Search Results (30)

Coding transcripts-derived small interfering RNAs (ct-siRNAs) have emerged as a special class of endogenous siRNAs and have been implicated in the regulation of gene expression in plants, particularly under conditions where RNA metabolic pathways are perturbed. When the RNA quality control (RQC) system is impaired, the aberrant mRNA fragments were converted to double stranded forms by RNA-directed RNA polymerase 6 (RDR6) with the assistance of Suppressor of Gene Silencing 3 (SGS3) and subsequently processed by DICER-LIKE proteins into 21-nt and 22-nt ct-siRNAs. The accumulation of ct-siRNAs and the resulting suppression of their cognate genes are usually associated with altered plant growth and stress response. In this review, we summarize our current understanding of the ct-siRNAs, particularly their biogenesis under different RNA metabolic defective conditions. Comparative analysis of these genetic contexts indicates that ct-siRNAs act through translation inhibition and/or mRNA cleavage, with regulatory outcomes influenced by siRNA length and genetic background. We further summarize the biological consequence of ct-siRNA accumulation, which are frequently associated with impaired plant growth and stress adaptation. Finally, we discuss current controversies on ct-siRNAs research and highlight key unsolved questions for future investigation. Collectively, this review highlights ct-siRNAs as a link between impaired RNA metabolisms and post-transcriptional gene silencing, with context-dependent effects on plant growth and stress responses. Full article

With the intensification of global climate change and the frequent occurrence of extreme drought events, forest production is facing severe challenges. This study imposed drought stress and exogenous abscisic acid (ABA) treatment on Larix gmelini seedlings, evaluated their physiological characteristics, and analyzed the transcriptional response mechanism using transcriptome sequencing. The results showed that drought stress induced organ-specific changes in superoxide dismutase (SOD) and peroxidase (POD) activities, malondialdehyde (MDA) accumulation, and soluble protein content. SOD activity in leaves significantly increased, while POD activity, MDA content, and soluble protein levels in roots exhibited more dynamic changes. After ABA application, SOD activity in leaves reached its peak at 24 h, which was opposite to the situation in roots and stems, where POD activity was highest at 24 h. At 48 h, MDA accumulation was most significant in roots, while the early response in leaves was minimal. At 24 h, the soluble protein increase was most significant in stems. In addition, at this time point, ABA application significantly increased the soluble protein content in all three organs. Transcriptome sequencing analysis further identified core response genes involved in the MAPK signaling pathway, plant hormone signal transduction, starch and sucrose metabolism, and flavonoid biosynthesis pathways, including SNRK2, MAPKKK17, PYL, PP2C, XRN4, TMEM, TIR1, and TGA. In summary, Larix gmelini seedlings alleviate the inhibitory effect of drought stress on growth through a synergistic mechanism, specifically by activating the antioxidant system, initiating the MAPK signaling pathway, regulating plant hormone signal transduction, and reshaping carbon metabolism pathways, thereby enhancing stress resistance. Full article

Plant RNA interference (RNAi) is a fundamental antiviral defense that relies on coordinated activities of DICER-like endonucleases (DCLs), Argonaute proteins (AGOs) and RNA-dependent RNA polymerases (RDRs). Over the past decades, studies using model and crop species have uncovered complex and often redundant roles for DCLs and RDRs in generating and amplifying virus-derived small interfering RNAs (vsiRNAs), in addition to connections with transcriptional gene silencing (TGS) and epigenetic defenses against DNA viruses. Concurrently, plant viruses have evolved diverse counterstrategies—proteinaceous RNA silencing suppressors (RSSs), exoribonuclease (XRN)-resistant noncoding RNAs, and indirect manipulation of host pathways—to evade RNAi. Driven by the co-evolutionary arms race, plants have developed sophisticated counter-countermeasures that modulate or overcome viral anti-RNAi activity. Accumulated evidence suggests that plants encode host factor genes that are activated to degrade or sequester viral components such as RSSs against viral infection. On the other hand, plants have also evolved endogenous host modulators of antiviral RNAi that can either reinforce the antiviral response or be co-opted by viruses to antagonize it, representing a furious dynamic molecular battling mechanism. Here, we review recent advances in the molecular functions of DCLs and RDRs across species, summarize newly discovered viral counter-defenses (including RNA-based suppressors), and discuss host counter-countermeasures. We research key areas—such as the roles of RDRγ-class proteins, RTL1 (RNase three-like 1)-mediated competition with DCLs, and the mechanistic impact of viral noncoding RNAs—and outline translational opportunities for improving virus resistance in crops through breeding, biotechnological approaches, and RNA-based applications. Full article

Eukaryotic cells possess surveillance mechanisms that detect and degrade defective transcripts. Aberrant transcripts include mRNAs with a premature termination codon (PTC), targeted by the nonsense-mediated decay (NMD) pathway, and mRNAs lacking a termination codon, targeted by the nonstop decay (NSD) pathway. The eukaryotic exosome, a ribonucleolytic complex, plays a crucial role in mRNA processing and turnover through its catalytic subunits PM/Scl100 (Rrp6 in yeast), DIS3 (Rrp44 in yeast), and DIS3L1. Additionally, eukaryotic cells have other ribonucleases, such as SMG6 and XRN1, that participate in RNA surveillance. However, the specific pathways through which ribonucleases recognize and degrade mRNAs remain elusive. In this study, we characterized the involvement of human ribonucleases, both nuclear and cytoplasmic, in the mRNA surveillance mechanisms of NMD and NSD. We performed knockdowns of SMG6, PM/Scl100, XRN1, DIS3, and DIS3L1, analyzing the resulting changes in mRNA levels of selected natural NMD targets by RT-qPCR. Additionally, we examined the levels of different human β-globin variants under the same conditions: wild-type, NMD-resistant, NMD-sensitive, and NSD-sensitive. Our results demonstrate that all the studied ribonucleases are involved in the decay of certain endogenous NMD targets. Furthermore, we observed that the ribonucleases SMG6 and DIS3 contribute to the degradation of all β-globin variants, with an exception for βNS in the former case. This is also the case for PM/Scl100, which affects all β-globin variants except the NMD-sensitive variants. In contrast, DIS3L1 and XRN1 show specificity for β-globin WT and NMD-resistant variants. These findings suggest that eukaryotic ribonucleases are target-specific rather than pathway-specific. In addition, our data suggest that ribonucleases play broader roles in mRNA surveillance and degradation mechanisms beyond just NMD and NSD. Full article

R-loops (RNA–DNA hybrids with displaced single-stranded DNA) have emerged as a potent source of DNA damage and genomic instability. The termination of defective RNA polymerase II (RNAPII) is one of the major sources of R-loop formation. 5′-3′-exoribonuclease 2 (XRN2) promotes genome-wide efficient RNAPII termination, and XRN2-deficient cells exhibit increased DNA damage emanating from elevated R-loops. Recently, we showed that DNA damage instigated by XRN2 depletion in human fibroblast cells resulted in enhanced poly(ADP-ribose) polymerase 1 (PARP1) activity. Additionally, we established a synthetic lethal relationship between XRN2 and PARP1. However, the underlying cellular stress response promoting this synthetic lethality remains elusive. Here, we delineate the molecular consequences leading to the synthetic lethality of XRN2-deficient cancer cells induced by PARP inhibition. We found that XRN2-deficient lung and breast cancer cells display sensitivity to two clinically relevant PARP inhibitors, Rucaparib and Olaparib. At a mechanistic level, PARP inhibition combined with XRN2 deficiency exacerbates R-loop and DNA double-strand break formation in cancer cells. Consistent with our previous findings using several different siRNAs, we also show that XRN2 deficiency in cancer cells hyperactivates PARP1. Furthermore, we observed enhanced replication stress in XRN2-deficient cancer cells treated with PARP inhibitors. Finally, the enhanced stress response instigated by compromised PARP1 catalytic function in XRN2-deficient cells activates caspase-3 to initiate cell death. Collectively, these findings provide mechanistic insights into the sensitivity of XRN2-deficient cancer cells to PARP inhibition and strengthen the underlying translational implications for targeted therapy. Full article

Subgenomic flaviviral RNAs (sfRNAs) are produced during flavivirus infections in both arthropod and vertebrate cells. They are undegraded products originating from the viral 3′ untranslated region (3′ UTR), a result of the action of the host 5′-3′ exoribonuclease, Xrn1, when it encounters specific RNA structures known as Xrn1-resistant RNAs (xrRNAs) within the viral 3′ UTR. Dengue viruses generate three to four distinct species of sfRNAs through the presence of two xrRNAs and two dumbbell structures (DBs). The tertiary structures of xrRNAs have been characterized to form a ringlike structure around the 5′ end of the viral RNA, effectively inhibiting the activity of Xrn1. The most important role of DENV sfRNAs is to inhibit host antiviral responses by interacting with viral and host proteins, thereby influencing viral pathogenicity, replicative fitness, epidemiological fitness, and transmission. In this review, we aimed to summarize the biogenesis, structures, and functions of DENV sfRNAs, exploring their implications for viral interference. Full article

XRN1 is an exoribonuclease that degrades mRNA in the cytoplasm along the 5′–3′ direction. A previous study indicated that it may be involved in the reproduction of Macrobrachium nipponense. Quantitative real-time PCR was used to detect the spatiotemporal expression pattern of Mn-XRN1. At the tissue level, Mn-XRN1 was significantly expressed in the ovary. During development, Mn-XRN1 was significantly expressed at the CS stage of the embryo, on the 10th day post-larval and in the O2 stage of ovarian reproduction. The in situ hybridization results showed the location of Mn-XRN1 in the ovary. The expression of Mn-VASA was significantly increased after in vivo injection of Mn-XRN1 dsRNA. This suggests that Mn-XRN1 negatively regulates the expression of Mn-VASA. Furthermore, we counted the number of M. nipponense at various stages of ovarian reproduction on different days after RNAi. The results showed that ovarian development was significantly accelerated. In general, the results of the present study indicate that Mn-XRN1 has an inhibitory effect on the ovarian maturation of M. nipponense. The inhibitory effect might be through negative regulation of Mn-VASA. Full article

Red clover necrotic mosaic virus (RCNMV) is a segmented positive-strand RNA virus consisting of RNA1 and RNA2. Previous studies demonstrated that efficient translation of RCNMV RNA2 requires de novo synthesis of RNA2 during infections, suggesting that RNA2 replication is required for its translation. We explored a potential mechanism underlying the regulation of replication-associated translation of RNA2 by examining RNA elements in its 5′ untranslated region (5′UTR). Structural analysis of the 5′UTR suggested that it can form two mutually exclusive configurations: a more thermodynamically stable conformation, termed the 5′-basal stem structure (5′BS), in which 5′-terminal sequences are base paired, and an alternative conformation, where the 5′-end segment is single stranded. Functional mutational analysis of the 5′UTR structure indicated that (i) 43S ribosomal subunits enter at the very 5′-end of RNA2; (ii) the alternative conformation, containing unpaired 5′-terminal nucleotides, mediates efficient translation; (iii) the 5′BS conformation, with a paired 5′-end segment, supresses translation; and (iv) the 5′BS conformation confers stability to RNA2 from 5′-to-3′ exoribonuclease Xrn1. Based on our results, we suggest that during infections, newly synthesized RNA2s transiently adopt the alternative conformation to allow for efficient translation, then refold into the 5′BS conformation, which supresses translation and promotes efficient RNA2 replication. The potential advantages of this proposed 5′UTR-based regulatory mechanism for coordinating RNA2 translation and replication are discussed. Full article

The condensation of nuclear promyelocytic leukemia bodies, cytoplasmic P-granules, P-bodies (PBs), and stress granules is reversible and dynamic via liquid–liquid phase separation. Although each condensate comprises hundreds of proteins with promiscuous interactions, a few key scaffold proteins are required. Essential scaffold domain sequence elements, such as poly-Q, low-complexity regions, oligomerizing domains, and RNA-binding domains, have been evaluated to understand their roles in biomolecular condensation processes. However, the underlying mechanisms remain unclear. We analyzed Nst1, a PB-associated protein that can intrinsically induce PB component condensations when overexpressed. Various Nst1 domain deletion mutants with unique sequence distributions, including intrinsically disordered regions (IDRs) and aggregation-prone regions, were constructed based on structural predictions. The overexpression of Nst1 deletion mutants lacking the aggregation-prone domain (APD) significantly inhibited self-condensation, implicating APD as an oligomerizing domain promoting self-condensation. Remarkably, cells overexpressing the Nst1 deletion mutant of the polyampholyte domain (PD) in the IDR region (Nst1_∆PD) rarely accumulate endogenous enhanced green fluorescent protein (EGFP)-tagged Dcp2. However, Nst1_∆PD formed self-condensates, suggesting that Nst1 requires PD to interact with Dcp2, regardless of its self-condensation. In Nst1_∆PD-overexpressing cells treated with cycloheximide (CHX), Dcp2, Xrn1, Dhh1, and Edc3 had significantly diminished condensation compared to those in CHX-treated Nst1-overexpressing cells. These observations suggest that the PD of the IDR in Nst1 functions as a hub domain interacting with other PB components. Full article

One of the major obstacles in treating brain cancers, particularly glioblastoma multiforme, is the occurrence of secondary tumor lesions that arise in areas of the brain and are inoperable while obtaining resistance to current therapeutic agents. Thus, gaining a better understanding of the cellular factors that regulate glioblastoma multiforme cellular movement is imperative. In our study, we demonstrate that the 5′-3′ exoribonuclease XRN2 is important to the invasive nature of glioblastoma. A loss of XRN2 decreases cellular speed, displacement, and movement through a matrix of established glioblastoma multiforme cell lines. Additionally, a loss of XRN2 abolishes tumor formation in orthotopic mouse xenograft implanted with G55 glioblastoma multiforme cells. One reason for these observations is that loss of XRN2 disrupts the expression profile of several cellular factors that are important for tumor invasion in glioblastoma multiforme cells. Importantly, XRN2 mRNA and protein levels are elevated in glioblastoma multiforme patient samples. Elevation in XRN2 mRNA also correlates with poor overall patient survival. These data demonstrate that XRN2 is an important cellular factor regulating one of the major obstacles in treating glioblastomas and is a potential molecular target that can greatly enhance patient survival. Full article

The XRN family of 5′-3′ Exoribonucleases is functionally conserved in eukaryotic organisms. However, the molecular evolution of XRN proteins in plants and their functions in plant response to environment stresses remain largely unexplored. In this study, we identified 23 XRN proteins in 6 representative plant species. Polygenetic analysis revealed that XRN2 was Arabidopsis-specific among these species, and additional branches outside the clades of XRN3 and XRN4 proteins, which we named as XRN5, were found in rice, maize, and soybean. However, XRN5 in soybean lost their entire 5′-3′ XRN Exoribonuclease domain. Protein conserved sequence analysis showed that XRN3/XRN2 contained potential bipartite nuclear-localization signals (NLS) while all the XRN4 proteins lost their second KR/RR motif of NLS, potentially leading to their cytoplasm localization. SIXRN3-2 contained one mutation in this second KR/RR motif, which may change their sub-cellular localization. The promoter cis-element analysis indicated that these XRN genes responded to multiple stresses and plant hormones diversely at transcriptional level. Finally, transcriptomic analysis suggested that OsXRN3 and ZmXRN3-1 were induced by low temperature, SIXRN4 and ZmXRN4 was inhibited by heat shock, and OsXRN5 and GmXRN5-2 were repressed by drought. However, in general, the expression patterns revealed the response diversity of XRNs to environment stimuli in different plant species. Taken together, this study characterized 23 XRNs with NLS variation that contributed to their sub-cellular localization and provided an overview of the XRNs response diversity to multiple environmental stresses, suggesting that XRNs could be used as potential gene editing candidates for precise stress-tolerant crop breeding. Full article

A comparison of overlapping proximity captures at the head region of the ribosomal 40S subunit (hr40S) in Saccharomyces cerevisiae from four adjacent perspectives, namely Asc1/RACK1, Rps2/uS5, Rps3/uS3, and Rps20/uS10, corroborates dynamic co-localization of proteins that control activity and fate of both ribosomes and mRNA. Co-locating factors that associate with the hr40S are involved in (i) (de)ubiquitination of ribosomal proteins (Hel2, Bre5-Ubp3), (ii) clamping of inactive ribosomal subunits (Stm1), (iii) mRNA surveillance and vesicular transport (Smy2, Syh1), (iv) degradation of mRNA (endo- and exonucleases Ypl199c and Xrn1, respectively), (v) autophagy (Psp2, Vps30, Ykt6), and (vi) kinase signaling (Ste20). Additionally, they must be harmonized with translation initiation factors (eIF3, cap-binding protein Cdc33, eIF2A) and mRNA-binding/ribosome-charging proteins (Scp160, Sro9). The Rps/uS-BioID perspectives revealed substantial Asc1/RACK1-dependent hr40S configuration indicating a function of the β-propeller in context-specific spatial organization of this microenvironment. Toward resolving context-specific constellations, a Split-TurboID analysis emphasized the ubiquitin-associated factors Def1 and Lsm12 as neighbors of Bre5 at hr40S. These shuttling proteins indicate a common regulatory axis for the fate of polymerizing machineries for the biosynthesis of proteins in the cytoplasm and RNA/DNA in the nucleus. Full article

3’-Phosphoadenosine 5’-monophosphate (pAp) is a byproduct of sulfate assimilation and coenzyme A metabolism. pAp can inhibit the activity of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) reductase and sulfotransferase and regulate gene expression under stress conditions by inhibiting XRN family of exoribonucleases. In metazoans, plants, yeast, and some bacteria, pAp can be converted into 5’-adenosine monophosphate (AMP) and inorganic phosphate by CysQ. In some bacteria and archaea, nanoRNases (Nrn) from the Asp-His-His (DHH) phosphoesterase superfamily are responsible for recycling pAp. In addition, histidinol phosphatase from the amidohydrolase superfamily can hydrolyze pAp. The bacterial enzymes for pAp turnover and their catalysis mechanism have been well studied, but these processes remain unclear in archaea. Pyrococcus yayanosii, an obligate piezophilic hyperthermophilic archaea, encodes a DHH family pApase homolog (PyapApase). Biochemical characterization showed that PyapApase can efficiently convert pAp into AMP and phosphate. The resolved crystal structure of apo-PyapApase is similar to that of bacterial nanoRNaseA (NrnA), but they are slightly different in the α-helix linker connecting the DHH and Asp-His-His associated 1 (DHHA1) domains. The longer α-helix of PyapApase leads to a narrower substrate-binding cleft between the DHH and DHHA1 domains than what is observed in bacterial NrnA. Through mutation analysis of conserved amino acid residues involved in coordinating metal ion and binding substrate pAp, it was confirmed that PyapApase has an ion coordination pattern similar to that of NrnA and slightly different substrate binding patterns. The results provide combined structural and functional insight into the enzymatic turnover of pAp, implying the potential function of sulfate assimilation in hyperthermophilic cells. Full article

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the “spacer” sequences. The canonical pathway for the removal of the ITS1 spacer involves cleavages at the 3′ end of 18S rRNA and at two sites inside ITS1. The process can generate either a long or a short 5.8S rRNA that differs in the number of ITS1 nucleotides retained at the 5.8S 5′ end. Here we document a novel pathway to the long 5.8S, which bypasses cleavage within ITS1. Instead, the entire ITS1 is degraded from its 5′ end by exonuclease Xrn1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA. Traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. However, results from this work show that the MRP-induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the link between RNase MRP and 5.8S 5′ end formation involves RNase MRP cleavage at unknown sites elsewhere in pre-rRNA or in RNA molecules other than pre-rRNA. Full article