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1. Deciphering transcript architectural complexity in bacteria and archaea

   https://doi.org/10.1128/mbio.02359-24  

   Mattick, John_S A; Bromley, Robin E; Watson, Kaylee J; Adkins, Ricky S; Holt, Christopher I; Lebov, Jarrett F; Sparklin, Benjamin C; Tyson, Tyonna S; Rasko, David A; Dunning_Hotopp, Julie C (October 2024, mBio)

   Parkhill, Julian (Ed.)

   ABSTRACT RNA transcripts are potential therapeutic targets, yet bacterial transcripts have uncharacterized biodiversity. We developed an algorithm for transcript prediction called tp.py using it to predict transcripts (mRNA and other RNAs) inEscherichia coliK12 and E2348/69 strains (Bacteria:gamma-Proteobacteria),Listeria monocytogenesstrains Scott A and RO15 (Bacteria:Firmicute),Pseudomonas aeruginosastrains SG17M and NN2 strains (Bacteria:gamma-Proteobacteria), andHaloferax volcanii(Archaea:Halobacteria). From >5 millionE. coliK12 and >3 millionE. coliE2348/69 newly generated Oxford Nanopore Technologies direct RNA sequencing reads, 2,487 K12 mRNAs and 1,844 E2348/69 mRNAs were predicted, with the K12 mRNAs containing more than half of the predictedE. coliK12 proteins. While the number of predicted transcripts varied by strain based on the amount of sequence data used, across all strains examined, the predicted average size of the mRNAs was 1.6–1.7 kbp, while the median size of the 5′- and 3′-untranslated regions (UTRs) were 30–90 bp. Given the lack of bacterial and archaeal transcript annotation, most predictions were of novel transcripts, but we also predicted many previously characterized mRNAs and ncRNAs, including post-transcriptionally generated transcripts and small RNAs associated with pathogenesis in theE. coliE2348/69LEEpathogenicity islands. We predicted small transcripts in the 100–200 bp range as well as >10 kbp transcripts for all strains, with the longest transcript for two of the seven strains being thenuooperon transcript, and for another two strains it was a phage/prophage transcript. This quick, easy, and reproducible method will facilitate the presentation of transcripts, and UTR predictions alongside coding sequences and protein predictions in bacterial genome annotation as important resources for the research community.IMPORTANCEOur understanding of bacterial and archaeal genes and genomes is largely focused on proteins since there have only been limited efforts to describe bacterial/archaeal RNA diversity. This contrasts with studies on the human genome, where transcripts were sequenced prior to the release of the human genome over two decades ago. We developed software for the quick, easy, and reproducible prediction of bacterial and archaeal transcripts from Oxford Nanopore Technologies direct RNA sequencing data. These predictions are urgently needed for more accurate studies examining bacterial/archaeal gene regulation, including regulation of virulence factors, and for the development of novel RNA-based therapeutics and diagnostics to combat bacterial pathogens, like those with extreme antimicrobial resistance. 

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2. The impact of epitranscriptomic marks on post-transcriptional regulation in plants

   https://doi.org/10.1093/bfgp/elaa021  

   Yu, Xiang; Sharma, Bishwas; Gregory, Brian D (December 2020, Briefings in Functional Genomics)

   null (Ed.)

   Abstract Ribonucleotides within the various RNA molecules in eukaryotes are marked with more than 160 distinct covalent chemical modifications. These modifications include those that occur internally in messenger RNA (mRNA) molecules such as N6-methyladenosine (m6A) and 5-methylcytosine (m5C), as well as those that occur at the ends of the modified RNAs like the non-canonical 5′ end nicotinamide adenine dinucleotide (NAD+) cap modification of specific mRNAs. Recent findings have revealed that covalent RNA modifications can impact the secondary structure, translatability, functionality, stability and degradation of the RNA molecules in which they are included. Many of these covalent RNA additions have also been found to be dynamically added and removed through writer and eraser complexes, respectively, providing a new layer of epitranscriptome-mediated post-transcriptional regulation that regulates RNA quality and quantity in eukaryotic transcriptomes. Thus, it is not surprising that the regulation of RNA fate mediated by these epitranscriptomic marks has been demonstrated to have widespread effects on plant development and the responses of these organisms to abiotic and biotic stresses. In this review, we highlight recent progress focused on the study of the dynamic nature of these epitranscriptome marks and their roles in post-transcriptional regulation during plant development and response to environmental cues, with an emphasis on the mRNA modifications of non-canonical 5′ end NAD+ capping, m6A and several other internal RNA modifications. 

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3. Genome-wide discovery and characterization of terpene synthases contributing to strawberry aroma metabolism

   https://doi.org/10.1093/plphys/kiag292  

   Madera, Mary A; Yasmin, Farida; Becker, Kristen L; Weissman, Jenevieve D; Famula, Randi A; Bjornson, Marta; Cole, Glenn S; Feldmann, Mitchell J; Knapp, Steven J; Zerbe, Philipp (July 2026, Plant Physiology)

   Abstract Strawberry (Fragaria × ananassa) is a globally cultivated fruit crop appreciated by consumers for its unique flavor and aroma. Major groups of volatile organic compounds that define strawberry aroma include terpenes, fatty acid esters, furanones, and benzenoids, with complex terpene blends contributing characteristic fruity, citrus, and floral notes. Here, we report the genome-wide identification and functional analysis of the terpene synthase (TPS) family that governs terpene chemical diversity in strawberry. Mining of the allo-octoploid genome of the commercial “Royal Royce” cultivar identified 75 TPS gene candidates. Biochemical characterization of 33 mono-, sesqui- and di-TPS enzymes via recombinant protein assays and/or microbial co-expression studies demonstrated TPS activities involved in the biosynthesis of nearly two-thirds of known strawberry terpenes as well as products not previously described in octoploid strawberry. Complementary metabolomic and transcriptomic studies across a diversity panel of strawberry accessions illustrated substantial variation in the composition and abundance of more than 30 common and accession-specific terpene aroma metabolites. Analysis of 2 commercial accessions, “Royal Royce” and “Mara de Bois,” with contrasting aroma profiles further revealed accession-specific alterations of terpene metabolism during fruit ripening. These findings expand our understanding of the biosynthetic genes and pathways underlying strawberry terpene metabolism and provide resources to develop breeding strategies toward improving desirable strawberry aroma traits. 

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4. MALAT1 regulates mRNA processing through sequence dependent RNA–RNA and RNA–protein interactions

   https://doi.org/10.1093/nar/gkaf784  

   Balaji, Adarsh; Hall, Simone; Johnson, Raul; Zhu, Jonathan; Ellis, Lauren; McHugh, Colleen A (August 2025, Nucleic Acids Research)

   Abstract Messenger RNAs (mRNAs) are subject to multiple layers of gene expression regulation, enabling the production of a large diversity of RNA transcripts and encoded proteins from a smaller number of genes in the human genome. We find that the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) non-coding RNA regulates mRNA processing through direct RNA–RNA and RNA–protein interactions. MALAT1 interacts with both the TAR DNA binding protein (TDP-43) and the spermine/spermidine acetyltransferase SAT1 pre-mRNA to enhance alternative splicing of SAT1 through direct, sequence-specific interactions. MALAT1 interaction with TDP-43 and SAT1 increases TDP-43 binding affinity for SAT1 pre-mRNA by coordinating tripartite RNA–RNA–protein interactions. These tripartite interactions enhance SAT1 alternative splicing. This mechanism of pre-mRNA processing may not be limited to MALAT1, TDP-43, and SAT1. Similarly, alternative splicing of the liprin-α3 PPFIA3 pre-mRNA at exon 16 by the cleavage stimulation factor subunit 2 protein is enhanced by sequence-specific interactions with MALAT1 RNA. We conclude that the abundant MALAT1 non-coding RNA contains modular RNA–RNA and RNA–protein binding regions that facilitate the processing of mRNA transcripts relevant for neuronal function. 

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5. Single-cell nascent RNA sequencing unveils coordinated global transcription

   https://doi.org/10.1038/s41586-024-07517-7  

   Mahat, Dig B; Tippens, Nathaniel D; Martin-Rufino, Jorge D; Waterton, Sean K; Fu, Jiayu; Blatt, Sarah E; Sharp, Phillip A (July 2024, Nature)

   Abstract Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers^1,2. Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity in cell populations³. However, fundamental questions about the temporal regulation of transcription and enhancer–gene coordination remain unanswered, primarily because of the absence of a single-cell perspective on active transcription. In this study, we present scGRO–seq—a new single-cell nascent RNA sequencing assay that uses click chemistry—and unveil coordinated transcription throughout the genome. We demonstrate the episodic nature of transcription and the co-transcription of functionally related genes. scGRO–seq can estimate burst size and frequency by directly quantifying transcribing RNA polymerases in individual cells and can leverage replication-dependent non-polyadenylated histone gene transcription to elucidate cell cycle dynamics. The single-nucleotide spatial and temporal resolution of scGRO–seq enables the identification of networks of enhancers and genes. Our results suggest that the bursting of transcription at super-enhancers precedes bursting from associated genes. By imparting insights into the dynamic nature of global transcription and the origin and propagation of transcription signals, we demonstrate the ability of scGRO–seq to investigate the mechanisms of transcription regulation and the role of enhancers in gene expression. 

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