December 8th, 2024
Recent Publications Harnessing the Power of Translatomics
Every week we provide a digest of a small number of recent interesting papers in the field of translatomics.
In this week’s Sunday papers, Coscujuela Tarrero et al. present a Nanodynamo, novel method to measure RNA life cycle dynamics in cells; Gong et al. introduces CircProPlus, an enhanced computational tool for identifying protein-coding circRNAs; and, Shaw et al. demonstrates the circuit between T-loop tRNA modifications in yeast.
Nanodynamo quantifies subcellular RNA dynamics revealing extensive coupling between steps of the RNA life cycle
Nature Communications, 2024
Coscujuela Tarrero, L., Famà, V., D’Andrea, G., Maestri, S., de Polo, A., Biffo, S., Furlan, M. and Pelizzola, M.
The different stages of the RNA life cycle are known to be tightly coordinated. Current results in the field can explain the interaction between one and another specific step of the RNA life cycle, while a comprehensive picture of all processes being coupled together is not yet uncovered.
To solve this challenge, the authors introduce Nanodynamo, a novel method to measure RNA life cycle dynamics in cells. This computational and experimental approach quantifies key stages, including transcription, processing, export, translation, and decay, using mathematical modelling, RNA metabolic labelling and Nanopore sequencing of native RNA for the transcripts profiling in polysomes and other cellular fractions.
Nanodynamo was applied for characterisation of transcriptional programs in SUM159 triple negative breast cancer cells. By clustering of genes based on the RNA quantities and magnitude of the kinetic rates, it turned out that 65% of the genes adopted co-transcriptional processing. It also revealed that genes efficiently translated tended to be efficiently degraded and the rate of polysomal degradation was faster than the rate of cytoplasmic degradation. Another finding was related to the size of polyA tails – the rapid nuclear deadenylation of polyA tails that occurs after transcription. Also transcripts with high rates of synthesis had particularly short polyA tails. Nanodynamo also demonstrated that splicing perturbation has broad consequences on the cell system that go beyond the expected repression of RNA processing. In fact, it involves a switch from co- to post-transcriptional RNA processing and impacts the export and translational machinery.
This research emphasises the potential of Nanodynamo to further elucidate regulatory determinants in gene expression and offers a framework to expand studies on RNA dynamics under various cellular and therapeutic conditions.
An updated resource for the detection of protein-coding circRNA with CircProPlus
Scientific Reports, 2024
Gong, X., Liu, Y., Wu, G., Xu, Z., Zeng, L., Tian, M., Zhang, R., Zeng, C. and Chen, Y.
Biological significance and coding potential of circular RNAs (circRNAs) are becoming a rising focus in the RNA world. The experimental gold standard for finding protein-coding circRNAs is to combine Ribo-seq data with circRNA-seq or RNA-seq data, however, the performance of circRNA detection and coding prediction software is still far from perfect.
The paper presents CircProPlus, an enhanced computational tool for identifying protein-coding circRNAs. CircProPlus builds on the previous CircPro framework and integrates three modules including circRNA detection, protein-coding potential score and junction reads derived from Ribo-seq.
In the new version, one of the major improvements is allowing user-defined input of circRNAs calculated elsewhere. CIRI2 is set by default for circRNA detection with an addition of other algorithms for better customisation. CPC2 and CPAT were introduced as protein-coding assessment tools instead of CPC. Ribo-seq reads are allowed to align to any site of circRNA other than back splice junction as it was in the previous version. The tool has benefited from parallelisation and optimisation of the overall workflow.
The tool outperforms previous models in identifying translatable circRNAs from RNase R treated and untreated samples in human breast tissue and mouse embryonic stem cells circRNA and Ribo-seq data. This advancement holds promise for better circRNA analysis, supporting potential applications in oncology, neurology, and other research areas where circRNAs play functional roles.
Combining Nanopore direct RNA sequencing with genetics and mass spectrometry for analysis of T-loop base modifications across 42 yeast tRNA isoacceptors
Nucleic Acid Research, 2024
Shaw, E.A., Thomas, N.K., Jones, J.D., Abu-Shumays, R.L., Vaaler, A.L., Akeson, M., Koutmou, K.S., Jain, M. and Garcia, D.M.
tRNAs are known to be heavily chemically modified, and these modifications are crucial for maintaining tRNA structure and regulating the protein synthesis. The authors utilised Nanopore direct RNA-seq and direct measurements of chemical modifications by mass spectrometry to study the link between modifications that are known to stabilize tRNA structure.
The authors focused on closely-spaced, conserved modifications T-loop modifications including 5-methyluridine (m5U54), pseudouridine (Ψ55), and 1-methyladenosine (m1A58). To predict their positions across 42 yeast tRNA isoacceptors, the researchers sequenced tRNA from yeast strains in which each one of the three enzymes responsible for modifying the T-loop was knocked out, as well as an in vitro transcribed (IVT) library of the same 42 isoacceptor sequences lacking all modifications.
With the help of this data, the presence of Ψ55 and m1A58 across all 42 cytosolic tRNA isoacceptors was inferred by exploring the systematic nucleotide miscall patterns in T-loops. As for m5U54, it did not result in a miscall pattern in Nanopore DRS suggesting that software improvements for base calling and ionic current analysis will be required for a better identification of its signatures in tRNA.
Due to the ability of DRS to simultaneously profile multiple modifications, it was found that Ψ55 strongly affected addition of m1A58 on 19 isoacceptors which is more than has been previously documented for this modification circuits.
These advancements show how orthogonal technologies combined with genetics enable precise detection of modification landscapes of individual, full-length tRNAs, at transcriptome-scale.