Biology subjects

Pryszcz, L.

Publications and source records attributed to Pryszcz, L..

4 recordsLinked to original sources

bioRxiv2024

Nano3P-seq: charting the coding and non-coding transcriptome at single molecule resolution

RNA polyadenylation is crucial for RNA maturation, stability and function, with polyA tail lengths significantly influencing mRNA translation, efficiency and decay. Here, we provide a step-by-step protocol to perform Nanopore 3 end-capture sequencing (Nano3P-seq), a nanopore-based cDNA sequencing method to simultaneously capture RNA abundances, tail composition and tail length estimates at single-molecule resolution. Taking advantage of a template switching-based protocol, Nano3P-seq can sequence any RNA molecule from its 3 end, regardless of its polyadenylation status, without the need for PCR amplification or RNA adapter ligation. We provide an updated Nano3P-seq protocol that is compatible with R10.4 flowcells, as well as compatible software for polyA tail length and content prediction, which we term PolyTailor. We demonstrate that PolyTailor provides accurate estimates of transcript abundances, tail lengths and content information, while capturing both coding and non-coding RNA biotypes, including mRNAs, snRNAs, and rRNAs. This method can be applied to any RNA sample of interest (e.g. poly(A)-selected, ribodepleted, total RNA), and can be completed in one day. The Nano3P-seq protocol can be performed by researchers with moderate experience in molecular biology techniques and nanopore sequencing library preparation, and basic knowledge of linux bash syntax and R programming. This protocol makes Nano3P-seq accessible and easy to implement by future users aiming to study the tail dynamics and heterogeneity of both coding and non-coding transcriptome in a comprehensive and reproducible manner. Key PapersBe[g]ik O, Diensthuber G, Liu H, Delgado-Tejedor A, Kontur C, Niazi AM, Valen E, Giraldez AJ, Beaudoin JD, Mattick JS, Novoa EM. Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing. Nature Methods 20, 75-85 (2023). https://doi.org/10.1038/s41592-022-01714-w Delgado-Tejedor A, Medina M, Begik O, Cozzuto L, Lopez J, Blanco B, Ponomarenko J, Novoa EM. Native RNA nanopore sequencing reveals antibiotic-induced loss of rRNA modifications in the A- and P-sites. NatComm 15, 10054 (2024). https://doi.org/10.1038/s41467-024-54368-x

molecular biology↗

bioRxiv2023

Enhanced detection of RNA modifications and mappability with high-accuracy nanopore RNA basecalling models

In recent years, nanopore direct RNA sequencing (DRS) has established itself as a valuable tool for studying the epitranscriptome, due to its ability to detect multiple modifications within the same full-length native RNA molecules. While RNA modifications can be identified in the form of systematic basecalling errors in DRS datasets, N6-methyladenosine (m6A) modifications produce relatively low errors compared to other RNA modifications, limiting the applicability of this approach to m6A sites that are modified at high stoichiometries. Here, we demonstrate that the use of alternative RNA basecalling models, trained with fully unmodified sequences, increases the error signal of m6A, leading to enhanced detection and improved sensitivity even at low stoichiometries. Moreover, we find that high-accuracy alternative RNA basecalling models can show up to 97% median basecalling accuracy, outperforming currently available RNA basecalling models, which show 91% median basecalling accuracy. Notably, the use of high-accuracy basecalling models is accompanied by a significant increase in the number of mapped reads -especially in shorter RNA fractions- and increased basecalling error signatures at pseudouridine ({Psi}) and N1-methylpseudouridine (m1{Psi}) modified sites. Overall, our work demonstrates that alternative RNA basecalling models can be used to improve the detection of RNA modifications, read mappability and basecalling accuracy in nanopore DRS datasets.

genomics↗

bioRxiv2023

De novo basecalling of m6A modifications at single molecule and single nucleotide resolution

RNA modifications hold pivotal roles in shaping the fate and function of RNA molecules. Although nanopore sequencing technologies have proven successful at transcriptome-wide detection of RNA modifications, current algorithms are limited to predicting modifications at a per-site level rather than within individual RNA molecules. Herein, we introduce m6ABasecaller, an innovative method enabling direct basecalling of m6A modifications from raw nanopore signals within individual RNA molecules. This approach facilitates de novo prediction of m6A modifications with precision down to the single nucleotide and single molecule levels, without the need of paired knockout or control conditions. Using the m6ABasecaller, we find that the median transcriptome-wide m6A modification stoichiometry is [~]10-15% in human, mouse and zebrafish. Furthermore, we show that m6A modifications affect polyA tail lengths, exhibit a propensity for co-occurrence within the same RNA molecules, and show relatively consistent stoichiometry levels across isoforms. We further validate the m6ABasecaller by treating mESC with increasing concentrations of STM2457, a METTL3 inhibitor as well as in inducible METTL3 knockout systems. Overall, this work demonstrates the feasibility de novo basecalling of m6A modifications, opening novel avenues for the application of nanopore sequencing to samples with limited RNA availability and for which control knockout conditions are unavailable, such as patient-derived samples.

molecular biology↗

bioRxiv2021

Adar-mediated A-to-I editing is required for establishment of embryonic body axes in zebrafish

Adenosine deaminases (ADARs) catalyze the deamination of adenosine to inosine, also known as A-to-I editing, in RNA. Although A-to-I editing occurs widely across animals, and is well studied, new biological roles are still being discovered. Here, we study the role of A-to-I editing in early zebrafish development. We demonstrate that Adar, the zebrafish orthologue of mammalian ADAR1, is essential for establishing the antero-posterior and dorso-ventral axes and patterning. Genome-wide editing discovery revealed pervasive editing in maternal and the earliest zygotic transcripts, the majority of which occurred in the 3-UTR. Interestingly, transcripts implicated in gastrulation as well as dorso-ventral and antero-posterior patterning were found to contain multiple editing sites. Adar knockdown or overexpression affected gene expression and global editing patterns at 12 hpf, but not earlier. Our study established that RNA editing by Adar is necessary for the earliest steps of embryonic patterning along the zebrafish antero-posterior and dorso-ventral axes.

developmental biology↗