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Ribosome assembly involves essential RNA modifications. This study used direct RNA sequencing to analyze modifications in Escherichia coli, revealing altered pseudouridine incorporation in specific assembly pathways, indicating a new regulatory layer in ribosome biogenesis.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Ribosome biogenesis is a complex, multi-step process involving ribosomal RNA (rRNA) modification and processing.
  • Ribosomal RNA modifications are crucial for ribosome assembly and function, but their dynamic regulation during assembly is not fully understood.

Purpose of the Study:

  • To investigate the impact of DbpA helicase activity on 23S rRNA modification and 3'-end processing during ribosome assembly.
  • To analyze rRNA modifications in mature 50S large subunits (LSU) and distinct LSU assembly intermediates.

Main Methods:

  • Utilized Oxford Nanopore direct RNA sequencing for simultaneous detection and quantification of 23S rRNA modifications.
  • Analyzed 3'-end processing of 23S and 5S rRNAs in wild-type and mutant (R331A DbpA) Escherichia coli cells.
  • Compared rRNA modification patterns in mature 50S LSU and 35S/45S assembly intermediates.

Main Results:

  • Most 23S rRNA modifications were incorporated similarly in intermediates and mature subunits, irrespective of DbpA activity.
  • N2-methyladenosine 2507 and pseudouridine (Ψ) 2508 showed altered incorporation in the 50S LSU from R331A DbpA expressing cells.
  • Ψ2608 incorporation was reduced in the 50S subunit from R331A DbpA cells compared to intermediates and wild-type 50S subunits, indicating pathway-specific regulation.

Conclusions:

  • DbpA helicase activity influences specific 23S rRNA modifications during ribosome assembly.
  • Pseudouridine 2608 incorporation is selectively reprogrammed across alternative in vivo ribosome assembly routes.
  • These findings reveal an additional regulatory layer in ribosome biogenesis controlled by rRNA modification dynamics.