New sequencing methods reveal the RNA secrets of extreme microorganisms

Regensburg researchers decode the step-by-step ribosome biosynthesis in archaea.

Archaea are microscopic life forms that rank among the three main forms of life on our planet, along with bacteria and eukaryotes (including humans and animals). They were only discovered at the end of the last century and are known to survive in extreme living conditions in places that we normally consider hostile to life. Recent studies also point to a close evolutionary history of archaea and eukaryotes, positioning them as our close relatives. However, the biology of archaea and their role in ecological cycles are poorly understood.

For many years, the scientists at the Chair of Microbiology, to which the unique German Archaea Center is affiliated, have been world leaders in researching these unusual organisms. In a study recently published in the journal RNA, the scientists have now used an innovative single-molecule sequencing method called nanopore sequencing to decipher a key biosynthetic pathway essential to the survival of these specialists. The nanopore sequencing technique, which allows a detailed analysis of very long sequence sections of DNA and RNA molecules, has now been developed by Prof. Dr. Dina Grohmann (University of Regensburg) and Dr. Sebastien Ferreira-Cerca, (since February 2023 at École Polytechnique de Paris and previously also at the University of Regensburg), used to study the maturation of ribosomal RNA (rRNA) in three different model archaea. Small enough to fit in a trouser pocket, this technology enables inexpensive experiments to be carried out even outside the laboratory. She also played a crucial role in decoding numerous genomes of the SARS virus.

Dr Felix Grünberger, who specializes in modern high-throughput sequencing techniques and who played a key role in shaping the project with his experimental and bioinformatic expertise, describes this technique as “revolutionary and directly decisive for the new findings of our study”. The Regensburg researchers were among the first laboratories to use the nanopore method in microbiology for sequencing RNA and were awarded the publication prize of the science journal RNA for their methodical developments in 2023.

Now the team has used the technique to study ribosome maturation. The ribosome is a complex macromolecular machine composed of ribosomal RNA and ribosomal proteins that is involved in the decoding of genetic information. As such, the ribosome is a key component of gene expression and cellular life. By examining the ribosomal RNA maturation pathway of three different archaea with different extreme lifestyles, the researchers were able to gain insight into the common and organism-specific steps of rRNA maturation. They were also able to obtain information about the decoration of the RNA molecules with additional chemical side chains, the so-called RNA modifications, which are known to refine the function of the translation machinery.

Exploring rRNA processing and modifications using nanopore-based RNA-seq

(A) Simplified pictogram of rRNA maturation in archaea: The primary transcript containing the 16S (dark gray), internal tRNA (brown; only in Euryarchaeota), and 23S (light gray) is folded at the bulge-helix-bulge (BHB), cleaved at multiple positions (orange triangles), circularized, and bases are modified (pink circles) until the mature rRNA products are formed that ultimately are an integral part of the ribosomal subunits. (B) Nanopore-based library preparation methods for direct sequencing of cDNAs (left, 1) and RNAs (right, 2): For cDNA sequencing, 3′-adapter-ligated RNAs and custom VN-primers were used as input for the DCS109 protocol established by Oxford Nanopore. For RNA sequencing, libraries were prepared from artificially polyadenylated RNAs according to the RNA002 protocol established by Oxford Nanopore. Adapters that carry the motor protein are highlighted with a red pacman symbol. (C) Exemplary direct cDNA coverage profile and (D) single-read plot of the rRNA locus 1 of H. volcanii. Cleavage sites at the bulge-helix-bulge are indicated by orange triangles and dashed vertical lines. (E) RNA base modifications can lead to deviations in the recorded current intensities from the expected signal and/or to systematic base-calling errors, which can be used to detect RNA modifications 

The results of this study demonstrate that ribosomal RNA maturation can be studied more efficiently by providing multidimensional information such as processing and RNA modification events in a single experiment, and therefore can also study organisms that are notoriously difficult to culture under normal laboratory conditions . Due to its simplicity, the developed technology and methodological pipeline could help to explore the biodiversity of (ribosomal) RNA maturation across all domains of life and uncover common features and cellular adaptations of (ribosomal) RNA maturation.

SourceInformationsdienst Wissenschaft

Grünberger F, Jüttner M, Knüppel R, Ferreira-Cerca S, Grohmann D. (2023) Nanopore-based RNA sequencing deciphers the formation, processing, and modification steps of rRNA intermediates in archaea. RNA 29(8):1255-1273. [article]

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