bims-micpro 2025-08-17 papers

Nucleic Acids Res. 2025 Aug 11. pii: gkaf774. [Epub ahead of print]53(15):

PIRT-Seq: a high-resolution whole-genome assay to identify protein-coding genes.

Emily C A Goodall, Freya Hodges, Weine Kok, Budi Permana, Thom Cuddihy, Zihao Yang, Nicole Kahler, Kenneth Shires, Karthik Pullela, Von Vergel L Torres, Jessica L Rooke, Antoine Delhaye, Jean-François Collet, Jack A Bryant, Brian M Forde, Matthew R Hemm, Ian R Henderson.

The advent of high-density mutagenesis and data-mining studies suggest the existence of further coding potential within bacterial genomes. Small or overlapping genes are prevalent across all domains of life but are often overlooked for annotation and function because of challenges in their detection. To overcome limitations in existing protein detection methods, we applied a genetics-based approach. We combined transposon insertion sequencing using a dual-selection transposon with a translation reporter to identify translated open reading frames throughout the genome at scale but independent of genome annotation. We applied our method to the well-characterised species Escherichia coli. This method revealed over 200 putative novel protein coding sequences (CDS). These are mostly short CDSs (<50 amino acids) and include proteins that are highly conserved and neighbour functionally important genes. Using chromosomal tags, we validated the expression of selected CDSs. We present this method (Protein Identification through Reporter Transposon-Sequencing: PIRT-Seq) as a complementary method to whole cell proteomics and ribosome trapping for condition-dependent identification of protein CDSs, and as a high-throughput method for testing conditional gene expression. We anticipate this technique will be a starting point for future high-throughput genetics investigations to determine the existence of unannotated genes in multiple bacterial species.

DOI: https://doi.org/10.1093/nar/gkaf774

bioRxiv. 2025 Aug 04. pii: 2025.08.04.668486. [Epub ahead of print]

Ribo-ITP expands the translatome of limited input samples.

Vighnesh Ghatpande, Uma Paul, MacKenzie A Howard, Can Cenik.

In the last decade, an unexpectedly large number of translated regions (translons) have been discovered using ribosome profiling and proteomics. Translons can regulate mRNA translation and encode micropeptides that contribute to multiprotein complex formation, Ca 2+ regulation in muscle, and signaling during embryonic development. However, identification of translons has been limited to cell lines or large organs due to high input requirements for conventional ribosome profiling and mass spectrometry. Here, we address this challenge using Ribo-ITP on difficult-to-collect samples like microdissected hippocampal tissues and single pre-implantation embryos. Comparative analysis of more than a thousand ribosome profiling datasets across a wide range of cell types revealed distinct sample specific expression patterns of the detected translons. To test the translational capacity of the identified translons, we engineered a translon-dependent GFP reporter system and detected expression of translons initiating at near-cognate start codons in mouse embryonic stem cells (mESCs). Mutating the translons in mESCs identified a small proportion that negatively impacted growth. Taken together, we present a proof-of-concept study to identify non-canonical translation events from low input samples which can be applied to cell and tissue types inaccessible to conventional methods.

DOI: https://doi.org/10.1101/2025.08.04.668486

bioRxiv. 2025 Jul 14. pii: 2025.07.14.664749. [Epub ahead of print]

5' untranslated regions tune Toxoplasma translation.

Michelle L Peters, Aditi Shukla, Zoe A Gotthold, Dylan M McCormick, Kehui Xiang, David P Bartel, Sebastian Lourido.

Some of the longest 5' untranslated regions (UTRs) documented in eukaryotes belong to parasites of the phylum Apicomplexa. Translational regulation plays prominent roles in the development of these parasites, including the agents of toxoplasmosis ( Toxoplasma gondii ) and malaria. To understand the function of 5' UTRs in apicomplexan translation, we performed high-resolution ribosome profiling of T. gondii in human fibroblasts. We show that parasite translation efficiency (TE) is largely controlled by 5' UTR features and tuned by the number of upstream AUGs (uAUGs). Examination of ribosome occupancy reveals that, despite widespread assembly of parasite monosomes on uAUGs, ribosomes seldom translate uORFs. These determinants of translation are reaffirmed in a massively parallel reporter assay examining the effect of more than 30,000 synthetic 5' UTRs in T. gondii . A model trained on these results accurately predicted the TE of newly designed 5' UTRs. Together, this work defines the regulatory language of T. gondii translation, providing a framework to understand the evolution of exceptionally long 5' UTRs in apicomplexans.

DOI: https://doi.org/10.1101/2025.07.14.664749