Dynamics of alternative polyadenylation in single root cells of Arabidopsis thaliana.

Introduction: Single-cell RNA-seq (scRNA-seq) technologies have been widely used to reveal the diversity and complexity of cells, and pioneering studies on scRNA-seq in plants began to emerge since 2019. However, existing studies on plants utilized scRNA-seq focused only on the gene expression regulation. As an essential post-transcriptional mechanism for regulating gene expression, alternative polyadenylation (APA) generates diverse mRNA isoforms with distinct 3' ends through the selective use of different polyadenylation sites in a gene. APA plays important roles in regulating multiple developmental processes in plants, such as flowering time and stress response. Methods: In this study, we developed a pipeline to identify and integrate APA sites from different scRNA-seq data and analyze APA dynamics in single cells. First, high-confidence poly(A) sites in single root cells were identified and quantified. Second, three kinds of APA markers were identified for exploring APA dynamics in single cells, including differentially expressed poly(A) sites based on APA site expression, APA markers based on APA usages, and APA switching genes based on 3' UTR (untranslated region) length change. Moreover, cell type annotations of single root cells were refined by integrating both the APA information and the gene expression profile. Results: We comprehensively compiled a single-cell APA atlas from five scRNA-seq studies, covering over 150,000 cells spanning four major tissue branches, twelve cell types, and three developmental stages. Moreover, we quantified the dynamic APA usages in single cells and identified APA markers across tissues and cell types. Further, we integrated complementary information of gene expression and APA profiles to annotate cell types and reveal subtle differences between cell types. Discussion: This study reveals that APA provides an additional layer of information for determining cell identity and provides a landscape of APA dynamics during Arabidopsis root development.

Pan-cancer analysis of m1A writer gene RRP8: implications for immune infiltration and prognosis in human cancers.

BACKGROUND: Ribosomal RNA Processing 8 (RRP8) is a gene associated with RNA modification and has been implicated in the development of several types of tumors in recent research. Nevertheless, the biological importance of RRP8 in pan-cancer has not yet been thoroughly and comprehensively investigated. METHODS: In this study, we conducted an analysis of various public databases to investigate the biological functions of RRP8. Our analysis included examining its correlation with pan-cancer prognosis, heterogeneity, stemness, immune checkpoint genes, and immune cell infiltration. Furthermore, we utilized the GDSC and CTRP databases to assess the sensitivity of RRP8 to small molecule drugs. RESULTS: Our findings indicate that RRP8 exhibits differential expression between tumor and normal samples, particularly impacting the prognosis of various cancers such as Adrenocortical carcinoma (ACC) and Kidney Chromophobe (KICH). The expression of RRP8 is intricately linked to tumor heterogeneity and stemness markers. Additionally, RRP8 shows a positive correlation with the presence of tumor-infiltrating cells, with TP53 being the predominant mutated gene in these malignancies. CONCLUSION: Our findings suggest that RRP8 may serve as a potential prognostic marker and therapeutic target in a variety of cancer types.

Full-Length Transcriptome Profiling of the Complete Mitochondrial Genome of Sericothrips houjii (Thysanoptera: Thripidae: Sericothripinae) Featuring Extensive Gene Rearrangement and Duplicated Control Regions.

The mitochondrial genome (mitogenome) of Thysanoptera has extensive gene rearrangement, and some species have repeatable control regions. To investigate the characteristics of the gene expression, transcription and post-transcriptional processes in such extensively gene-rearranged mitogenomes, we sequenced the mitogenome and mitochondrial transcriptome of Sericothrips houjii to analyze. The mitogenome was 14,965 bp in length and included two CRs contains 140 bp repeats between COIII-trnN (CR1) and trnT-trnP (CR2). Unlike the putative ancestral arrangement of insects, S. houjii exhibited only six conserved gene blocks encompassing 14 genes (trnL2-COII, trnD-trnK, ND2-trnW, ATP8-ATP6, ND5-trnH-ND4-ND4L and trnV-lrRNA). A quantitative transcription map showed the gene with the highest relative expression in the mitogenome was ND4-ND4L. Based on analyses of polycistronic transcripts, non-coding RNAs (ncRNAs) and antisense transcripts, we proposed a transcriptional model of this mitogenome. Both CRs contained the transcription initiation sites (TISs) and transcription termination sites (TTSs) of both strands, and an additional TIS for the majority strand (J-strand) was found within antisense lrRNA. The post-transcriptional cleavage processes followed the "tRNA punctuation" model. After the cleavage of transfer RNAs (tRNAs), COI and ND3 matured as bicistronic mRNA COI/ND3 due to the translocation of intervening tRNAs, and the 3' untranslated region (UTR) remained in the mRNAs for COII, COIII, CYTB and ND5. Additionally, isoform RNAs of ND2, srRNA and lrRNA were identified. In summary, the relative mitochondrial gene expression levels, transcriptional model and post-transcriptional cleavage process of S. houjii are notably different from those insects with typical mitochondrial gene arrangements. In addition, the phylogenetic tree of Thripidae including S. houjii was reconstructed. Our study provides insights into the phylogenetic status of Sericothripinae and the transcriptional and post-transcriptional regulation processes of extensively gene-rearranged insect mitogenomes.

Characterization of RNA Processing Genes in Colon Cancer for Predicting Clinical Outcomes.

Objective: Colon cancer is associated with multiple levels of molecular heterogeneity. RNA processing converts primary transcriptional RNA to mature RNA, which drives tumourigenesis and its maintenance. The characterisation of RNA processing genes in colon cancer urgently needs to be elucidated. Methods: In this study, we obtained 1033 relevant samples from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases to explore the heterogeneity of RNA processing phenotypes in colon cancer. Firstly, Unsupervised hierarchical cluster analysis detected 4 subtypes with specific clinical outcomes and biological features via analysis of 485 RNA processing genes. Next, we adopted the least absolute shrinkage and selection operator (LASSO) as well as Cox regression model with penalty to characterise RNA processing-related prognostic features. Results: An RNA processing-related prognostic risk model based on 10 genes including FXR1, MFAP1, RBM17, SAGE1, SNRPA1, SRRM4, ADAD1, DDX52, ERI1, and EXOSC7 was identified finally. A composite prognostic nomogram was constructed by combining this feature with the remaining clinical variables including TNM, age, sex, and stage. Genetic variation, pathway activation, and immune heterogeneity with risk signatures were also analysed via bioinformatics methods. The outcomes indicated that the high-risk subgroup was associated with higher genomic instability, increased proliferative and cycle characteristics, decreased tumour killer CD8+ T cells and poorer clinical prognosis than the low-risk group. Conclusion: This prognostic classifier based on RNA-edited genes facilitates stratification of colon cancer into specific subgroups according to TNM and clinical outcomes, genetic variation, pathway activation, and immune heterogeneity. It can be used for diagnosis, classification and targeted treatment strategies comparable to current standards in precision medicine. It provides a rationale for elucidation of the role of RNA editing genes and their clinical significance in colon cancer as prognostic markers.

Neuronal RNA processing: cross-talk between transcriptional regulation and RNA-binding proteins.

In the nervous system, alternative RNA processing is particularly prevalent, which results in the expression of thousands of transcript variants found in no other tissue. Neuron-specific RNA-binding proteins co-transcriptionally regulate alternative splicing, alternative polyadenylation, and RNA editing, thereby shaping the RNA identity of nervous system cells. Recent evidence suggests that interactions between RNA-binding proteins and cis-regulatory elements such as promoters and enhancers play a role in the determination of neuron-specific expression profiles. Here, we discuss possible mechanisms through which transcription and RNA processing cross-talk to generate the uniquely complex neuronal transcriptome, with a focus on alternative 3'-end formation.

An atypical form of 60S ribosomal subunit in Diamond-Blackfan anemia linked to RPL17 variants.

Diamond-Blackfan anemia syndrome (DBA) is a ribosomopathy associated with loss-of-function variants in more than 20 ribosomal protein (RP) genes. Here, we report the genetic, functional, and biochemical dissection of 2 multigenerational pedigrees with variants in RPL17, a large ribosomal subunit protein-encoding gene. Affected individuals had clinical features and erythroid proliferation defects consistent with DBA. Further, RPL17/uL22 depletion resulted in anemia and micrognathia in zebrafish larvae, and in vivo complementation studies indicated that RPL17 variants were pathogenic. Lymphoblastoid cell lines (LCLs) derived from patients displayed a ribosomal RNA maturation defect reflecting haploinsufficiency of RPL17. The proteins encoded by RPL17 variants were not incorporated into ribosomes, but 10%-20% of 60S ribosomal subunits contained a short form of 5.8S rRNA (5.8SC), a species that is marginal in normal cells. These atypical 60S subunits were actively engaged in translation. Ribosome profiling showed changes of the translational profile, but those are similar to LCLs bearing RPS19 variants. These results link an additional RP gene to DBA. They show that ribosomes can be modified substantially by RPL17 haploinsufficiency but support the paradigm that translation alterations in DBA are primarily related to insufficient ribosome production rather than to changes in ribosome structure or composition.

Nuclear retention coupled with sequential polyadenylation dictates post-transcriptional m6A modification in the nucleus.

N6-methyladenosine (m6A) modification is deemed to be co-transcriptionally installed on pre-mRNAs, thereby influencing various downstream RNA metabolism events. However, the causal relationship between m6A modification and RNA processing is often unclear, resulting in premature or even misleading generalizations on the function of m6A modification. Here, we develop 4sU-coupled m6A-level and isoform-characterization sequencing (4sU-m6A-LAIC-seq) and 4sU-GLORI to quantify the m6A levels for both newly synthesized and steady-state RNAs at transcript and single-base-resolution levels, respectively, which enable dissecting the relationship between m6A modification and alternative RNA polyadenylation. Unexpectedly, our results show that many m6A addition events occur post-transcriptionally, especially on transcripts with high m6A levels. Importantly, we find higher m6A levels on shorter 3' UTR isoforms, which likely result from sequential polyadenylation of longer 3' UTR isoforms with prolonged nuclear dwelling time. Therefore, m6A modification can also take place post-transcriptionally to intimately couple with other key RNA metabolism processes to establish and dynamically regulate epi-transcriptomics in mammalian cells.

Elevated WTAP promotes hyperinflammation by increasing m6A modification in inflammatory disease models.

Emerging evidence has linked the dysregulation of N6-methyladenosine (m6A) modification to inflammation and inflammatory diseases, but the underlying mechanism still needs investigation. Here, we found that high levels of m6A modification in a variety of hyperinflammatory states are p65-dependent because Wilms tumor 1-associated protein (WTAP), a key component of the "writer" complex, is transcriptionally regulated by p65, and its overexpression can lead to increased levels of m6A modification. Mechanistically, upregulated WTAP is more prone to phase separation to facilitate the aggregation of the writer complex to nuclear speckles and the deposition of m6A marks on transcriptionally active inflammatory transcripts, thereby accelerating the proinflammatory response. Further, a myeloid deficiency in WTAP attenuates the severity of LPS-induced sepsis and DSS-induced IBD. Thus, the proinflammatory effect of WTAP is a general risk-increasing mechanism, and interrupting the assembly of the m6A writer complex to reduce the global m6A levels by targeting the phase separation of WTAP may be a potential and promising therapeutic strategy for alleviating hyperinflammation.

Assembly mechanism of Integrator's RNA cleavage module.

The modular Integrator complex is a transcription regulator that is essential for embryonic development. It attenuates coding gene expression via premature transcription termination and performs 3'-processing of non-coding RNAs. For both activities, Integrator requires endonuclease activity that is harbored by an RNA cleavage module consisting of INTS4-9-11. How correct assembly of Integrator modules is achieved remains unknown. Here, we show that BRAT1 and WDR73 are critical biogenesis factors for the human cleavage module. They maintain INTS9-11 inactive during maturation by physically blocking the endonuclease active site and prevent premature INTS4 association. Furthermore, BRAT1 facilitates import of INTS9-11 into the nucleus, where it is joined by INTS4. Final BRAT1 release requires locking of the mature cleavage module conformation by inositol hexaphosphate (IP6). Our data explain several neurodevelopmental disorders caused by BRAT1, WDR73, and INTS11 mutations as Integrator assembly defects and reveal that IP6 is an essential co-factor for cleavage module maturation.

A biallelic variant of the RNA exosome gene, EXOSC4, associated with neurodevelopmental defects impairs RNA exosome function and translation.

The RNA exosome is an evolutionarily conserved complex required for both precise RNA processing and decay. Pathogenic variants in EXOSC genes, which encode structural subunits of this complex, are linked to several autosomal recessive disorders. Here, we describe a missense allele of the EXOSC4 gene that causes a collection of clinical features in two affected siblings. This missense variant (NM_019037.3: exon3:c.560T>C) changes a leucine residue within a conserved region of EXOSC4 to proline (p.Leu187Pro). The two affected individuals show prenatal growth restriction, failure to thrive, global developmental delay, intracerebral and basal ganglia calcifications, and kidney failure. Homozygosity for the damaging variant was identified by exome sequencing with Sanger sequencing to confirm segregation. To explore the functional consequences of this amino acid change, we modeled EXOSC4-L187P in the corresponding budding yeast protein, Rrp41 (Rrp41-L187P). Cells that express Rrp41-L187P as the sole copy of the essential Rrp41 protein show growth defects. Steady-state levels of both Rrp41-L187P and EXOSC4-L187P are decreased compared to controls, and EXOSC4-L187P shows decreased copurification with other RNA exosome subunits. RNA exosome target transcripts accumulate in rrp41-L187P cells, including the 7S precursor of 5.8S rRNA. Polysome profiles show a decrease in actively translating ribosomes in rrp41-L187P cells as compared to control cells with the incorporation of 7S pre-rRNA into polysomes. This work adds EXOSC4 to the structural subunits of the RNA exosome that have been linked to human disease and defines foundational molecular defects that could contribute to the adverse phenotypes caused by EXOSC pathogenic variants.

Full-length direct RNA sequencing reveals extensive remodeling of RNA expression, processing and modification in aging Caenorhabditis elegans.

Organismal aging is marked by decline in cellular function and anatomy, ultimately resulting in death. To inform our understanding of the mechanisms underlying this degeneration, we performed standard RNA sequencing and Nanopore direct RNA sequencing over an adult time course in Caenorhabditis elegans. Long reads allowed for identification of hundreds of novel isoforms and age-associated differential isoform accumulation, resulting from alternative splicing and terminal exon choice. Genome-wide analysis reveals a decline in RNA processing fidelity and a rise in inosine and pseudouridine editing events in transcripts from older animals. In this first map of pseudouridine modifications for C. elegans, we find that they largely reside in coding sequences and that the number of genes with this modification increases with age. Collectively, this analysis discovers transcriptomic signatures associated with age and is a valuable resource to understand the many processes that dictate altered gene expression patterns and post-transcriptional regulation in aging.

Long-Term In Vitro Culture Alters Gene Expression Pattern of Genes Involved in Ontological Groups Representing Cellular Processes.

The oviduct provides an optimal environment for the final preparation, transport, and survival of gametes, the fertilization process, and early embryonic development. Most of the studies on reproduction are based on in vitro cell culture models because of the cell's accessibility. It creates opportunities to explore the complexity of directly linked processes between cells. Previous studies showed a significant expression of genes responsible for cell differentiation, maturation, and development during long-term porcine oviduct epithelial cells (POECs) in vitro culture. This study aimed at establishing the transcriptomic profile and comprehensive characteristics of porcine oviduct epithelial cell in vitro cultures, to compare changes in gene expression over time and deliver information about the expression pattern of genes highlighted in specific GO groups. The oviduct cells were collected after 7, 15, and 30 days of in vitro cultivation. The transcriptomic profile of gene expression was compared to the control group (cells collected after the first day). The expression of COL1A2 and LOX was enhanced, while FGFBP1, SERPINB2, and OVGP1 were downregulated at all selected intervals of cell culture in comparison to the 24-h control (p-value < 0.05). Adding new detailed information to the reproductive biology field about the diversified transcriptome profile in POECs may create new future possibilities in infertility treatments, including assisted reproductive technique (ART) programmes, and may be a valuable tool to investigate the potential role of oviduct cells in post-ovulation events.

SnoRNAs: Exploring Their Implication in Human Diseases.

Small nucleolar RNAs (snoRNAs) are earning increasing attention from research communities due to their critical role in the post-transcriptional modification of various RNAs. These snoRNAs, along with their associated proteins, are crucial in regulating the expression of a vast array of genes in different human diseases. Primarily, snoRNAs facilitate modifications such as 2'-O-methylation, N-4-acetylation, and pseudouridylation, which impact not only ribosomal RNA (rRNA) and their synthesis but also different RNAs. Functionally, snoRNAs bind with core proteins to form small nucleolar ribonucleoproteins (snoRNPs). These snoRNAs then direct the protein complex to specific sites on target RNA molecules where modifications are necessary for either standard cellular operations or the regulation of pathological mechanisms. At these targeted sites, the proteins coupled with snoRNPs perform the modification processes that are vital for controlling cellular functions. The unique characteristics of snoRNAs and their involvement in various non-metabolic and metabolic diseases highlight their potential as therapeutic targets. Moreover, the precise targeting capability of snoRNAs might be harnessed as a molecular tool to therapeutically address various disease conditions. This review delves into the role of snoRNAs in health and disease and explores the broad potential of these snoRNAs as therapeutic agents in human pathologies.

Multiplexed single-cell characterization of alternative polyadenylation regulators.

Most mammalian genes have multiple polyA sites, representing a substantial source of transcript diversity regulated by the cleavage and polyadenylation (CPA) machinery. To better understand how these proteins govern polyA site choice, we introduce CPA-Perturb-seq, a multiplexed perturbation screen dataset of 42 CPA regulators with a 3' scRNA-seq readout that enables transcriptome-wide inference of polyA site usage. We develop a framework to detect perturbation-dependent changes in polyadenylation and characterize modules of co-regulated polyA sites. We find groups of intronic polyA sites regulated by distinct components of the nuclear RNA life cycle, including elongation, splicing, termination, and surveillance. We train and validate a deep neural network (APARENT-Perturb) for tandem polyA site usage, delineating a cis-regulatory code that predicts perturbation response and reveals interactions between regulatory complexes. Our work highlights the potential for multiplexed single-cell perturbation screens to further our understanding of post-transcriptional regulation.

Interferon-Regulated Expression of Cellular Splicing Factors Modulates Multiple Levels of HIV-1 Gene Expression and Replication.

Type I interferons (IFN-Is) are pivotal in innate immunity against human immunodeficiency virus I (HIV-1) by eliciting the expression of IFN-stimulated genes (ISGs), which encompass potent host restriction factors. While ISGs restrict the viral replication within the host cell by targeting various stages of the viral life cycle, the lesser-known IFN-repressed genes (IRepGs), including RNA-binding proteins (RBPs), affect the viral replication by altering the expression of the host dependency factors that are essential for efficient HIV-1 gene expression. Both the host restriction and dependency factors determine the viral replication efficiency; however, the understanding of the IRepGs implicated in HIV-1 infection remains greatly limited at present. This review provides a comprehensive overview of the current understanding regarding the impact of the RNA-binding protein families, specifically the two families of splicing-associated proteins SRSF and hnRNP, on HIV-1 gene expression and viral replication. Since the recent findings show specifically that SRSF1 and hnRNP A0 are regulated by IFN-I in various cell lines and primary cells, including intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs), we particularly discuss their role in the context of the innate immunity affecting HIV-1 replication.

RNA modifying enzymes shape tRNA biogenesis and function.

Transfer RNAs (tRNAs) are the most highly modified cellular RNAs, both with respect to the proportion of nucleotides that are modified within the tRNA sequence and with respect to the extraordinary diversity in tRNA modification chemistry. However, the functions of many different tRNA modifications are only beginning to emerge. tRNAs have two general clusters of modifications. The first cluster is within the anticodon stem-loop including several modifications essential for protein translation. The second cluster of modifications is within the tRNA elbow, and roles for these modifications are less clear. In general, tRNA elbow modifications are typically not essential for cell growth, but nonetheless several tRNA elbow modifications have been highly conserved throughout all domains of life. In addition to forming modifications, many tRNA modifying enzymes have been demonstrated or hypothesized to also play an important role in folding tRNA acting as tRNA chaperones. In this review, we summarize the known functions of tRNA modifying enzymes throughout the lifecycle of a tRNA molecule, from transcription to degradation. Thereby, we describe how tRNA modification and folding by tRNA modifying enzymes enhance tRNA maturation, tRNA aminoacylation, and tRNA function during protein synthesis, ultimately impacting cellular phenotypes and disease.

Nuclear miRNAs: Gene Regulation Activities.

MicroRNAs (miRNAs) are small non-coding RNAs which contribute to the regulation of many physiological and pathological processes. Conventionally, miRNAs perform their activity in the cytoplasm where they regulate gene expression by interacting in a sequence-specific manner with mature messenger RNAs. Recent studies point to the presence of mature miRNAs in the nucleus. This review summarizes current findings regarding the molecular activities of nuclear miRNAs. These molecules can regulate gene expression at the transcriptional level by directly binding DNA on the promoter or the enhancer of regulated genes. miRNAs recruit different protein complexes to these regions, resulting in activation or repression of transcription, through a number of molecular mechanisms. Hematopoiesis is presented as a paradigmatic biological process whereby nuclear miRNAs possess a relevant regulatory role. Nuclear miRNAs can influence gene expression by affecting nuclear mRNA processing and by regulating pri-miRNA maturation, thus impacting the biogenesis of miRNAs themselves. Overall, nuclear miRNAs are biologically active molecules that can be critical for the fine tuning of gene expression and deserve further studies in a number of physiological and pathological conditions.

SARS-CoV-2 Selectively Induces the Expression of Unproductive Splicing Isoforms of Interferon, Class I MHC, and Splicing Machinery Genes.

RNA processing is a highly conserved mechanism that serves as a pivotal regulator of gene expression. Alternative processing generates transcripts that can still be translated but lead to potentially nonfunctional proteins. A plethora of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), strategically manipulate the host's RNA processing machinery to circumvent antiviral responses. We integrated publicly available omics datasets to systematically analyze isoform-level expression and delineate the nascent peptide landscape of SARS-CoV-2-infected human cells. Our findings explore a suggested but uncharacterized mechanism, whereby SARS-CoV-2 infection induces the predominant expression of unproductive splicing isoforms in key IFN signaling, interferon-stimulated (ISGs), class I MHC, and splicing machinery genes, including IRF7, HLA-B, and HNRNPH1. In stark contrast, cytokine and chemokine genes, such as IL6 and TNF, predominantly express productive (protein-coding) splicing isoforms in response to SARS-CoV-2 infection. We postulate that SARS-CoV-2 employs an unreported tactic of exploiting the host splicing machinery to bolster viral replication and subvert the immune response by selectively upregulating unproductive splicing isoforms from antigen presentation and antiviral response genes. Our study sheds new light on the molecular interplay between SARS-CoV-2 and the host immune system, offering a foundation for the development of novel therapeutic strategies to combat COVID-19.

Rapid Ribonuclease P Kinetics Measured by Stopped-Flow Fluorescence and Fluorescence Anisotropy.

Stopped-flow fluorescence spectroscopy is a highly sensitive method for measuring rapid enzyme kinetics. A wide range of fluorophores can be employed, and fluorescence and fluorescence polarization can be measured. Thus, binding, conformational changes, and catalysis can, in principle, be measured, making it helpful in probing the entire kinetic landscape of a reaction. In this chapter, we use the bacterial RNA processing enzyme ribonuclease P (RNase P) as a model system to illustrate the determination of the kinetic constants for substrate binding and cleavage, thus allowing mechanistic questions regarding the effects of reaction conditions, mutations, or drug binding to be answered.

Structural Insights into the Rrp4 Subunit from the Crystal Structure of the Thermoplasma acidophilum Exosome.

The exosome multiprotein complex plays a critical role in RNA processing and degradation. This system governs the regulation of mRNA quality, degradation in the cytoplasm, the processing of short noncoding RNA, and the breakdown of RNA fragments. We determined two crystal structures of exosome components from Thermoplasma acidophilum (Taci): one with a resolution of 2.3 Å that reveals the central components (TaciRrp41 and TaciRrp42), and another with a resolution of 3.5 Å that displays the whole exosome (TaciRrp41, TaciRrp42, and TaciRrp4). The fundamental exosome structure revealed the presence of a heterodimeric complex consisting of TaciRrp41 and TaciRrp42. The structure comprises nine subunits, with TaciRrp41 and TaciRrp42 arranged in a circular configuration, while TaciRrp4 is located at the apex. The RNA degradation capabilities of the TaciRrp4:41:42 complex were verified by RNA degradation assays, consistent with prior findings in other archaeal exosomes. The resemblance between archaeal exosomes and bacterial PNPase suggests a common mechanism for RNA degradation. Despite sharing comparable topologies, the surface charge distributions of TaciRrp4 and other archaea structures are surprisingly distinct. Different RNA breakdown substrates may be responsible for this variation. These newfound structural findings enhance our comprehension of RNA processing and degradation in biological systems.