A transthyretin-like protein acts downstream of miR397 and LACCASE to regulate grain yield in rice.

Increasing grain yield is a major goal of breeders due to the rising global demand for food. We previously reported that the miR397-LACCASE (OsLAC) module regulates brassinosteroid (BR) signaling and grain yield in rice (Oryza sativa). However, the precise roles of laccase enzymes in the BR pathway remain unclear. Here, we report that OsLAC controls grain yield by preventing the turnover of TRANSTHYRETIN-LIKE (OsTTL), a negative regulator of BR signaling. Overexpressing OsTTL decreased BR sensitivity in rice, while loss-of-function of OsTTL led to enhanced BR signaling and increased grain yield. OsLAC directly binds to OsTTL and regulates its phosphorylation-mediated turnover. The phosphorylation site Ser226 of OsTTL is essential for its ubiquitination and degradation. Overexpressing the dephosphorylation-mimic form of OsTTL (OsTTLS226A) resulted in more severe defects than did overexpressing OsTTL. These findings provide insight into the role of an ancient laccase in BR signaling and suggest that the OsLAC-OsTTL module could serve as a target for improving grain yield.

Blockade of the lncRNA-DOT1L-LAMP5 axis enhances autophagy and promotes degradation of MLL fusion proteins.

BACKGROUND: Mixed-lineage leukemia (MLL) fusion gene caused by chromosomal rearrangement is a dominant oncogenic driver in leukemia. Due to having diverse MLL rearrangements and complex characteristics, MLL leukemia treated by currently available strategies is frequently associated with a poor outcome. Therefore, there is an urgent need to identify novel therapeutic targets for hematological malignancies with MLL rearrangements. METHODS: qRT-PCR, western blot, and spearman correction analysis were used to validate the regulation of LAMP5-AS1 on LAMP5 expression. In vitro and in vivo experiments were conducted to assess the functional relevance of LAMP5-AS1 in MLL leukemia cell survival. We utilized chromatin isolation by RNA purification (ChIRP) assay, RNA pull-down assay, chromatin immunoprecipitation (ChIP), RNA fluorescence in situ hybridization (FISH), and immunofluorescence to elucidate the relationship among LAMP5-AS1, DOT1L, and the LAMP5 locus. Autophagy regulation by LAMP5-AS1 was evaluated through LC3B puncta, autolysosome observation via transmission electron microscopy (TEM), and mRFP-GFP-LC3 puncta in autophagic flux. RESULTS: The study shows the crucial role of LAMP5-AS1 in promoting MLL leukemia cell survival. LAMP5-AS1 acts as a novel autophagic suppressor, safeguarding MLL fusion proteins from autophagic degradation. Knocking down LAMP5-AS1 significantly induced apoptosis in MLL leukemia cell lines and primary cells and extended the survival of mice in vivo. Mechanistically, LAMP5-AS1 recruits the H3K79 histone methyltransferase DOT1L to LAMP5 locus, directly activating LAMP5 expression. Importantly, blockade of LAMP5-AS1-LAMP5 axis can represses MLL fusion proteins by enhancing their degradation. CONCLUSIONS: The findings underscore the significance of LAMP5-AS1 in MLL leukemia progression through the regulation of the autophagy pathway. Additionally, this study unveils the novel lncRNA-DOT1L-LAMP5 axis as promising therapeutic targets for degrading MLL fusion proteins.

MicroRNA397 promotes rice flowering by regulating the photorespiration pathway.

The precise timing of flowering plays a pivotal role in ensuring successful plant reproduction and seed production. This process is intricately governed by complex genetic networks that integrate internal and external signals. This study delved into the regulatory function of microRNA397 (miR397) and its target gene LACCASE-15 (OsLAC15) in modulating flowering traits in rice (Oryza sativa). Overexpression of miR397 led to earlier heading dates, decreased number of leaves on the main stem, and accelerated differentiation of the spikelet meristem. Conversely, overexpression of OsLAC15 resulted in delayed flowering and prolonged vegetative growth. Through biochemical and physiological assays, we uncovered that miR397-OsLAC15 had a profound impact on carbohydrate accumulation and photosynthetic assimilation, consequently enhancing the photosynthetic intensity in miR397-overexpressing rice plants. Notably, we identified that OsLAC15 is at least partially localized within the peroxisome organelle, where it regulates the photorespiration pathway. Moreover, we observed that a high CO2 concentration could rescue the late flowering phenotype in OsLAC15-overexpressing plants. These findings shed valuable insights into the regulatory mechanisms of miR397-OsLAC15 in rice flowering and provided potential strategies for developing crop varieties with early flowering and high-yield traits through genetic breeding.

New insight into circRNAs: characterization, strategies, and biomedical applications.

Circular RNAs (circRNAs) are a class of covalently closed, endogenous ncRNAs. Most circRNAs are derived from exonic or intronic sequences by precursor RNA back-splicing. Advanced high-throughput RNA sequencing and experimental technologies have enabled the extensive identification and characterization of circRNAs, such as novel types of biogenesis, tissue-specific and cell-specific expression patterns, epigenetic regulation, translation potential, localization and metabolism. Increasing evidence has revealed that circRNAs participate in diverse cellular processes, and their dysregulation is involved in the pathogenesis of various diseases, particularly cancer. In this review, we systematically discuss the characterization of circRNAs, databases, challenges for circRNA discovery, new insight into strategies used in circRNA studies and biomedical applications. Although recent studies have advanced the understanding of circRNAs, advanced knowledge and approaches for circRNA annotation, functional characterization and biomedical applications are continuously needed to provide new insights into circRNAs. The emergence of circRNA-based protein translation strategy will be a promising direction in the field of biomedicine.

Calcium-dependent activation of CPK12 facilitates its cytoplasm-to-nucleus translocation to potentiate plant hypoxia sensing by phosphorylating ERF-VII transcription factors.

Calcium-dependent protein kinases (CDPKs/CPKs) are key regulators of plant stress signaling that translate calcium signals into cellular responses by phosphorylating diverse substrate proteins. However, the molecular mechanism by which plant cells relay calcium signals in response to hypoxia remains elusive. Here, we show that one member of the CDPK family in Arabidopsis thaliana, CPK12, is rapidly activated during hypoxia through calcium-dependent phosphorylation of its Ser-186 residue. Phosphorylated CPK12 shuttles from the cytoplasm to the nucleus, where it interacts with and phosphorylates the group VII ethylene-responsive transcription factors (ERF-VII) that are core regulators of plant hypoxia sensing, to enhance their stabilities. Consistently, CPK12 knockdown lines show attenuated tolerance of hypoxia, whereas transgenic plants overexpressing CPK12 display improved hypoxia tolerance. Nonethelss, loss of function of five ERF-VII proteins in an erf-vii pentuple mutant could partially suppress the enhanced hypoxia-tolerance phenotype of CPK12-overexpressing lines. Moreover, we also discovered that phosphatidic acid and 14-3-3κ protein serve as positive and negative modulators of the CPK12 cytoplasm-to-nucleus translocation, respectively. Taken together, these findings uncover a CPK12-ERF-VII regulatory module that is key to transducing calcium signals from the cytoplasm into the nucleus to potentiate hypoxia sensing in plants.

Poaceae-specific ß-1,3;1,4-d-glucans link jasmonate signalling to OsLecRK1-mediated defence response during rice-brown planthopper interactions.

Brown planthopper (BPH, Nilaparvata lugens), a highly destructive insect pest, poses a serious threat to rice (Oryza sativa) production worldwide. Jasmonates are key phytohormones that regulate plant defences against BPH; however, the molecular link between jasmonates and BPH responses in rice remains largely unknown. Here, we discovered a Poaceae-specific metabolite, mixed-linkage ß-1,3;1,4-d-glucan (MLG), which contributes to jasmonate-mediated BPH resistance. MLG levels in rice significantly increased upon BPH attack. Overexpressing OsCslF6, which encodes a glucan synthase that catalyses MLG biosynthesis, significantly enhanced BPH resistance and cell wall thickness in vascular bundles, whereas knockout of OsCslF6 reduced BPH resistance and vascular wall thickness. OsMYC2, a master transcription factor of jasmonate signalling, directly controlled the upregulation of OsCslF6 in response to BPH feeding. The AT-rich domain of the OsCslF6 promoter varies in rice varieties from different locations and natural variants in this domain were associated with BPH resistance. MLG-derived oligosaccharides bound to the plasma membrane-anchored LECTIN RECEPTOR KINASE1 OsLecRK1 and modulated its activity. Thus, our findings suggest that the OsMYC2-OsCslF6 module regulates pest resistance by modulating MLG production to enhance vascular wall thickness and OsLecRK1-mediated defence signalling during rice-BPH interactions.

METTL3 protects METTL14 from STUB1-mediated degradation to maintain m6 A homeostasis.

N6 -Methyladenosine (m6 A) is an important RNA modification catalyzed by methyltransferase-like 3 (METTL3) and METTL14. m6 A homeostasis mediated by the methyltransferase (MTase) complex plays key roles in various biological processes. However, the mechanism underlying METTL14 protein stability and its role in m6 A homeostasis remain elusive. Here, we show that METTL14 stability is regulated by the competitive interaction of METTL3 with the E3 ligase STUB1. STUB1 directly interacts with METTL14 to mediate its ubiquitination at lysine residues K148, K156, and K162 for subsequent degradation, resulting in a significant decrease in total m6 A levels. The amino acid regions 450-454 and 464-480 of METTL3 are essential to promote METTL14 stabilization. Changes in STUB1 expression affect METTL14 protein levels, m6 A modification and tumorigenesis. Collectively, our findings uncover an ubiquitination mechanism controlling METTL14 protein levels to fine-tune m6 A homeostasis. Finally, we present evidence that modulating STUB1 expression to degrade METTL14 could represent a promising therapeutic strategy against cancer.

MYB30 integrates light signals with antioxidant biosynthesis to regulate plant responses during postsubmergence recovery.

Submergence is an abiotic stress that limits agricultural production world-wide. Plants sense oxygen levels during submergence and postsubmergence reoxygenation and modulate their responses. Increasing evidence suggests that completely submerged plants are often exposed to low-light stress, owing to the depth and turbidity of the surrounding water; however, how light availability affects submergence tolerance remains largely unknown. Here, we showed that Arabidopsis thaliana MYB DOMAIN PROTEIN30 (MYB30) is an important transcription factor that integrates light signaling and postsubmergence stress responses. MYB DOMAIN PROTEIN30 protein abundance decreased upon submergence and accumulated during reoxygenation. Under submergence conditions, CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), a central regulator of light signaling, caused the ubiquitination and degradation of MYB30. In response to desubmergence, however, light-induced MYB30 interacted with MYC2, a master transcription factor involved in jasmonate signaling, and activated the expression of the VITAMIN C DEFECTIVE1 (VTC1) and GLUTATHIONE SYNTHETASE1 (GSH1) gene families to enhance antioxidant biosynthesis. Consistent with this, the myb30 knockout mutant showed increased sensitivity to submergence, which was partially rescued by overexpression of VTC1 or GSH1. Thus, our findings uncover the mechanism by which the COP1-MYB30 module integrates light signals with cellular oxidative homeostasis to coordinate plant responses to postsubmergence stress.

Insights into the roles of long noncoding RNAs in the communication between plants and the environment.

In addition to coding proteins, RNA molecules, especially long noncoding RNAs (lncRNAs), have well-established functions in regulating gene expression. The number of studies focused on the roles played by different types of lncRNAs in a variety of plant biological processes has markedly increased. These lncRNA roles involve plant vegetative and reproductive growth and responses to biotic and abiotic stresses. In this review, we examine the classification, mechanisms, and functions of lncRNAs and then emphasize the roles played by these lncRNAs in the communication between plants and the environment mainly with respect to the following environmental factors: temperature, light, water, salt stress, and nutrient deficiencies. We also discuss the consensus among researchers and the remaining challenges and underscore the exciting ways lncRNAs may affect the biology of plants.

The snoRNA-like lncRNA LNC-SNO49AB drives leukemia by activating the RNA-editing enzyme ADAR1.

Long noncoding RNAs (lncRNAs) are usually 5' capped and 3' polyadenylated, similar to most typical mRNAs. However, recent studies revealed a type of snoRNA-related lncRNA with unique structures, leading to questions on how they are processed and how they work. Here, we identify a novel snoRNA-related lncRNA named LNC-SNO49AB containing two C/D box snoRNA sequences, SNORD49A and SNORD49B; and show that LNC-SNO49AB represents an unreported type of lncRNA with a 5'-end m7G and a 3'-end snoRNA structure. LNC-SNO49AB was found highly expressed in leukemia patient samples, and silencing LNC-SNO49AB dramatically suppressed leukemia progression in vitro and in vivo. Subcellular location indicated that the LNC-SNO49AB is mainly located in nucleolus and interacted with the nucleolar protein fibrillarin. However, we found that LNC-SNO49AB does not play a role in 2'-O-methylation regulation, a classical function of snoRNA; instead, its snoRNA structure affected the lncRNA stability. We further demonstrated that LNC-SNO49AB could directly bind to the adenosine deaminase acting on RNA 1(ADAR1) and promoted its homodimerization followed by a high RNA A-to-I editing activity. Transcriptome profiling shows that LNC-SNO49AB and ADAR1 knockdown respectively share very similar patterns of RNA modification change in downstream signaling pathways, especially in cell cycle pathways. These findings suggest a previously unknown class of snoRNA-related lncRNAs, which function via a manner in nucleolus independently on snoRNA-guide rRNA modification. This is the first report that a lncRNA regulates genome-wide RNA A-to-I editing by enhancing ADAR1 dimerization to facilitate hematopoietic malignancy, suggesting that LNC-SNO49AB may be a novel target in therapy directed to leukemia.

Chromatin-associated orphan snoRNA regulates DNA damage-mediated differentiation via a non-canonical complex.

Small nucleolar RNAs (snoRNAs) are commonly acknowledged as a class of homogeneous non-coding RNAs that guide ribosomal RNA modifications. However, snoRNAs referred to as orphans have largely unknown functions. Here, we systematically profile chromatin-associated snoRNAs (casnoRNAs) in mammalian cells and identify a subgroup of orphan casnoRNAs responding to DNA damage stress, among which SNORA73 shows the most marked reduction in chromatin enrichment. Downregulated SNORA73 maintains cancer genome stability and differentiation block in hematopoietic malignancy. Mechanistically, casnoRNA the 5' end non-canonical structure of SNORA73 is critical for its function and binding to poly (ADP-ribose) polymerase 1 (PARP1). SNORA73 inhibits PARP1 auto-PARylation to affect cancer genome stability by forming a small nucleolar ribonucleoprotein (snoRNP) with PARP1 and canonical H/ACA proteins DKC1/NHP2. Our findings reveal the role of an orphan snoRNA serving as casnoRNA and highlights a link between non-canonical structure of snoRNA and their functional diversity.

Genome-wide analysis and functional annotation of chromatin-enriched noncoding RNAs in rice during somatic cell regeneration.

BACKGROUND: Plants have the remarkable ability to generate callus, a pluripotent cell mass that acquires competence for subsequent tissue regeneration. Global chromatin remodeling is required for this cell fate transition, but how the process is regulated is not fully understood. Chromatin-enriched noncoding RNAs (cheRNAs) are thought to play important roles in maintaining chromatin state. However, whether cheRNAs participate in somatic cell regeneration in plants has not yet been clarified. RESULTS: To uncover the characteristics and functions of cheRNAs during somatic cell reprogramming in plants, we systematically investigate cheRNAs during callus induction, proliferation and regeneration in rice. We identify 2284 cheRNAs, most of which are novel long non-coding RNAs or small nucleolar RNAs. These cheRNAs, which are highly conserved across plant species, shuttle between chromatin and the nucleoplasm during somatic cell regeneration. They positively regulate the expression of neighboring genes via specific RNA motifs, which may interact with DNA motifs around cheRNA loci. Large-scale mutant analysis shows that cheRNAs are associated with plant size and seed morphology. Further detailed functional investigation of two che-lncRNAs demonstrates that their loss of function impairs cell dedifferentiation and plant regeneration, highlighting the functions of cheRNAs in regulating the expression of neighboring genes via specific motifs. These findings support cis- regulatory roles of cheRNAs in influencing a variety of rice traits. CONCLUSIONS: cheRNAs are a distinct subclass of regulatory non-coding RNAs that are required for somatic cell regeneration and regulate rice traits. Targeting cheRNAs has great potential for crop trait improvement and breeding in future.

The parent-of-origin lncRNA MISSEN regulates rice endosperm development.

The cereal endosperm is a major factor determining seed size and shape. However, the molecular mechanisms of endosperm development are not fully understood. Long noncoding RNAs (lncRNAs) function in various biological processes. Here we show a lncRNA, MISSEN, that plays an essential role in early endosperm development in rice (Oryza sativa). MISSEN is a parent-of-origin lncRNA expressed in endosperm, and negatively regulates endosperm development, leading to a prominent dent and bulge in the seed. Mechanistically, MISSEN functions through hijacking a helicase family protein (HeFP) to regulate tubulin function during endosperm nucleus division and endosperm cellularization, resulting in abnormal cytoskeletal polymerization. Finally, we revealed that the expression of MISSEN is inhibited by histone H3 lysine 27 trimethylation (H3K27me3) modification after pollination. Therefore, MISSEN is the first lncRNA identified as a regulator in endosperm development, highlighting the potential applications in rice breeding.

Identification and Characterization of Plant Circular RNAs.

Recent studies have reported that circular RNAs (circRNAs) are a newly discovered type of ubiquitous, abundant and stable noncoding RNAs (ncRNAs) that play important roles in various biological processes in eukaryotic organisms. However, the biological functions of circRNAs in plants remain largely unknown and need further studies. Identification of plant circRNAs from plant circRNA database or sequencing analysis is a first step to investigate their functions. Here, we provide a series of protocols for circRNA identification including circular forms, composition features and location even in plant tissues which are rich in polysaccharides and polyphenols and difficult to extract RNAs.

N6-methyladenosine methyltransferases: functions, regulation, and clinical potential.

N6-methyladenosine (m6A) has emerged as an abundant modification throughout the transcriptome with widespread functions in protein-coding and noncoding RNAs. It affects the fates of modified RNAs, including their stability, splicing, and/or translation, and thus plays important roles in posttranscriptional regulation. To date, m6A methyltransferases have been reported to execute m6A deposition on distinct RNAs by their own or forming different complexes with additional partner proteins. In this review, we summarize the function of these m6A methyltransferases or complexes in regulating the key genes and pathways of cancer biology. We also highlight the progress in the use of m6A methyltransferases in mediating therapy resistance, including chemotherapy, targeted therapy, immunotherapy and radiotherapy. Finally, we discuss the current approaches and clinical potential of m6A methyltransferase-targeting strategies.

Genome-Wide Analysis Identified a Set of Conserved lncRNAs Associated with Domestication-Related Traits in Rice.

Crop domestication, which gives rise to a number of desirable agronomic traits, represents a typical model system of plant evolution. Numerous genomic evidence has proven that noncoding RNAs such as microRNAs and phasiRNAs, as well as protein-coding genes, are selected during crop domestication. However, limited data shows plant long noncoding RNAs (lncRNAs) are also involved in this biological process. In this study, we performed strand-specific RNA sequencing of cultivated rice Oryza sativa ssp. japonica and O. sativa ssp. indica, and their wild progenitor O. rufipogon. We identified a total of 8528 lncRNAs, including 4072 lncRNAs in O. rufipogon, 2091 lncRNAs in japonica rice, and 2365 lncRNAs in indica rice. The lncRNAs expressed in wild rice were revealed to be shorter in length and had fewer exon numbers when compared with lncRNAs from cultivated rice. We also identified a number of conserved lncRNAs in the wild and cultivated rice. The functional study demonstrated that several of these conserved lncRNAs are associated with domestication-related traits in rice. Our findings revealed the feature and conservation of lncRNAs during rice domestication and will further promote functional studies of lncRNAs in rice.

Ubiquitin-dependent Argonauteprotein MEL1 degradation is essential for rice sporogenesis and phasiRNA target regulation.

MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice (Oryza sativa) Argonaute (AGO) protein, has been reported to function specifically at premeiotic and meiotic stages of germ cell development and is associated with a novel class of germ cell-specific small noncoding RNAs called phased small RNAs (phasiRNAs). MEL1 accumulation is temporally and spatially regulated and is eliminated after meiosis. However, the metabolism and turnover (i.e. the homeostasis) of MEL1 during germ cell development remains unknown. Here, we show that MEL1 is ubiquitinated and subsequently degraded via the proteasome pathway in vivo during late sporogenesis. Abnormal accumulation of MEL1 after meiosis leads to a semi-sterile phenotype. We identified a monocot-specific E3 ligase, XBOS36, a CULLIN RING-box protein, that is responsible for the degradation of MEL1. Ubiquitination at four K residues at the N terminus of MEL1 by XBOS36 induces its degradation. Importantly, inhibition of MEL1 degradation either by XBOS36 knockdown or by MEL1 overexpression prevents the formation of pollen at the microspore stage. Further mechanistic analysis showed that disrupting MEL1 homeostasis in germ cells leads to off-target cleavage of phasiRNA target genes. Our findings thus provide insight into the communication between a monocot-specific E3 ligase and an AGO protein during plant reproductive development.