Biology subjects

Leppek, K.

Publications and source records attributed to Leppek, K..

4 recordsLinked to original sources

bioRxiv2026

Eukaryotic tRNA ligases mediate RNA break repair

RNA is continuously exposed to damage during physiological metabolism and stress, yet cellular responses to RNA damage remain less understood than DNA repair pathways. RNA strand breaks are particularly deleterious because they generate chemically incompatible RNA ends. Eukaryotic tRNA ligases have been implicated in RNA processing and repair, but whether they function as general RNA repair enzymes remains unresolved. Here, we show that the evolutionarily divergent tRNA ligases, human RTCB and fungal Trl1, mediate RNA break repair (RBR) targeting ribosomes and other ribonucleoprotein (RNP) complexes. Using direct RNA nanopore sequencing, we map these repair events at nucleotide resolution, demonstrating that tRNA ligases repair breaks in ribosomal RNA and restore translational activity of repaired ribosomes. We further identify repair across additional structured cellular RNAs. We show that loss of RBR activity leads to RNA fragmentation in human cells and impairs cell viability upon oxidative stress. Together, these findings uncover a broader role for eukaryotic tRNA ligases in repairing RNA breaks and maintaining transcriptome integrity.

biochemistry↗

bioRxiv2026

Synthetic circRNAs employ IRES activity for translation in cells and in cell-free translation systems

Gene regulation through translation is critical for spatiotemporal protein expression. Internal ribosomal entry sites (IRESes) mediate mRNA-specific translation by recruiting ribosomes to 5' untranslated regions. Circular RNAs (circRNAs), naturally occurring and stable RNAs, are increasingly used as synthetic tools for sustained therapeutic protein translation by IRES-driven initiation. However, the functionality of different IRESes in synthetic circRNAs remains sparsely characterized. We systematically examine circRNA reporter translation by viral and cellular IRESes in human cells and in diverse in vitro translation systems. Improved circRNA purification by urea-PAGE and RNase R-treatment removes contaminants that induce RNA sensing. Viral CVB3 and HCV, as well as cellular Hoxa9, Chrdl1, Cofilin and c-Myc IRESes, effectively drive circRNA translation. We also establish circRNA translation in a recently developed human cell-free extract that recapitulates IRES-dependent regulation, and allows for precise engineering of HCV IRES-mediated translation. These findings inform IRES selection for synthetic circRNA translation relevant for circRNA-based medicine. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/715045v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1a60d08org.highwire.dtl.DTLVardef@a1a7d6org.highwire.dtl.DTLVardef@112042aorg.highwire.dtl.DTLVardef@1cf6c34_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop a new RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that "superfolder" mRNAs can be designed to improve both stability and expression that are further enhanced through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

molecular biology↗

bioRxiv2020

VELCRO-IP RNA-seq explores ribosome expansion segment function in translation genome-wide

SUMMARYRoles for ribosomal RNA (rRNA) in gene regulation remain largely unexplored. With hundreds of rDNA units scattered across multiple chromosomal loci, it is not possible to genetically modify rRNA in mammalian cells, hindering understanding of ribosome function. Emerging evidence suggests that expansion segments (ESs), tentacle-like rRNA extensions that vary in sequence and size across eukaryotic evolution, may provide platforms for the binding of proteins and mRNAs. Here, we develop VELCRO-IP RNA-seq: a versatile methodology to generate species-adapted ESs and map specific mRNA regions across the transcriptome that preferentially associate with ESs. By applying VELCRO-IP RNA-seq to a mammalian ES, ES9S, we identified a large array of mRNAs that are selectively recruited to ribosomes via an ES. We further characterize a set of specific 5’ UTRs that facilitate cap-independent translation through ES9S-mediated ribosome recruitment. These data provide a novel technology for studying the enigmatic ESs of the ribosome in gene-specific translation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗