Last October, leaders in the RNA field, post-docs, and students came together at EMBL Heidelberg with the aim of disseminating and discussing the most recent developments in the field of RNA. The programme of the long-standing EMBO | EMBL Symposium ‘The complex life of RNA’ included topics on transcription, RNA processing and modification, mRNA export and localisation, mRNA surveillance and decay, translation, and more.

During the conference, 245 posters were displayed along the Advanced Training Centre’s helices, enabling participants to showcase their research at the multiple poster sessions. Four winners were chosen by popular vote, and we’re excited to present their work to you today. Congratulations to Nurseda, Joe, Matina-Jasemi, and Marcos!

RNAylated proteins as a platform to develop next-generation RNA therapeutics

Presenter: Nurseda Yilmaz Demirel

Authors: Nurseda Yilmaz Demirel, Katharina Höfer

Abstract:

RNA therapeutics hold significant promise as they offer a new way to treat or prevent diseases such as cancer, genetic disorders, and viral infections, as highlighted by the success of mRNA-based COVID-19 vaccines. However, several limitations need to be addressed to utilize their complete potential, including the instability of exogenous RNA. Eukaryotic cells can distinguish between self and non-self RNA effectively. Therefore, when exogenous RNA is delivered to the eukaryotic systems, it can activate various pathways resulting in RNA degradation and mRNA translation inhibition. Enhancing the stability of RNA can be achieved through the strategic introduction of RNA modifications. Recently, a novel RNA modification termed “RNAylation”, discovered by the Höfer lab, has a high potential to be utilized for increasing RNA stability. In this reaction, RNA is covalently attached to a target protein in a single-step enzymatic reaction, catalyzed by the bacteriophage T4 ADP-ribosyltransferase ModB. 

We have shown that this novel conjugation strategy improves RNA stability by protecting RNA from nuclease degradation. Furthermore, we have created a modular synthetic tool utilizing RNAylation to generate RNA-protein conjugates in vitro. This technology enables the exchange of RNA of interest and protein components depending on the application purpose, unlocking the potential to control the localization and activity of RNA-protein conjugates. Additionally, an efficientpurification strategy, which is needed for the therapeutic application of RNAylated proteins, has been developed to obtain pure RNA-protein conjugates. Moreover, we successfully delivered RNAylated proteins to eukaryotic cells as a proof of concept. Therefore, in this study, we set the foundation to generate a novel, flexible, and applicable platform to produce RNA-protein conjugates in vitro that can be applied to eukaryotic systems in vivo as therapeutics. 

Due to the confidentiality of the unpublished data, we cannot share the poster.

The visual proteomics of gene expression in a minimal bacterium

Presenter: Joe Dobbs

Authors: Joe Dobbs, Rasmus Jensen, Liang Xue, Julia Mahamid

Abstract:

Recent advances in cryogenic electron tomography (cryo-ET) are now allowing researchers to structurally characterize macromolecular complexes in situ at increasingly high resolution, investigate interaction networks and protein communities, and derive functional insight from dynamic and heterogenous assemblies unsuitable for traditional structural methods. Here, we present our investigation into the molecular sociology of gene expression, focusing on the interaction of transcriptional and translational machineries in the minimal prokaryote Mycoplasma pneumoniae. M. pneumoniae has been used recently by our group in studies of the ribosome, providing the first detailed snapshots of translation elongation inside the cell, and our new analysis is expanded to encompass the entire cycle of prokaryotic translation, visualizing and quantifying translation initiation, elongation, and recycling complexes. Our findings implicate noncanonical translation initiation. Traditionally, the Shine-Dalgarno (SD) sequence, a common motif in prokaryotic mRNAs, has been thought to be the primary driver of mRNA delivery to the bacterial ribosome, but bioinformatics analyses have demonstrated that across all prokaryotic genomes only a minority of open reading frames (ORFs) are preceded by this sequence element. M. pneumoniae, in particular, has a low percentage  (8.1%) of SD-sequence ORFs, and most of these occur in the coding region: we show that the bacterium has a high frequency of transcription-translation coupling during translation initiation (in addition to elongation), and that the interaction between RNA polymerase and the ribosome occurs differently in cells of different sizes. Furthermore, we suggest, with antibiotic perturbations and structural evidence, that some of the mechanisms involved are substantially different from those typically understood to be occurring in prokaryotes.

Due to the confidentiality of the unpublished data, we cannot share the poster.

Structural basis of mRNA decay by the human exosome-ribosome supercomplex

Presenter: Matina-Jasemi Loukeri

Authors: Alexander Kögel, Achim Keidel, Matina-Jasemi Loukeri, Christopher Kuhn, Lukas Langer, Ingmar Schäfer, Elena Conti

Abstract:

The interplay between translation and mRNA decay is widespread in human cells and epitomized by the role of the RNA-degrading exosome in mRNA quality-control pathways linked to translating ribosomes. The cytoplasmic exosome consists of an exoribonuclease core complex and an associated helicase complex, SKI238, which interacts with the ribosome 40S subunit. SKI238 can extract 80S-bound mRNA and is expected to transfer it to the exoribonuclease core via a bridging factor, HBS1L3/SKI7. Using biochemical and structural approaches, we uncovered how the human exosome is recruited by HBS1L3/SKI7 to a ribosome-bound SKI238 complex. Capturing the structure during active decay revealed a continuous path whereby the 80S-bound RNA substrate threads from the ribosome through the SKI2 helicase into the exoribonuclease active site of the cytoplasmic exosome. In this active ‘open’ state of the helicase complex, the so-called gatekeeping module of SKI238 engages the ribosome 40S subunit at a position that has interesting implications in the context of ribosome stalling, a functionally relevant scenario considering that SKI2 has been found enriched on transcripts upon ribosome stalling events in cells. Indeed, our biochemical analyses confirmed that the gatekeeping module can bind collided disomes. The cytoplasmic exosome and ribosome thus appear to work together as a single structural and functional unit in co-translational mRNA decay, coordinating their activities in a transient supercomplex.

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Programmable RNA writing with trans-splicing

Presenter: Marcos Manero Carranza

Authors: Cian Franz Schmitt-Ulms, Cian Franz Schmitt-Ulms, Wenyuan Zhou, Alisan Kayabolen, Keira Donnelly, Marcos Manero Carranza, Sabrina Pia Nuccio, Kazuki Kato, Hiroshi Nishimasu, Jonathan Gootenberg, Omar Abudayyeh

Abstract:

The repurposing of CRISPR effectors has allowed for the development of a toolbox capable of performing programmable and efficient genetic interventions for a wide range of purposes. Most of these tools directly modify the DNA sequence or alter gene expression level, leaving RNA modification comparatively unexplored. However, RNA editing offers the opportunity to introduce stable or transient modifications without the risk of permanent off-target effects. This could be applied for sensing, labelling, or repairing of RNA transcripts, but installation of arbitrary edits into the transcriptome is currently infeasible. Trans-splicing-based RNA editing technologies can install diverse edits by introducing exogenous templates via competition with the endogenous exon in the pre-spliced mRNA. However, currently they suffer from low efficiency, even after extensive screening, which hinders their applicability. Here, we describe Programmable RNA Editing & Cleavage for Insertion, Substitution, and Erasure (PRECISE), a versatile RNA editing method for writing RNA of arbitrary length and sequence into existing pre-mRNAs via 5′ or 3′ trans-splicing. We demonstrate PRECISE editing across 11 distinct endogenous transcripts of widely varying expression levels, showcasing more than 50 types of edits, including all possible transversions and transitions and a wide range of insertions and deletions. We show high efficiency replacement of MECP2 last exon, addressing most mutations that drive the Rett Syndrome and replacement of exon 1 of HTT, removing the hallmark repeat expansions of Huntington‘s disease. Furthermore, we combine payload engineering and ribozymes for protein-free, high-efficiency trans-splicing in editing HTT exon 1 via AAV delivery and show that PRECISE achieves editing in in non-dividing neurons and patient-derived Huntington′s disease fibroblasts. Ultimately, our results provide the scientific community with a novel tool that broadens the scope of RNA editing, is straightforward to deliver, lacks permanent off-targets, and can enable any type of edit, including those not otherwise possible with current RNA editors.

Due to the confidentiality of the unpublished data, we cannot share the poster.

The EMBO | EMBL Symposium ‘The complex life of RNA‘ took place from 15 – 18 October 2024 at EMBL Heidelberg and virtually. Want to join the next edition? Mark your calendars for 13 – 16 October 2026!