Permuted 23S rRNA is integrated in 50S ribosome particles in Thermococcus barophilus

This study reveals that in *Thermococcus barophilus*, the 23S rRNA undergoes a unique circular permutation resulting from the deletion of helix H98, yet this altered form is successfully integrated into functional 50S ribosomal subunits and 70S monosomes.

The Gist

The Big Picture: A Factory Assembly Line

Imagine a cell as a busy factory. Inside this factory, there are tiny machines called ribosomes that build proteins (the workers that do the actual jobs). To build these machines, the factory needs blueprints made of RNA.

Usually, these blueprints come in long, messy rolls of tape (called precursors) that need to be cut, trimmed, and glued together to make the perfect, final blueprint.

This paper is about a strange discovery in a tiny, heat-loving microbe called Thermococcus barophilus. The scientists found that this microbe doesn't just cut and trim its blueprints; it actually rewinds the tape and cuts out a middle section before assembling the machine. It's like taking a movie, cutting out the middle scene, and then splicing the end of the movie to the beginning so the story starts with the climax.

The Story in Three Acts

1. The "Circular" Mystery

In many ancient microbes (Archaea), the factory makes a special kind of intermediate blueprint. Instead of a straight line, the tape is glued into a circle. Think of it like a rubber band.

• The Standard Process: The factory has a specific pair of scissors (an enzyme called EndA) that recognizes a specific knot in the rubber band (called a BHB motif). It cuts the knot, and then a glue gun (an enzyme called RtcB) snaps the ends together to make a perfect circle.
• The Discovery: The scientists looked at the blueprints of Thermococcus barophilus and found these circular rubber bands, just as expected. But they also found something weird: some of these circles had been cut and re-glued in a different way, creating "alternative" circles.

2. The Great "Permutation" (The Magic Trick)

Here is the main surprise. When the factory turns these circular rubber bands back into straight lines to build the ribosome, something strange happens to the 23S rRNA (the large part of the machine).

• The Expectation: You would expect the machine to start at the very beginning of the tape and end at the very end.
• The Reality: The tape is permuted. Imagine a scroll of a story.
  • Normal: Start at Page 1, read to Page 100.
  • Permuted: The factory cuts out Page 50 (a specific section called Helix H98) and throws it away. Then, it takes the end of the story (Pages 90–100) and glues it to the front. Now, the story starts with the ending and ends with the middle.

The scientists proved this by looking at the actual "ends" of the RNA. The 23S RNA in this microbe starts with a section (Helix H99) that usually sits in the middle of the molecule. It's as if the ribosome was built with the engine block attached to the roof of the car instead of the bottom.

3. Does the Car Still Drive?

The big question was: "If you rearrange the blueprint and delete a piece, does the machine still work?"

• The Test: The scientists separated the ribosomes from the rest of the cell. They checked if these "permuted" blueprints were actually inside the working machines.
• The Result: Yes! The permuted 23S RNA is fully integrated into the 50S subunit (the large half of the ribosome) and the complete 70S machine. The machine is functional, even though it's missing a piece (Helix H98) and starts in the middle of the story.

Why is this important? (The "So What?")

1. It's a Unique Trick: This "circular permutation" (swapping the start and end) was seen in one other microbe (Pyrococcus furiosus), but not in others. It seems to be a special trick used by this specific family of heat-loving microbes.
2. Deleting the "Extra" Part: The piece that gets deleted (Helix H98) is like an "expansion segment." In humans and other complex life, this part is huge and important. But in these simple microbes, it seems useless. By cutting it out and rearranging the rest, the microbe creates a more compact, efficient machine.
3. The "Glue" Stays: Interestingly, the spot where the circle was glued together (the BHB junction) stays in the final machine. It's like leaving a visible patch of tape on a finished sculpture. The scientists modeled this and found that this "patch" fits perfectly into a gap in the 3D structure of the ribosome, suggesting the cell knows exactly how to handle it.

Summary Analogy

Imagine you are building a LEGO castle.

• Normal way: You follow the instructions from step 1 to step 100.
• Thermococcus way: The instructions tell you to take the instructions for steps 50–60 and throw them in the trash. Then, it tells you to take the instructions for steps 80–100 and paste them to the front of the book. You build the castle starting with the roof, then the walls, and finish with the foundation.
• The Result: The castle stands perfectly fine, and it actually looks better because you got rid of the useless steps (steps 50–60) that didn't fit the design.

In short: This paper reveals that nature is more creative than we thought. Even in the microscopic world, organisms can rearrange their genetic blueprints, delete unnecessary parts, and still build perfectly functional machines.

Technical Summary

1. Problem Statement

Ribosome biogenesis in Archaea involves the processing of precursor rRNAs (pre-rRNAs) into mature functional molecules. A conserved mechanism in many archaea involves the formation of circular intermediates (circ-pre-rRNAs) via the recognition and cleavage of a Bulge-Helix-Bulge (BHB) motif by the endonuclease EndA, followed by ligation. While this circularization is well-documented, the subsequent maturation steps to generate linear, functional rRNAs are poorly characterized. Specifically, it was unclear whether the circular intermediates in Thermococcus barophilus (a member of the Thermococcales order) are simply precursors that are fully processed back to canonical linear forms, or if they result in unique structural variations. Previous studies in Pyrococcus furiosus suggested a circular permutation of the 23S rRNA, but this was not observed in other archaeal species like Haloferax volcanii, leaving the prevalence and functional integration of such permutations in question.

2. Methodology

The authors employed a multi-faceted approach combining bioinformatics, molecular biology, and structural modeling:

• Genomic Analysis: Analyzed 34 complete reference genomes of the Thermococcales order to map rDNA operon organization and identify conserved non-coding RNAs (ncRNAs) and BHB motifs.
• Transcriptomics (RNA-seq): Performed deep Illumina short-read sequencing on total RNA from wild-type T. barophilus cells in both exponential and stationary growth phases.
  • Used STAR aligner with chimeric alignment settings to detect circularization junctions (reads spanning two non-contiguous genomic regions).
• Molecular Validation:
  • Primer Extension: Mapped the 5' and 3' ends of 16S and 23S rRNAs using radio-labeled primers on total RNA and ribosome-enriched fractions.
  • RACE (Rapid Amplification of cDNA Ends): Performed 5' and 3' RACE to precisely map the nucleotide boundaries of the mature rRNAs.
  • PCR: Amplified circular junctions from cDNA to confirm their existence.
• Ribosome Fractionation: Used Ribo Mega-SEC (Size Exclusion Chromatography) to separate ribosomal subunits (30S, 50S) and monosomes (70S) from whole-cell extracts. Western blotting with antibodies against ribosomal proteins (S4e, L10e) confirmed the presence of ribosomal particles in specific fractions.
• Structural Modeling: Mapped the identified permuted 23S rRNA sequence onto the cryo-EM structure of the Thermococcus kodakarensis 50S subunit (PDB 6SKF) using Coot and ChimeraX to verify structural compatibility.

3. Key Contributions and Results

A. Identification of Circular Junctions and Alternative Splicing

• Canonical Junctions: Confirmed the presence of canonical 16S and 23S circ-pre-rRNA junctions at predicted BHB motifs.
• Abundance Discrepancy: The 23S canonical junction reads were 29 times more abundant than 16S junction reads, despite being transcribed from the same operon.
• Alternative Junctions: Identified three novel, abundant alternative 23S circular junctions (A, B, C) located within the terminal helix (H1) of the 23S rRNA. These introduce variability at the 3' end of the mature rRNA.

B. Discovery of Circularly Permuted 23S rRNA

• Primer Extension & RACE Findings:
  • 16S rRNA: Confirmed to have standard 5' and 3' ends as annotated in the genome.
  • 23S rRNA: The major form is circularly permuted.
    • The 5' end is located at nucleotide 2926 (end of helix H98), meaning the sequence corresponding to helices H99–H101 is now at the 5' terminus.
    • The 3' end is located at nucleotide 2888 (start of helix H98).
    • Consequence: This permutation results in the deletion of helix H98 from the mature linear molecule.
    • Retention of Junction: The mature permuted rRNA retains the 44-nucleotide sequence corresponding to the BHB circularization junction.

C. Functional Integration into Ribosomes

• Ribosome Association: Primer extension analysis of SEC-fractionated ribosomes (30S, 50S, and 70S) showed that the permuted 23S rRNA is the dominant form incorporated into functional ribosomal particles.
• Structural Validation: Modeling the permuted 23S rRNA (lacking H98 but containing the BHB junction) into the T. kodakarensis 50S cryo-EM map successfully explained previously unassigned density and accounted for the absence of density corresponding to helix H98.

D. Conservation and Evolutionary Context

• H98 Distribution: Helix H98 is present in Thermococcales and Asgard archaea but absent in most other Euryarchaeota, TACK, and DPANN groups.
• Hypothesis: The study suggests that circular permutation in Thermococcales may be a specific mechanism to remove helix H98, which might be non-functional or detrimental in the context of the mature ribosome for these specific organisms.

4. Significance

• Redefining Archaeal Ribosome Maturation: This study provides the first definitive evidence that the mature, functional 23S rRNA in T. barophilus is a circularly permuted molecule integrated into the 50S subunit, challenging the assumption that circ-pre-rRNAs are merely transient intermediates fully processed back to canonical linear forms.
• Mechanism of H98 Deletion: It elucidates a unique maturation pathway where the reopening of the circular precursor occurs at the boundaries of helix H98, effectively excising this expansion segment (homologous to eukaryotic ES39) from the final ribosome.
• Structural Implications: The retention of the BHB junction in the mature rRNA suggests that this sequence does not disrupt ribosome function and may occupy a specific structural niche, as supported by the cryo-EM modeling.
• Methodological Impact: The study highlights the necessity of combining transcriptomic data with rigorous molecular mapping (RACE/Primer Extension) and structural modeling to accurately characterize rRNA maturation, as standard genome annotations often fail to capture these complex permutations.

In conclusion, the paper demonstrates that in Thermococcus barophilus, the 23S rRNA undergoes a circular permutation event that deletes helix H98 and retains the circularization junction, resulting in a functional ribosome subunit that differs significantly from the genomic annotation.