Biology subjects

Patel, B. I.

Publications and source records attributed to Patel, B. I..

3 recordsLinked to original sources

bioRxiv2025

Evaluation of Dorado v5.2.0 de novo basecalling models for the detection of tRNA modifications using RNA004 chemistry

Direct RNA sequencing with Oxford Nanopore Technologies (ONT) captures nucleotide-specific current signals that reflect both sequence and chemical modifications, offering the potential to detect RNA modifications directly from native RNA molecules. To interpret such signals, ONT provides modification-aware basecalling models that estimate the probability of selected modifications at each nucleotide. In May 2025, ONT released updated modification-calling models (Dorado v5.2.0) for pseudouridine ({Psi}), inosine, m6A and m5C, alongside new models for 2'O-ribose-methylations, necessitating independent validation. Here, we benchmark Dorado v5.2.0 against v5.1.0 using ex cellulo tRNAs from Schizosaccharomyces pombe, leveraging their well-defined modification landscape. We generated modification probability profiles at single-nucleotide resolution and quantified model performance using curated sets of annotated and validated modification sites. Our results reveal that, despite notable improvements in {Psi} detection, most modification callers remain challenged by the dense and heterogeneous modification environments of tRNAs. This work provides the first comprehensive evaluation of Dorado v5.2.0 on native tRNAs and establishes a methodological framework for benchmarking future ONT modification models in complex RNA modification contexts.

molecular biology↗

bioRxiv2025

MoDorado: Enhanced detection of tRNA modifications in nanopore sequencing by off-label use of modification callers

Rapid and accurate identification of tRNA modifications is crucial for understanding their role in protein translation and disease. However, their detection on tRNAs is challenging due to their high modification density. Recently, modification calling models for nanopore direct RNA sequencing became available for pseudouridine ({Psi}), m6A, inosine and m5C, as part of the Dorado basecaller. Applying the {Psi} model to tRNAs, we have mapped both known and novel {Psi} sites in Schizosaccharomyces pombe and assigned the responsible pseudouridine synthetases. Furthermore, we have developed MoDorado, an algorithm to detect modifications beyond those used in model training ("off-label use") by measuring prediction differences of pre-trained machine learning models. By leveraging the {Psi}/m6A/inosine/m5C models, MoDorado detected seven additional modifications (ncm5U, mcm5U, mcm5s2U, m7G, queuosine, m1A, and i6A), thus generating a tRNA modification map of S. pombe. This work demonstrates the potential of pre-trained models in determining the intricate landscape of tRNA modifications.

molecular biology↗

bioRxiv2024

The oncogene SLC35F2 is a high-specificity transporter for the micronutrients queuine and queuosine

The nucleobase queuine (q) and its nucleoside queuosine (Q) are micronutrients derived from bacteria that are acquired from the gut microbiome and/or diet in humans. Following cellular uptake, Q is incorporated at the wobble base (position 34) of tRNAs with a GUN anticodon, which is important for efficient translation. Early studies suggested that cytosolic uptake of queuine is mediated by a selective transporter that is regulated by mitogenic signals, but the identity of this transporter has remained elusive. Here, through a cross-species bioinformatic search and genetic validation, we have identified the solute carrier family member SLC35F2 as a unique transporter for both queuine and queuosine in Schizosaccharomyces pombe and Trypanosoma brucei. Furthermore, gene disruption in HeLa cells revealed that SLC35F2 is the sole transporter for queuosine in HeLa cells (Km 174 nM) and a high-affinity transporter for the queuine nucleobase (Km 67 nM), with the presence of another low-affinity transporter (Km 259 nM) in these cells. Competition uptake studies show that SLC35F2 is not a general transporter for other canonical ribonucleobases or ribonucleosides, but selectively imports q and Q. The identification of SLC35F2, an oncogene, as the transporter of both q and Q advances our understanding of how intracellular levels of queuine and queuosine are regulated and how their deficiency contributes to a variety of pathophysiological conditions, including neurological disorders and cancer. Significance StatementThe discovery of SLC35F2 as the eukaryotic transporter of queuine and queuosine is key to understanding how these micronutrients are salvaged from the human gut and distributed to different body tissues. Queuosine modification of tRNAs enhances the accuracy and efficiency of codon-anticodon pairing and regulates a range of biological and pathophysiological states, including oxidative stress responses, cancer, learning, memory, and gut homeostasis.

biochemistry↗