mRNA Cleavage Research Articles

Post-transcriptional control drives Aurora kinase A expression in human cancers

Aurora kinase A (AURKA) is a major regulator of the cell cycle. A prominent association exists between high expression of AURKA and cancer, and impairment of AURKA levels can trigger its oncogenic activity. In order to explore the contribution of post-transcriptional regulation to AURKA expression in different cancers, we carried out a meta-analysis of -omics data of 18 cancer types from The Cancer Genome Atlas (TCGA). Our study confirmed a general trend for increased AURKA mRNA in cancer compared to normal tissues and revealed that AURKA expression is highly dependent on post-transcriptional control in several cancers. Correlation and clustering analyses of AURKA mRNA and protein expression, and expression of AURKA-targeting hsa-let-7a miRNA, unveiled that hsa-let-7a is likely involved to varying extents in controlling AURKA expression in cancers. We then measured differences in the short/long ratio (SLR) of the two alternative cleavage and polyadenylation (APA) isoforms of AURKA mRNA across cancers compared to the respective healthy counterparts. We suggest that the interplay between APA and hsa-let-7a targeting of AURKA mRNA may influence AURKA expression in some cancers. hsa-let-7a and APA may also independently contribute to altered AURKA levels. Therefore, we argue that AURKA mRNA and protein expression are often discordant in cancer as a result of dynamic post-transcriptional regulation.

ALTERED MERISTEM PROGRAM1 impairs RNA silencing by repressing the biogenesis of a subset of inverted repeat-derived siRNAs.

RNA silencing negatively regulates gene expression at the transcriptional and post-transcriptional levels through DNA methylation, histone modification, mRNA cleavage, and translational inhibition. Small interfering RNAs (siRNAs) of 21 to 24 nucleotides are processed from double-stranded RNAs by Dicer-like enzymes and play essential roles in RNA silencing in plants. Here, we demonstrated that ALTERED MERISTEM PROGRAM1 (AMP1) and its putative paralog LIKE AMP1 (LAMP1) impair RNA silencing by repressing the biogenesis of a subset of inverted repeat (IR)-derived siRNAs in Arabidopsis (Arabidopsis thaliana). AMP1 and LAMP1 inhibit Pol II-dependent IR gene transcription by suppressing ARGONAUTE 1 (AGO1) protein levels. Genetic analysis indicates that AMP1 acts upstream of RNA polymerase IV subunit 1 (NRPD1), RNA-dependent RNA polymerase 2 (RDR2), and Dicer-Like 4 (DCL4), which are required for IR-induced RNA silencing. We also show that AMP1 and LAMP1 inhibit siRNA-mediated silencing in a different mechanism from AGO4 and DCL3. Together, these results reveal two previously unknown players in siRNA biogenesis from IRs - AGO1, which promotes IR transcription, and AMP1, which inhibits IR transcription indirectly through the repression of AGO1 expression.

Overexpression of tae-miR9670 enhances cadmium tolerance in wheat by targeting mTERFs without yield penalty

Cadmium (Cd) is a widely distributed heavy metal that poses significant hazards to both crop productivity and human health. MicroRNAs (miRNAs) play pivotal roles in plant growth, development and responses to environmental stresses, yet little is known about their roles in regulating Cd tolerance in wheat. In this study, we identified tae-miR9670, a Triticeae-specific miRNA, as responsive to Cd exposure in wheat through miRNAome analysis. Tae-miR9670 can target genes that encode mitochondrial transcription termination factors (mTERFs), mediating their mRNA cleavage and suppressing their expression. Overexpression of tae-miR9670 significantly enhanced Cd tolerance in wheat seedlings, as demonstrated by increased biomass and reduced levels of malondialdehyde (MDA), H2O2, and Cd content. Consequently, multiple downstream genes involved in ROS scavenging, detoxification and heavy metal transport were upregulated in tae-miR9670 overexpression plants. Moreover, the grain Cd content in mature plants overexpressing tae-miR9670 was reduced by over 60% compared to wild-type controls. Our results also indicated that overexpressing tae-miR9670 in wheat preserved yield-related traits, thereby overcoming the trade-off between stress resistance and grain yield. Overall, our findings provide new insights into the role of tae-miR9670 in Cd tolerance in wheat and its potential application in breeding low-Cd cultivars.

DNA-templated nanosheets for enhanced chemodynamic therapy and gene therapy to inhibit tumor recurrence and metastasis

Recurrence and metastasis stand as the primary contributors to mortality among patients with triple-negative breast cancer post-surgery, presenting a formidable clinical obstacle. Chemodynamic therapy (CDT), leveraging metal-ion-mediated Fenton-like reactions within the tumor microenvironment (TME), emerges as a promising avenue for addressing cancer metastasis. Despite recent progress, challenges such as tumor cell antioxidant defenses and epithelial-mesenchymal transition (EMT) impede the efficacy of CDT. Here, we introduce a novel approach using DNA-templated nanosheets (Dz-MnO2) that combine the functions of Mn2+-mediated CDT and DNAzyme-mediated gene therapy to suppress tumor growth and metastasis. The Dz-MnO2 nanosheets respond effectively to the TME, releasing Mn2+ and DNAzyme. The DNAzyme exhibits mRNA cleavage activity, specifically targeting oncogenic transcripts to reduce tumor progression. Mn2+ not only facilitates a Fenton-like reaction, enhancing the chemodynamic treatment effect, but also serves as a cofactor for DNAzyme, improving its catalytic efficiency. Concurrently, the nanosheets robustly silence the Twist1 gene, mitigating the EMT process and reinforcing CDT efficacy by suppressing apoptosis resistance. Results indicate that Dz-MnO2 nanosheets efficiently polarize M2-tumor-associated macrophages (TAMs) into M1-TAMs by locally mitigating tumor hypoxia via catalyzing the decomposition of H2O2 into O2. This collaborative strategy presents a promising approach to enhance CDT, effectively inhibiting tumor recurrence and metastasis.

Spatially Localized Entropy-Driven Evolution of Nucleic Acid-Based Constitutional Dynamic Networks for Intracellular Imaging and Spatiotemporal Programmable Gene Therapy.

The primer-guided entropy-driven high-throughput evolution of the DNA-based constitutional dynamic network, CDN, is introduced. The entropy gain associated with the process provides a catalytic principle for the amplified emergence of the CDN. The concept is applied to develop a programmable, spatially localized DNA circuit for effective in vitro and in vivo theranostic, gene-regulated treatment of cancer cells. The localized circuit consists of a DNA tetrahedron core modified at its corners with four tethers that include encoded base sequences exhibiting the capacity to emerge and assemble into a [2 × 2] CDN. Two of the tethers are caged by a pair of siRNA subunits, blocking the circuit into a mute, dynamically inactive configuration. In the presence of miRNA-21 as primer, the siRNA subunits are displaced, resulting in amplified release of the siRNAs silencing the HIF-1α mRNA and fast dynamic reconfiguration of the tethers into a CDN. The resulting CDN is, however, engineered to be dynamically reconfigured by miRNA-155 into an equilibrated mixture enriched with a DNAzyme component, catalyzing the cleavage of EGR-1 mRNA. The DNA tetrahedron nanostructure stimulates enhanced permeation into cancer cells. The miRNA-triggered entropy-driven reconfiguration of the spatially localized circuit leads to the programmable, cooperative bis-gene-silencing of HIF-1α and EGR-1 mRNAs, resulting in the effective and selective apoptosis of breast cancer cells and effective inhibition of tumors in tumor bearing mice.

Widespread changes to the translational landscape in a maize microRNA biogenesis mutant.

MicroRNAs are short, non-coding RNAs that repress gene expression in both plants and animals and have diverse functions related to growth, development, and stress responses. The ribonuclease, DICER-LIKE1 (DCL1) is required for two steps in plant miRNA biogenesis: cleavage of the primary miRNAs (pri-miRNAs) to release a hairpin structure, called the precursor miRNA (pre-miRNA) and cleavage of the pre-miRNA to generate the miRNA/miRNA* duplex. The mature miRNA guides the RNA-induced silencing complex to target RNAs with complementary sequences, resulting in translational repression and/or RNA cleavage of target mRNAs. However, the relative contribution of translational repression versus mRNA degradation by miRNAs remains unknown at the genome-level in crops, especially in maize. The maize fuzzy tassel (fzt) mutant contains a hypomorphic mutation in DCL1 resulting in broad developmental defects. While most miRNAs are reduced in fzt, the levels of miRNA-targeted mRNAs are not dramatically increased, suggesting that translational regulation by miRNAs may be common. To gain insight into the repression mechanism of plant miRNAs, we combined ribosome profiling and RNA-sequencing to globally survey miRNA activities in maize. Our data indicate that translational repression contributes significantly to regulation of most miRNA targets and that approximately one-third of miRNA targets are regulated primarily at the translational level. Surprisingly, ribosomes appear altered in fzt mutants suggesting that DCL1 may also have a role in ribosome biogenesis. Thus, DICER-LIKE1 shapes the translational landscape in plants through both miRNA-dependent and miRNA-independent mechanisms.

The Growing Class of Novel RNAi Therapeutics.

The clinical use of RNA interference (RNAi) molecular mechanisms has introduced a novel, growing class of RNA therapeutics capable of treating diseases by controlling target gene expression at the posttranscriptional level. With the newly approved nedosiran (Rivfloza), there are now six RNAi-based therapeutics approved by the United States Food and Drug Administration (FDA). Interestingly, five of the six FDA-approved small interfering RNA (siRNA) therapeutics [patisiran (Onpattro), lumasiran (Oxlumo), inclisiran (Leqvio), vutrisiran (Amvuttra), and nedosiran] were revealed to act on the 3'-untranslated regions of target mRNAs, instead of coding sequences, thereby following the common mechanistic action of genome-derived microRNAs (miRNA). Furthermore, three of the FDA-approved siRNA therapeutics [patisiran, givosiran (Givlaari), and nedosiran] induce target mRNA degradation or cleavage via near-complete rather than complete base-pair complementarity. These features along with previous findings confound the currently held characteristics to distinguish siRNAs and miRNAs or biosimilars, of which all converge in the RNAi regulatory pathway action. Herein, we discuss the RNAi mechanism of action and current criteria for distinguishing between miRNAs and siRNAs while summarizing the common and unique chemistry and molecular pharmacology of the six FDA-approved siRNA therapeutics. The term "RNAi" therapeutics, as used previously, provides a coherently unified nomenclature for broader RNAi forms as well as the growing number of therapeutic siRNAs and miRNAs or biosimilars that best aligns with current pharmacological nomenclature by mechanism of action. SIGNIFICANCE STATEMENT: The common and unique chemistry and molecular pharmacology of six FDA-approved siRNA therapeutics are summarized, in which nedosiran is newly approved. We point out rather a surprisingly mechanistic action as miRNAs for five siRNA therapeutics and discuss the differences and similarities between siRNAs and miRNAs that supports using a general and unified term "RNAi" therapeutics to align with current drug nomenclature criteria in pharmacology based on mechanism of action and embraces broader forms and growing number of novel RNAi therapeutics.

Switching action modes of miR408-5p mediates auxin signaling in rice

MicroRNAs (miRNAs) play fundamental roles in many developmental and physiological processes in eukaryotes. MiRNAs in plants generally regulate their targets via either mRNA cleavage or translation repression; however, which approach plays a major role and whether these two function modes can shift remains elusive. Here, we identify a miRNA, miR408-5p that regulates AUXIN/INDOLE ACETIC ACID 30 (IAA30), a critical repressor in the auxin pathway via switching action modes in rice. We find that miR408-5p usually inhibits IAA30 protein translation, but in a high auxin environment, it promotes the decay of IAA30 mRNA when it is overproduced. We further demonstrate that IDEAL PLANT ARCHITECTURE1 (IPA1), an SPL transcription factor regulated by miR156, mediates leaf inclination through association with miR408-5p precursor promoter. We finally show that the miR156-IPA1-miR408-5p-IAA30 module could be controlled by miR393, which silences auxin receptors. Together, our results define an alternative auxin transduction signaling pathway in rice that involves the switching of function modes by miR408-5p, which contributes to a better understanding of the action machinery as well as the cooperative network of miRNAs in plants.

DCas13-mediated translational repression for accurate gene silencing in mammalian cells

Current gene silencing tools based on RNA interference (RNAi) or, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)‒Cas13 systems have critical drawbacks, such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPR‒Cas13). Thus, a more specific method of gene knockdown is needed. Here, we develop CRISPRδ, an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13). Owing to its tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes during translation initiation and does not affect mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy regardless of the translation initiation mechanism: cap-dependent, internal ribosome entry site (IRES)-dependent, or repeat-associated non-AUG (RAN) translation. Strikingly, genome-wide ribosome profiling reveals the ultrahigh gene silencing specificity of CRISPRδ. Moreover, the fusion of a translational repressor to dCas13 further improves the performance. Our method provides a framework for translational repression-based gene silencing in eukaryotes.

Pleiotropic Effects of miR5504 Underlying Plant Height, Grain Yield and Quality in Rice.

MicroRNAs (miRNAs) are known to play critical roles in regulating rice agronomic traits through mRNA cleavage or translational repression. Our previous study indicated that miR5504 regulates plant height by affecting cell proliferation and expansion. Here, the two independent homozygous mir5504 mutants (CR1 and CR2) and overexpression lines (OE1 and OE2) were further used to investigate the functions of miR5504. The panicle length, 1000-grain weight and grain yield per plant of miR5504-OE lines were identical to those of Nipponbare (NIP), but the 1000-grain weight of mir5504 mutants was reduced by about 10% and 9%, respectively. Meanwhile, the grain width and thickness of mir5504 mutants decreased significantly by approximately 10% and 11%, respectively. Moreover, the cytological results revealed a significant decrease in cell number along grain width direction and cell width in spikelet in mir5504, compared with those in NIP. In addition, several major storage substances of the rice seeds were measured. Compared to NIP, the amylose content of the mir5504 seeds was noticeably decreased, leading to an increase of nearly 10 mm in gel consistency (GC) in mir5504 lines. Further investigation confirmed that LOC_Os08g16914 was the genuine target of miR5504: LOC_Os08g16914 over-expression plants phenocopied the mir5504 mutants. This study provides insights into the role of miR5504 in rice seed development.

CPSF3 inhibition blocks pancreatic cancer cell proliferation through disruption of core histone mRNA processing.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited effective treatment options, potentiating the importance of uncovering novel drug targets. Here, we target cleavage and polyadenylation specificity factor 3 (CPSF3), the 3' endonuclease that catalyzes mRNA cleavage during polyadenylation and histone mRNA processing. We find that CPSF3 is highly expressed in PDAC and is associated with poor prognosis. CPSF3 knockdown blocks PDAC cell proliferation and colony formation in vitro and tumor growth in vivo. Chemical inhibition of CPSF3 by the small molecule JTE-607 also attenuates PDAC cell proliferation and colony formation, while it has no effect on cell proliferation of nontransformed immortalized control pancreatic cells. Mechanistically, JTE-607 induces transcriptional readthrough in replication-dependent histones, reduces core histone expression, destabilizes chromatin structure, and arrests cells in the S-phase of the cell cycle. Therefore, CPSF3 represents a potential therapeutic target for the treatment of PDAC.

Generation of DNAzyme in Bacterial Cells by a Bacterial Retron System.

DNAzymes are catalytically active single-stranded DNAs in which DNAzyme 10-23 (Dz 10-23) consists of a catalytic core and a substrate-binding arm that reduces gene expression through sequence-specific mRNA cleavage. However, the in vivo application of Dz 10-23 depends on exogenous delivery, which leads to its inability to be synthesized and stabilized in vivo, thus limiting its application. As a unique reverse transcription system, the bacterial retron system can synthesize single-stranded DNA in vivo using ncRNA msr/msd as a template. The objective of this work is to reduce target gene expression using Dz 10-23 generated in vivo by the retron system. In this regard, we successfully generated Dz 10-23 by cloning the Dz 10-23 coding sequence into the retron msd gene and tested its ability to reduce specific gene expression by examining the mRNA levels of cfp encoding cyan fluorescence protein and other functional genes such as mreB and ftsZ. We found that Dz had different repressive effects when targeting different mRNA regions, and in general, the repressive effect was stronger when targeting downstream of mRNAs. Our results also suggested that the reduction effect was due to cleavage of the substrate mRNA by Dz 10-23 rather than the antisense effect of the substrate-binding arm. Therefore, this study not only provided a retron-based method for the intracellular generation of Dz 10-23 but also demonstrated that Dz 10-23 could reduce gene expression by cleaving target mRNAs in cells. We believe that this new strategy would have great potential in the regulation of gene expression.

Therapeutic siRNAs: small molecules with bigger function

In the realm of molecular medicine, therapeutic small-interfering RNA (siRNA) is a promising class of molecules with exciting new opportunities in healthcare. An essential component of RNA interference (RNAi) is siRNA. In clinical setup, siRNA may be engineered to specifically target genes linked to certain diseases, providing a focused and accurate method of modulating gene expression. Many important factors characterize the use of therapeutic siRNA. Target gene expression is selectively silenced by therapeutic siRNA through the use of the RNAi pathway. Guided by corresponding messenger RNA (mRNA) sequences, the double-stranded siRNA molecule is integrated into the RNA-induced silencing complex (RISC). This mechanism causes translational repression or mRNA cleavage, which in turn causes the relevant protein to be downregulated. Therapeutic siRNA is a promising avenue in several areas. siRNA can be used in oncology to target oncogenes or other important regulators, inhibiting cancer cell proliferation. Furthermore, by modifying the expression of genes linked to disease, siRNA may be used to treat viral infections, neurological conditions, and other rare and hereditary diseases. The selectivity of therapeutic siRNA is one of its main advantages. siRNA sequences can be engineered and customized to target specific genes linked to different diseases like cancer or genetic disorders. This focused strategy improves the accuracy of the therapeutic intervention and reduces off-target effects. In a recent article by Nissen et al., siRNA targeting LP(a) in a trial with 48 participants without cardiovascular disease and with lipoprotein(a) concentrations of 75 nmol/L or greater (or ≥30 mg/dL), was used to evaluate its tolerability and efficacy to reduce LP(a). Results showed that Lepodisiran was found to be well tolerated and resulted in dose-dependent, long-lasting reduction in serum lipoprotein(a) concentrations in phase 1 trial with elevated lipoprotein(a) levels. Therapeutic siRNA-based diagnosis is rapidly advancing, and several siRNA-based medications are now entering clinical trials. These trials aim to assess the pharmacokinetics, safety, and effectiveness of siRNA therapeutics in human subjects, offering crucial new information about their potential as a treatment option. Despite the therapeutic potential of siRNA, clinical translation of siRNA faces a challenge in its effective delivery. Naked siRNA molecules are more likely to degrade with a poor half-life and poor cellular uptake. Therefore, to increase the stability and delivery of therapeutic siRNA to target cells, a variety of delivery systems, such as lipid nanoparticles, viral vectors, and polymer-based carriers, are being investigated.
 

RNase H-dependent DNA thresholder modulated by cancer marker concentration.

Threshold antisense oligonucleotide constructs were designed to cleave mRNA within different biomarker concentrations. The mRNA cleavage is activated by 2.6, 7.5 or 39.5 nM of biomarker depending on the construct design. The constructs can be used to differentiate cancer from normal cells by the level of oncogene expression followed by silencing of a targeted gene.

PemK's Arg24 is a crucial residue for PemIK toxin-antitoxin system to induce the persistence of Weissella cibaria against ciprofloxacin stress.

The toxin-antitoxin (TA) system plays a key role in bacteria escaping antibiotic stress with persistence, however, the mechanisms by which persistence is controlled remain poorly understood. Weissella cibaria, a novel probiotic, can enters a persistent state upon encountering ciprofloxacin stress. Conversely, it resumes from the persistence when ciprofloxacin stress is relieved or removed. Here, it was found that PemIK TA system played a role in transitioning between these two states. And the PemIK was consisted of PemK, an endonuclease toxic to mRNA, and antitoxin PemI which neutralized its toxicity. The PemK specifically cleaved the U↓AUU in mRNA encoding enzymes involved in glycolysis, TCA cycle and respiratory chain pathways. This cleavage event subsequently disrupted the crucial cellular processes such as hydrogen transfer, electron transfer, NADH and FADH2 synthesis, ultimately leading to a decrease in ATP levels and an increase in membrane depolarization and persister frequency. Notably, Arg24 was a critical active residue for PemK, its mutation significantly reduced the mRNA cleavage activity and the adverse effects on metabolism. These insights provided a clue to comprehensively understand the mechanism by which PemIK induced the persistence of W. cibaria to escape ciprofloxacin stress, thereby highlighting another novel aspect PemIK respond for antibiotic stress.

Genome-Wide Identification of Cotton MicroRNAs Predicted for Targeting Cotton Leaf Curl Kokhran Virus-Lucknow

Cotton leaf curl Kokhran virus (CLCuKoV) (genus, Begomovirus; family, Geminiviridae) is one of several plant virus pathogens of cotton (Gossypium hirsutum L.) that cause cotton leaf curl disease in Pakistan. Begomoviruses are transmitted by the whitefly Bemisia tabaci cryptic species group and cause economic losses in cotton and other crops worldwide. The CLCuKoV strain, referred to as CLCuKoV-Bur, emerged in the vicinity of Burewala, Pakistan, and was the primary causal virus associated with the second CLCuD epidemic in Pakistan. The monopartite ssDNA genome of (2.7 Kb) contains six open reading frames that encode four predicted proteins. RNA interference (RNAi)-mediated antiviral immunity is a sequence-specific biological process in plants and animals that has evolved to combat virus infection. The objective of this study was to design cotton locus-derived microRNA (ghr-miRNA) molecules to target strains of CLCuKoV, with CLCuKoV-Lu, as a typical CLCuD-begomovirus genome, predicted by four algorithms, miRanda, RNA22, psRNATarget, and RNA hybrid. Mature ghr-miRNA sequences (n = 80) from upland cotton (2n = 4x = 52) were selected from miRBase and aligned with available CLCuKoV-Lu genome sequences. Among the 80 cotton locus-derived ghr-miRNAs analyzed, ghr-miR2950 was identified as the most optimal, effective ghr-miRNA for targeting the CLCuKoV-Lu genome (nucleotide 82 onward), respectively, based on stringent criteria. The miRNA targeting relies on the base pairing of miRNA–mRNA targets. Conservation and potential base pairing of binding sites with the ghr-miR2950 were validated by multiple sequence alignment with all available CLCuKoV sequences. A regulatory interaction network was constructed to evaluate potential miRNA–mRNA interactions with the predicted targets. The efficacy of miRNA targeting of CLCuKoV was evaluated in silico by RNAi-mediated mRNA cleavage. This predicted targets for the development of CLCuD-resistant cotton plants.

SCARPET: site-specific quantification of methylated and nonmethylated adenosines reveals m6A stoichiometry.

m6A has different stoichiometry at different positions in different mRNAs. However, the exact stoichiometry of m6A is difficult to measure. Here, we describe SCARPET (site-specific cleavage and radioactive-labeling followed by purification, exonuclease digestion, and thin-layer chromatography), a simple and streamlined biochemical assay for quantifying m6A at any specific site in any mRNA. SCARPET involves a site-specific cleavage of mRNA immediately 5' of an adenosine site in an mRNA. This site is radiolabeled with 32P, and after a series of steps to purify the RNA and to remove nonspecific signals, the nucleotide is resolved by TLC to visualize A and m6A at this site. Quantification of these spots reveals the m6A stoichiometry at the site of interest. SCARPET can be applied to poly(A)-enriched RNA, or preferably purified mRNA, which produces more accurate m6A stoichiometry measurements. We show that sample processing steps of SCARPET can be performed in a single day, and results in a specific and accurate measurement of m6A stoichiometry at specific sites in mRNA. Using SCARPET, we measure exact m6A stoichiometries in specific mRNAs and show that Zika genomic RNA lacks m6A at previously mapped sites. SCARPET will be useful for testing specific sites for their m6A stoichiometry and to assess how m6A stoichiometry changes in different conditions and cellular contexts.

B. subtilis MutS2 splits stalled ribosomes into subunits without mRNA cleavage.

Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through the recruitment of SmrB, a nuclease that cleaves the mRNA. In B. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains, and we reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation through the ribosome quality control pathway. However, unlike SmrB, which cleaves mRNA in E. coli, we see no evidence that MutS2 mediates mRNA cleavage or promotes ribosome rescue by tmRNA. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in B. subtilis and raise questions about how these pathways function differently in diverse bacteria.

MRNA cleavage by 21-nucleotide phasiRNAs determines temperature-sensitive male sterility in rice.

Temperature-sensitive male sterility is one of the core components for hybrid rice (Oryza sativa) breeding based on the 2-line system. We previously found that knockout of ARGONAUTE 1d (AGO1d) causes temperature-sensitive male sterility in rice by influencing phased small interfering RNA (phasiRNA) biogenesis and function. However, the specific phasiRNAs and their targets underlying the temperature-sensitive male sterility in the ago1d mutant remain unknown. Here, we demonstrate that the ago1d mutant displays normal female fertility but complete male sterility at low temperature. Through a multiomics analysis of small RNA (sRNA), degradome, and transcriptome, we found that 21-nt phasiRNAs account for the greatest proportion of the 21-nt sRNA species in rice anthers and are sensitive to low temperature and markedly downregulated in the ago1d mutant. Moreover, we found that 21-nt phasiRNAs are essential for the mRNA cleavage of a set of fertility- and cold tolerance-associated genes, such as Earlier Degraded Tapetum 1 (EDT1), Tapetum Degeneration Retardation (TDR), OsPCF5, and OsTCP21, directly or indirectly determined by AGO1d-mediated gene silencing. The loss of function of 21-nt phasiRNAs can result in upregulation of their targets and causes varying degrees of defects in male fertility and grain setting. Our results highlight the essential functions of 21-nt phasiRNAs in temperature-sensitive male sterility in rice and suggest their promising application in 2-line hybrid rice breeding in the future.

Alternative cleavage and polyadenylation of the Ccnb1 mRNA defines accumulation of cyclin protein during the meiotic cell cycle.

Progression through the mitotic and meiotic cell cycle is driven by fluctuations in the levels of cyclins, the regulatory subunits controlling the localization and activity of CDK1 kinases. Cyclin levels are regulated through a precise balance of synthesis and degradation. Here we demonstrate that the synthesis of Cyclin B1 during the oocyte meiotic cell cycle is defined by the selective translation of mRNA variants generated through alternative cleavage and polyadenylation (APA). Using gene editing in mice, we introduced mutations into the proximal and distal polyadenylation elements of the 3' untranslated region (UTR) of the Ccnb1 mRNA. Through in vivo loss-of-function experiments, we demonstrate that the translation of mRNA with a short 3' UTR specifies Cyclin B1 protein levels that set the timing of meiotic re-entry. In contrast, translation directed by a long 3' UTR is necessary to direct Cyclin B1 protein accumulation during the MI/MII transition. These findings establish that the progression through the cell cycle is dependent on the selective translation of multiple mRNA variants generated by APA.