Microscopy: Confocal microscopy to visualize Lge1 nuclear puncta formation and Bre1 subcellular distribution (nuclear vs. cytoplasmic).
• Evolutionary Rescue Experiments: Expression of C-terminal domains from Arabidopsis thaliana (At SKIP) and Homo sapiens (Hs SKIP) in yeast prp45ΔCTR strains, including the creation of chimeric proteins (Sc N-terminus + At/Hs C-terminus).
• In Silico Modeling: AlphaFold-Multimer used to predict interaction interfaces between Prp45 and Lge1.

3. Key Contributions

• Discovery of a Non-Splicing Function: The paper identifies a novel, evolutionarily conserved role for the Prp45/SKIP C-terminal domain in regulating histone H2B monoubiquitination, a critical mark of active transcription elongation.
• Mechanism of Lge1 Stabilization: The study elucidates that the Prp45 C-terminus physically interacts with Lge1 (the scaffold for the Bre1 ubiquitin ligase complex) and is essential for stabilizing Lge1 protein levels.
• Cross-Species Conservation: Demonstrates that the C-terminal IDR/helical domain of Prp45/SKIP is functionally conserved from yeast to plants and humans, capable of rescuing yeast phenotypes when fused to the yeast N-terminus.
• Link to Cell Morphology: Connects the loss of H2B ubiquitination due to Prp45 truncation to a specific "Large" (Lge1-like) cell morphology phenotype, suggesting a role in cell size homeostasis.

4. Key Results

• C-Terminal Essentiality: Truncation of the Prp45 C-terminus (Prp45ΔCTR) causes severe growth defects at 37°C and a "Large" cell phenotype, similar to lge1Δ or bre1Δ mutants.
• Proteomic Interactions: Proximity labeling revealed that the Prp45 C-terminus is required for interactions with the H2B ubiquitination machinery, specifically Lge1 and Bre1. These interactions are lost upon C-terminal truncation.
• Lge1 Destabilization: In prp45ΔCTR cells, Lge1 protein levels are significantly reduced (approx. 90% loss) despite unchanged LGE1 mRNA levels, indicating post-translational destabilization. This leads to the dissolution of Lge1 nuclear condensates (puncta) and the mislocalization of Bre1 from the nucleus to the cytoplasm.
• H2B Ubiquitination Defect: prp45ΔCTR cells exhibit a ~90% reduction in H2BK123 monoubiquitination.
• Genetic Suppression: The growth defect and H2B ubiquitination loss in prp45ΔCTR are partially rescued by deleting UBP8 (a deubiquitinase), confirming that the phenotype is driven by reduced H2B ubiquitination.
• Trans-Complementation: Expressing the Prp45 C-terminal domain (residues 170-379) in trans in prp45ΔCTR cells restores Lge1 stability, H2B ubiquitination, and cell morphology.
• Evolutionary Conservation:
  • The C-terminal domain of Arabidopsis SKIP and Human SKIP cannot rescue prp45ΔCTR alone but can fully rescue the phenotype when fused to the yeast Prp45 N-terminus (chimera).
  • This confirms that the C-terminal domain's function in H2B ubiquitination is evolutionarily conserved, despite low sequence identity, provided it is presented in the correct structural context (likely via dimerization or interaction with the N-terminus).
• Synthetic Lethality: prp45ΔCTR shows synthetic lethality with bre1Δ and lge1Δ, suggesting the C-terminus has additional roles (likely in splicing) that become critical when H2B ubiquitination is already compromised.

5. Significance

• Bridging Splicing and Chromatin: This work provides a direct mechanistic link between the spliceosome and chromatin modification. It demonstrates that a splicing factor (Prp45) directly regulates the stability of a chromatin modifier (Lge1), thereby influencing the epigenetic landscape (H2B ubiquitination) required for transcription elongation.
• Role of Intrinsically Disordered Regions (IDRs): The study highlights the functional importance of the disordered C-terminal domain of Prp45/SKIP. Rather than being a passive linker, this IDR acts as a critical scaffold for protein-protein interactions that stabilize essential complexes (Lge1-Bre1).
• Evolutionary Insight: The ability of plant and human SKIP C-termini to functionally replace the yeast domain suggests that the mechanism of coupling splicing factors to H2B ubiquitination via the C-terminal IDR is an ancient, conserved regulatory node.
• Cell Cycle Implications: The restoration of cell size homeostasis upon rescuing H2B ubiquitination suggests that Prp45-mediated chromatin modification is vital for proper cell cycle progression and size control, potentially explaining the "Large" phenotype observed in splicing mutants.

In conclusion, the authors redefine Prp45/SKIP not just as a splicing factor, but as a critical regulator of the H2B ubiquitination machinery, acting through its C-terminal domain to stabilize the Lge1 scaffold and ensure proper chromatin dynamics during transcription.