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The continuous degradation and synthesis of prokaryotic mRNAs not only give rise to the metabolic changes that are required as cells grow and divide but also rapid adaptation to new environmental conditions. In bacteria, RNAs can be degraded by mechanisms that act independently, but in parallel, and that target different sites with different efficiencies. The accessibility of sites for degradation depends on several factors, including RNA higher-order structure, protection by translating ribosomes and polyadenylation status. Furthermore, RNA degradation mechanisms have shown to be determinant for the post-transcriptional control of gene expression. RNases mediate the processing, decay and quality control of RNA. RNases can be divided into endonucleases that cleave the RNA internally or exonucleases that cleave the RNA from one of the extremities. Just in Escherichia coli there are >20 different RNases. RNase E is a single-strand-specific endonuclease critical for mRNA decay in E. coli. The enzyme interacts with the exonuclease polynucleotide phosphorylase (PNPase), enolase and RNA helicase B (RhlB) to form the degradosome. However, in Bacillus subtilis, this enzyme is absent, but it has other main endonucleases such as RNase J1 and RNase III. RNase III cleaves double-stranded RNA and family members are involved in RNA interference in eukaryotes. RNase II family members are ubiquitous exonucleases, and in eukaryotes, they can act as the catalytic subunit of the exosome. RNases act in different pathways to execute the maturation of rRNAs and tRNAs, and intervene in the decay of many different mRNAs and small noncoding RNAs. In general, RNases act as a global regulatory network extremely important for the regulation of RNA levels.

Acinetobacter baumannii represents a burgeoning threat to human health and consistently ranks as a critical pathogen by the World Health Organization due to extensive antimicrobial resistance among clinical isolates. While much effort has focused on understanding how A. baumannii acquires antimicrobial resistance traits, our knowledge of key processes governing gene expression in this organism is lacking. In particular, very little is known regarding post-transcriptional regulation in A. baumannii . Here, we demonstrate that Hfq, a highly conserved RNA chaperone, coordinates the regulatory activities for nearly 100 small regulatory RNAs (sRNAs), including many that have not been described before. We also find that several Hfq-associated sRNAs directly regulate mRNA transcripts, which encode antibiotic resistance determinants and virulence factors. Collectively, our study provides evidence for the existence of a complex post-transcriptional regulatory network in A. baumannii and offers new insights into how the organism uses Hfq and sRNAs to coordinate gene expression.

The mechanisms by which transcription factors (TFs) and co-regulators control gene expression remain ill-defined. An interconnected network of TFs regulates an epigenetic switch between white and opaque states in Candida albicans and serves as a model to understand the transcriptional regulation of eukaryotic cell fate. Here, we examine the role of Ssn6, one of the eight core regulators of the white-opaque switch, and reveal key roles for both the structured TPR domain and the disordered C-terminal domain. We demonstrate that Ssn6 is readily incorporated into transcriptional condensates where it disrupts the liquid-like properties of these condensates, both in the presence and absence of the co-repressor Tup1. Together with studies in higher eukaryotes, these results suggest a conserved role for TPR-containing proteins in regulating gene expression via the modulation of the physical properties of transcriptional condensates.

Transcriptional terminators are key to defining transcript boundaries, ensuring mRNA maturation, and maintaining expression stability, yet they remain underexplored in filamentous fungi. Here, we systematically benchmarked 15 heterologous 3′ terminators in Aspergillus oryzae by measuring protein output via mCherry fluorescence and transcriptional readthrough based on a quantitative readthrough index (RTI) derived from RT-qPCR.Across the tested terminators, mCherry expression varied more than 5-fold and RTI spanned over 50-fold, indicating substantial functional diversity. Expression strength and termination efficiency were only weakly correlated (Spearman ρ = −0.39, p = 0.165), indicating that these two properties are largely independent and can be optimized separately.Strong terminators -TactA, Txyn1, TmutA and TtrpC combined good mCherry expression (>ΔT) and small readthrough (RT < 0.15). In contrast, short synthetic sequences (≤70 bp) showed low mCherry output accompanied by transcriptional leakage. Interestingly, widely used toolkit elements such as Ttef1 and Tcyc1 performed poorly in A. oryzae, highlighting the importance of empirical characterization over assumed transferability.Polyadenylation site mapping by 3′ rapid amplification of cDNA ends (3′RACE) reveals high-performing terminators that possess focused cleavage sites, continuous poly(A) tails, and recognizable A[AT]TAAA-like motifs with moderately AU-rich upstream regions, whereas weak terminators exhibited dispersed cleavage and sparse upstream sequence elements. Targeted disruption of the canonical AATAAA hexamer in TactA did not significantly impair expression or termination efficiency, demonstrating that the canonical PAS is not strictly required when AU-rich upstream sequence elements are present. Terminator performance rankings were robust across three carbon sources and were confirmed using a secreted NanoLuc luciferase reporter, demonstrating that functional behavior is largely sequence intrinsic. These findings demonstrate a functional terminator architecture and suggest a practical guideline for terminator selection in A. oryzae.

Background Genomic organization and gene expression regulation in trypanosomes are remarkable because protein-coding genes are organized into codirectional gene clusters with unrelated functions. Moreover, there is no dedicated promoter for each gene, resulting in polycistronic gene transcription, with posttranscriptional control playing a major role. Nonetheless, these parasites harbor epigenetic modifications at critical regulatory genome features that dynamically change among parasite stages, which are not fully understood. Results Here, we investigated the impact of chromatin changes in a scenario commanded by posttranscriptional control exploring the parasite Trypanosoma cruzi and its differentiation program using FAIRE-seq approach supported by transmission electron microscopy. We identified differences in T. cruzi genome compartments, putative transcriptional start regions, and virulence factors. In addition, we also detected a developmental chromatin regulation at tRNA loci (tDNA), which could be linked to the intense chromatin remodeling and/or the translation regulatory mechanism required for parasite differentiation. We further integrated the open chromatin profile with public transcriptomic and MNase-seq datasets. Strikingly, a positive correlation was observed between active chromatin and steady-state transcription levels. Conclusion Taken together, our results indicate that chromatin changes reflect the unusual gene expression regulation of trypanosomes and the differences among parasite developmental stages, even in the context of a lack of canonical transcriptional control of protein-coding genes.

T-cell intracellular antigen 1 (TIA1) is an RNA-binding protein that is expressed in many tissues and in the vast majority of species, although it was first discovered as a component of human cytotoxic T lymphocytes. TIA1 has a dual localization in the nucleus and cytoplasm, where it plays an important role as a regulator of gene-expression flux. As a multifunctional master modulator, TIA1 controls biological processes relevant to the physiological functioning of the organism and the development and/or progression of several human pathologies. This review summarizes our current knowledge of the molecular aspects and cellular processes involving TIA1, with relevance for human pathophysiology.

Vectors developed from adeno-associated virus (AAV) are powerful tools for in vivo transgene delivery in both humans and animal models, and several AAV-delivered gene therapies are currently approved for clinical use. However, AAV-mediated gene therapy still faces several challenges, including limited vector packaging capacity and the need for a safe, effective method for controlling transgene expression during and after delivery. Riboswitches, RNA elements which control gene expression in response to ligand binding, are attractive candidates for regulating expression of AAV-delivered transgene therapeutics because of their small genomic footprints and non-immunogenicity compared to protein-based expression control systems. In addition, the ligand-sensing aptamer domains of many riboswitches can be exchanged in a modular fashion to allow regulation by a variety of small molecules, proteins, and oligonucleotides. Riboswitches have been used to regulate AAV-delivered transgene therapeutics in animal models, and recently developed screening and selection methods allow rapid isolation of riboswitches with novel ligands and improved performance in mammalian cells. This review discusses the advantages of riboswitches in the context of AAV-delivered gene therapy, the subsets of riboswitch mechanisms which have been shown to function in human cells and animal models, recent progress in riboswitch isolation and optimization, and several examples of AAV-delivered therapeutic systems which might be improved by riboswitch regulation.

Over the past few decades, it has become increasingly clear that small RNAs (sRNAs) play important regulatory roles in bacteria. These RNA species often work in concert with RNA chaperones such as Hfq that facilitate their base-pairing with target mRNAs. The mapping of sRNA interaction networks through the identification of the RNA species that sRNAs base-pair with can therefore provide critical insights into the potential regulatory roles sRNAs play. Indeed, sRNA interaction networks can be complex, with some bacteria producing more than a hundred different sRNAs, each capable of targeting anywhere from a single mRNA to several hundred different mRNAs. Here, we highlight two high-throughput approaches, RNA interaction by ligation and sequencing and intracellular RNA interaction by ligation and sequencing, that enable transcriptome-wide identification of sRNA interaction partners. Both methods capture RNA-RNA interactions and exploit chaperone-associated RNA-RNA ligation to capture native sRNA-target duplexes, providing powerful and scalable strategies for determining bacterial sRNA interaction networks.

Conservons are operons that encode unusual regulatory systems found in bacteria of the phylum Actinomycetota. These regulatory systems are composed of four core proteins: a sensor histidine kinase-like protein (CvnA homolog), an MglB-type roadblock protein (CvnB homolog), a protein containing a domain of unknown function (CvnC homolog), and a small Ras-like GTPase (CvnD homolog). Based on their conserved small GTPase components and their phylogenetic distribution, we propose that the systems encoded by conservons should be known as ctinobacterial rotein ystems (AGPSs). The signal transduction path through AGPSs remains poorly understood, and some AGPSs have additional accessory proteins (CvnE and CvnF homologs) of unknown function. In this work, we show that AGPS accessory proteins are present when the cognate histidine kinase protein (CvnA homolog) lacks an extracytoplasmic sensory domain. It was previously shown that the Cvn8 AGPS of controls the expression of multiple pathways for specialized metabolism. The Cvn8 AGPS also contains an accessory protein, CvnF8. Through protein modeling, we found that CvnF8 may share an interaction interface with the histidine kinase CvnA8, prompting the hypothesis that CvnF8 may act as a modulator of CvnA8 activity. Consistent with this hypothesis, we found that when co-expressed in a heterologous host, CvnA8 and CvnF8 were purified as a stable complex. In a purified system, CvnF8 strongly stimulated the ATPase activity and autophosphorylation of CvnA8. Taken together, these findings indicate that CvnF family accessory proteins likely serve as sensors and/or modulators of histidine kinases of AGPSs found broadly in Actinomycetota. Many lineages of bacteria in the phylum Actinomycetota contain conserved operons (conservons) that encode an unusual type of regulatory system whose function is poorly understood. These lineages include pathogens such as and members of the genus that produce valuable natural products. These regulatory systems are composed of four proteins, including a sensor histidine kinase and a small Ras-like GTPase. We propose that these regulatory systems be known as actinobacterial G protein systems (AGPSs). We show that some AGPSs include accessory proteins that are only found with partner histidine kinases that lack sensory domains. We demonstrate that one such accessory protein can control the activity of its cognate histidine kinase. Our findings indicate that these CvnF-family accessory proteins likely serve as sensory inputs for AGPSs found broadly in Actinomycetota. This work sheds light on the initial steps of signal transduction within these unusual regulatory systems.

Bacteria respond to stress by rapidly regulating gene expression. Regulation can occur through the control of messenger RNA (mRNA) production (transcription elongation), stability of mRNAs, or translation of mRNAs. Bacteria can use small RNAs (sRNAs) to regulate gene expression at each of these steps, but we often do not understand how this works at a molecular level. In this study, we find that sRNAs in Escherichia coli regulate gene expression at the level of transcription elongation by promoting or inhibiting transcription termination by a protein called Rho. These results help us understand new molecular mechanisms of gene expression regulation in bacteria.