Kolberg, T. | Search bioRxiv

bioRxiv2026

A little feat in translation: miniaturized tRNAs in nematode and arachnid mitochondria

Transfer RNAs carry a highly conserved, cloverleaf-like secondary structure, which is essential to fulfill their task as adaptor molecules in translation. However, in mitochondria of metazoans, tRNA molecules were identified that deviate from this consensus structure and lack either the D- or T-arm, or, in some extreme cases, even both arms. These deviations are predominantly found in nematodes and arachnids, where in many cases the entire set of 22 mt tRNA genes are predicted to encode for such aberrant tRNAs or where even the complete loss of tRNA genes is proposed. Due to this unusual composition, we analyzed and characterized the mt tRNA pool of one representative of both groups. We identified the whole set of mt tRNAs and reannotated several tRNAs that differ significantly from previous genome-based predictions. In some cases, the sequence reads indicate putative tRNA editing events, showing that predictions exclusively based on mitogenome data have only a limited reliability. Our data also provide first insights into the modification pattern of such hairpin-like tRNAs.

Molecular Biology↗

bioRxiv2026

Rational design of mechanically active RNAs: de novo engineering of functional exoribonuclease-resistant RNAs

Mechanically active RNAs represent an emerging class of biomolecules whose function derives from resisting molecular forces. Among them, exoribonuclease-resistant RNAs (xr-RNAs) achieve this by folding into a ring-like topology that physically blocks 5 [->] 3 degra-dation. However, despite years of structural insight, the rational design of such mechanically functional RNA devices has remained elusive. Here, we describe a mechanics-aware RNA design approach that enables de novo engineering of functional xrRNAs. We first identify structural determinants of force resistance by perturbing pseudoknot architecture in a model xrRNA and quantifying resulting efficiencies in the stalling of exoribonuclease XRN1. We then implement these rules in a design framework that integrates explicit topological constraints with molecular dynamics-guided optimization. The resulting synthetic xrRNAs reproduce the ring-like architecture and stall exoribonuclease XRN1 with wild-type-like efficiency. Our top-performing constructs exhibit minimal sequence similarity to known xrRNAs and evade detection by covariance models, yet remain fully functional in vitro. Together, our results show that mechanical function can be rationally designed independent of evolutionary ancestry, laying the groundwork for the design of RNA elements that modulate decay and fine-tune the mechanical stability of engineered transcripts.

biochemistry↗