bims-micpro 2026-04-26 papers

Plant Direct. 2026 Apr;10(4): e70163

A Fluorescence-Based Transient Expression Assay for the Analysis of Upstream Open Reading Frames in Plants.

Benjamin E Haas, Faaiza Saif, Emily Bolger, Lynn Doran, Emma Rosales, Abigail Kim, Steven J Burgess, Stephen P Long.

Upstream open reading frames (uORFs) are regulatory elements present in the 5' leaders of mRNA that can significantly impact downstream gene expression in eukaryotes. In crop engineering, editing of uORFs can provide an avenue to upregulate expression of native genes without the need to add persistent transgenic copies. Even with genome-wide methods to identify translated uORFs such as ribosome profiling, their functional characterization depends on validation through reporter gene assays and mutagenesis studies. Current screening methods for plants use luciferases or protoplasts to measure differential gene expression between wild-type and mutated transcript leaders, which requires tissue processing and/or substrate addition. Here, we present a time- and cost-efficient alternative to investigate transcript leaders by co-expression of two fluorescent proteins in Nicotiana benthamiana leaf tissue and test our assay on genes involved in photoprotection, editing of which could provide a pathway to increase CO2 assimilation during sun-shade transitions.

Keywords: MoClo; Nicotiana benthamiana; VPZ; agroinfiltration; fluorescence; loop assembly; transient expression; uORF; upstream open reading frame

DOI: https://doi.org/10.1002/pld3.70163

Nucleic Acids Res. 2026 Apr 13. pii: gkag252. [Epub ahead of print]54(7):

Harnessing toxin-mediated ribosome stalling as a complementary tool to annotate bacterial ORFs.

Eduardo A Troian, Valdir C Barth, Unnati Chauhan, Haiyan Zheng, Caifeng Zhao, Jumei Zeng, Robert N Husson, Nancy A Woychik.

The Mycobacterium tuberculosis (Mtb) VapC4 endoribonuclease toxin exclusively cleaves and inactivates tRNACys, which leads to extensive ribosome stalling at Cys codons. Serendipitously, the precise position of stalled ribosomes is revealed within our 5' RNA-seq datasets used to identify and validate the tRNA target of the toxin, precluding the need for Ribo-seq. Here we show how mapping of stalled ribosomes can be harnessed as an innovative tool for reliable detection of new Cys-containing Mtb open reading frames (ORFs). Using proteogenomics we unmasked 96 unannotated ORFs; of which 54% are small ORFs ≤50 amino acids. We validated 69% of the 96 ORFs by mass spectrometry, including four whose spectra was matched to synthetic controls Also, 25% of these unannotated ORFs were identified by previously published Ribo-RET. Some of the 96 ORFs are Cys-responsive attenuators or encode stable Cys-containing proteins that map immediately before, or within, genes in the opposite, or same, orientation. These ORF sequences can also reveal functional clues, e.g. zinc-binding motifs or encode novel EsxB-like proteins. Our findings demonstrate that toxin-mediated ribosome stalling can serve as a robust genome annotation tool that is applicable to mycobacteria and other bacteria, with unique advantages that complement existing genome annotation methods.

DOI: https://doi.org/10.1093/nar/gkag252

Microb Cell. 2026 ;13 131-147

TOR-dependent regulation of the yeast homolog of the juvenile Batten Disease-associated gene CLN3.

Vijaykumar Pillalamarri, Samuel W M Gatesy, Amanda E Grassel, Lucienna Wolf, Justin P Whalley, David M Mueller.

The Juvenile form of Batten disease is a neurodegenerative disease with symptoms starting in the first decade and ending in death in the third decade of life. The gene defective in this form of Batten disease, CLN3, is conserved in eukaryotes, suggesting that the gene product serves a basic function in the cell, though the function is unknown. We have investigated the expression and regulation of the yeast homolog BTN1. Reanalysis of publicly available gene expression data suggests that transcription of BTN1 increases in response to oxidative stress, treatment with rapamycin or arsenate, amino acid starvation, and sporulation conditions. Similar to GCN4, there are upstream open reading frames (uORF) in front of BTN1, suggesting translational regulation. We developed reporter strains in which the HIS3 open reading frame replaced that of the BTN1 gene, with and without the uORFs. These reporters show that one or more of the uORFs decrease the expression of the HIS3 reporter. When expressed in the reporter strain using a high copy vector, GCN3, tRNA Arg , and tRNA Leu , increase expression, suggesting the involvement of the TORC1 pathway. BIT61 abuts BTN1 but is encoded on the opposite strand; 3' RACE analysis indicates that the mRNA of BIT61 overlaps with that of BTN1. BIT61 is involved in the TORC2 pathway, which interacts with the TORC1 pathway, suggesting a possible cis-acting mechanism of co-regulation. Lastly, we demonstrate that a yeast strain with a null mutation in BTN1 is sensitive to selective amino acid starvation, further supporting the association of BTN1 with TORC1.

Keywords: BTN1; Batten Disease; CLN3; amino acid starvation; translational regulation

DOI: https://doi.org/10.15698/mic2026.03.872

Metabol Open. 2026 Mar;29 100439

Adipogenin-seipin, lipid droplet architecture and the expanding metabolic frontier: Implications for metabolic disorders and cancer.

Maria Dalamaga.

Recent work by Li et al. identifying adipogenin as a structural cofactor of seipin introduces a new paradigm in lipid droplet (LD) biology, shifting attention from enzymatic control of lipid synthesis toward organelle architecture as a determinant of metabolic disease. By stabilizing a dodecameric seipin complex, adipogenin redirects triacylglycerol flux from LD nucleation to droplet expansion, thereby promoting LD enlargement, adipocyte hypertrophy and adipose tissue growth. This mechanism refines the adipose tissue expandability hypothesis by highlighting the importance of endoplasmic reticulum-LD interfaces and their associated microproteins in determining lipid storage capacity. Beyond adipose tissue, LDs have emerged as multifunctional organelles in cancer, supporting metabolic flexibility, redox homeostasis, hypoxia adaptation and resistance to cytotoxic therapies. Although adipogenin expression appears restricted to adipocytes, the structural principle it exemplifies, i.e. that small ER-embedded cofactors may modulate seipin assemblies and LD dynamics, may extend to malignant cells through yet-unidentified microproteins. Collectively, these observations position LD architecture as a conceptual model linking obesity, associated metabolic disorders, as well as cancer pathogenesis, suggesting that targeting organelle-level regulation, rather than lipid metabolism alone, may open new avenues for potential therapeutic interventions.

Keywords: Adipogenin; Cancer; Lipid droplet; Obesity; Seipin

DOI: https://doi.org/10.1016/j.metop.2025.100439