tREPs─A New Class of Functional tRNA-Encoded Peptides

Abstract

We asked if transfer RNA (tRNA) ever got an opportunity of translating its own sequence during evolution, what would have been the function of such tR NA-encoded p eptides (tREPs)? If not, could one artificially synthesize tREPs to study the corresponding functional outcomes? Here, we report a novel, first-in-the-class, chemically synthesized tREP-18 molecule originating from the Escherichia coli tRNA sequence showing potent antileishmanial property. As a first step, E. coli tRNAs were computationally translated into peptide sequence equivalents and a database of full-length hypothetical tREPs was created. The tREP sequences were sent into sequence, structure, and energy filters to narrow down potential peptides for experimental validation. Based on the functional predictions, tREPs were screened against antiparasitic targets, leading to the identification of tREP-18 as a potential antiparasitic peptide. The in vitro assay of chemically synthesized tREP-18 on the Ag83 strain of Leishmania donovani showed its potent antileishmanial property (IC50 value of 22.13 nM). The atomic force microscopy and scanning electron microscopy images indicated significant alteration in the cytoskeletal architecture of tREP-18-treated parasites. Also, tREP-18 seems to destabilize the mitochondrial membrane potential of parasites, disrupting their cellular integrity and leading to parasitic death. The cellular assays of the tREP-18 peptide on the BS12 strain, a clinical isolate of post-kala azar dermal leishmaniasis, demonstrated its significant efficacy at an IC50 value of 15 nM. The tREP-18 peptide showed a toxic effect on the amastigote stage of the parasite, showing macrophage pathogen clearance at a concentration of 22.5 nM. This study provides the proof of the concept of making a new class of functional peptides from tRNA sequences. It also opens a huge untapped tRNA-peptide space toward novel discoveries and applications. In the future, it would be interesting to perform tREP edits and redesign tREPs toward specific applications.

Repurposing the dark genome. II - Reverse Proteins

“…Subsequently, dark genome sequences were artificially expressed in E.coli, D.melanogaster and S.cerevisiae leading to antimalarial, antimicrobial and anti-cancer molecules (Joshi et al, 2013, Raj et al, 2015, Deepthi et al, 2017. Recently, tRNA sequences were used to produce tREPs (tRNA encoded peptides) showing a strong anti-leishmania properties (Chakrabarti et al, 2022).…”

“…cerevisiae leading to antimalarial, antimicrobial and anti-cancer molecules (Joshi et al, 2013, Raj et al, 2015, Deepthi et al, 2017). Recently, tRNA sequences were used to produce tREPs (tRNA encoded peptides) showing a strong anti-leishmania properties (Chakrabarti et al, 2022). in this study, sequences of known protein-coding genes in E .…”

Repurposing The Dark Genome. III - Intronic Proteins

“…Similarly, tRNA sequences of E. coli were expressed into tREPs showing strong antileishmania properties (Chakrabarti et al, 2022). Recently it was observed that antisense Sequences (Garg & Dhar, 2023) and reverse Sequences (Nayak & Dhar, 2023) can be artificially encoded into stable and functional protein molecules.…”

IRSOM2: a web server for predicting bifunctional RNAs

“…Unfortunately, there is no dataset of monofunctional RNA available. In particular, it is challenging to annotate RNAs as ‘strictly non-coding’, as even tRNAs have recently been shown to encode peptides ( 16 ). This limits the present study to the computation of the TPR and the false negative rate (FNR), calculated as:…”

“…To a lesser extent, other classes of ncRNAs may also be translated ( 15 ), such as circular RNAs (circRNAs), transcripts from untranslated regions (UTR), primary microRNAs (pri-miRNAs), ribosomal RNAs (rRNAs), and some archaeal and bacterial small RNAs (sRNAs). Most recently, transfer RNAs (tRNAs) have also been shown to be translated into functional peptides ( 16 ). In terms of application, these transcripts are notably of medical and agronomic interests, as they have been shown to be tightly associated with cancer ( 17 ) and to play various regulatory roles in plants ( 18 ).…”