Loss-of-Function of ATS1 Enhances Arabidopsis Salt Tolerance.

Despite the importance of lipid metabolism in various biological processes, little is known about the functionality of ATS1, a plastid glycerol-3-phosphate acyltransferase catalyzing the initial step of the prokaryotic glycerolipids biosynthetic pathway, in plant response to salt stress. In this study, both the loss-of-function mutants and the overexpression lines of ATS1 were analyzed for salt tolerance properties. The results showed that ATS1 overexpression lines had lower seed germination, shoot biomass, chlorophyll content, the proportion of relatively normal pod, and higher root/shoot ratio and anthocyanidin content compared with the wild type. Physiological and biochemical analysis revealed that ats1 mutants had more unsaturated fatty acids to stabilize the plasma membrane under salt damage. Additionally, less induction of three main antioxidant enzymes activity and lower MDA content in ats1 mutants indicated that mutation of the ATS1 gene could reduce the damage extent. Furthermore, the ats1 mutants maintained the K+/Na+ homeostasis by upregulating HAK5 expression to increase K+ absorption and down-regulating HKT1 expression to prevent Na+ uptake. This study suggested that the ATS1 gene negatively affects salt resistance in Arabidopsis.

ARS2 instructs early transcription termination-coupled RNA decay by recruiting ZC3H4 to nascent transcripts.

The RNA-binding ARS2 protein is centrally involved in both early RNA polymerase II (RNAPII) transcription termination and transcript decay. Despite its essential nature, the mechanisms by which ARS2 enacts these functions have remained unclear. Here, we show that a conserved basic domain of ARS2 binds a corresponding acidic-rich, short linear motif (SLiM) in the transcription restriction factor ZC3H4. This interaction recruits ZC3H4 to chromatin to elicit RNAPII termination, independent of other early termination pathways defined by the cleavage and polyadenylation (CPA) and Integrator (INT) complexes. We find that ZC3H4, in turn, forms a direct connection to the nuclear exosome targeting (NEXT) complex, hereby facilitating rapid degradation of the nascent RNA. Hence, ARS2 instructs the coupled transcription termination and degradation of the transcript onto which it is bound. This contrasts with ARS2 function at CPA-instructed termination sites where the protein exclusively partakes in RNA suppression via post-transcriptional decay.

The Effects of Combined 1-Methylcyclopropene and Melatonin Treatment on the Quality Characteristics and Active Oxygen Metabolism of Mango Fruit during Storage.

In this study, mango fruit (Tainong No. 1) was treated with either 0.1 mg/L 1-methylcyclopropene (1-MCP) alone or with a combination of 0.1 mg/L 1-MCP and 0.2 mM melatonin (MT). The mango fruit was then stored for 10 days at 25 °C and 85-90% relative humidity. Quality characteristics and the active oxygen metabolism of postharvest mangoes were evaluated every 2 days. Compared to untreated mango fruit, those with the treatments of 1-MCP alone or 1-MCP + MT had a better appearance and higher levels of soluble sugar, ascorbic acid, and titratable acidity. Moreover, these treatments prevented the loss of fruit firmness, successfully delayed the escalation of a* and b* values, and reduced malondialdehyde content and superoxide anion generation rate. After 10 days of storage, mango fruit treated by 1-MCP alone or 1-MCP + MT exhibited increased activities of antioxidant enzymes such as ascorbate peroxidase, catalase, superoxide dismutase, and other peroxidases; nevertheless, the two treatment protocols maintained higher mango total phenolic content only at the later stage of storage. These findings suggest that mango fruit treated with 1-MCP alone or with 1-MCP + MT improves the quality characteristics and antioxidant activities. Moreover, compared to 1-MCP treatment alone, 1-MCP + MT-treated mangoes exhibited higher quality and a stronger regulation of active metabolism during storage.

3' End sequencing of pA+ and pA- RNAs.

The identity and metabolism of RNAs are often governed by their 5' and 3' ends. Single gene loci produce a variety of transcript isoforms, varying primarily in their RNA 3' end status and consequently facing radically different cellular fates. Knowledge about RNA termini is therefore key to understanding the diverse RNA output from individual transcription units. In addition, the 3' end of a nascent RNA at the catalytic center of RNA polymerase provides a precise and strand-specific measure of the transcription process. Here, we describe a modified RNA 3' end sequencing method, that utilizes the in vivo metabolic labeling of RNA followed by its purification and optional in vitro polyadenylation to provide a comprehensive view of all RNA 3' ends. The strategy offers the advantages of (i) nucleotide resolution mapping of RNA 3' ends, (ii) increased sequencing depth of lowly abundant RNA and (iii) inference of RNA 3' end polyadenylation status. We have used the method to study RNA decay and transcription termination mechanisms with the potential utility to a wider range of biological questions.

Integrator is a genome-wide attenuator of non-productive transcription.

Termination of RNA polymerase II (RNAPII) transcription in metazoans relies largely on the cleavage and polyadenylation (CPA) and integrator (INT) complexes originally found to act at the ends of protein-coding and small nuclear RNA (snRNA) genes, respectively. Here, we monitor CPA- and INT-dependent termination activities genome-wide, including at thousands of previously unannotated transcription units (TUs), producing unstable RNA. We verify the global activity of CPA occurring at pA sites indiscriminately of their positioning relative to the TU promoter. We also identify a global activity of INT, which is largely sequence-independent and restricted to a ~3-kb promoter-proximal region. Our analyses suggest two functions of genome-wide INT activity: it dampens transcriptional output from weak promoters, and it provides quality control of RNAPII complexes that are unfavorably configured for transcriptional elongation. We suggest that the function of INT in stable snRNA production is an exception from its general cellular role, the attenuation of non-productive transcription.

The RNA exosome shapes the expression of key protein-coding genes.

The ribonucleolytic exosome complex is central for nuclear RNA degradation, primarily targeting non-coding RNAs. Still, the nuclear exosome could have protein-coding (pc) gene-specific regulatory activities. By depleting an exosome core component, or components of exosome adaptor complexes, we identify ∼2900 transcription start sites (TSSs) from within pc genes that produce exosome-sensitive transcripts. At least 1000 of these overlap with annotated mRNA TSSs and a considerable portion of their transcripts share the annotated mRNA 3' end. We identify two types of pc-genes, both employing a single, annotated TSS across cells, but the first type primarily produces full-length, exosome-sensitive transcripts, whereas the second primarily produces prematurely terminated transcripts. Genes within the former type often belong to immediate early response transcription factors, while genes within the latter are likely transcribed as a consequence of their proximity to upstream TSSs on the opposite strand. Conversely, when genes have multiple active TSSs, alternative TSSs that produce exosome-sensitive transcripts typically do not contribute substantially to overall gene expression, and most such transcripts are prematurely terminated. Our results display a complex landscape of sense transcription within pc-genes and imply a direct role for nuclear RNA turnover in the regulation of a subset of pc-genes.

A Two-Layered Targeting Mechanism Underlies Nuclear RNA Sorting by the Human Exosome.

Degradation of transcripts in human nuclei is primarily facilitated by the RNA exosome. To obtain substrate specificity, the exosome is aided by adaptors; in the nucleoplasm, those adaptors are the nuclear exosome-targeting (NEXT) complex and the poly(A) (pA) exosome-targeting (PAXT) connection. How these adaptors guide exosome targeting remains enigmatic. Employing high-resolution 3' end sequencing, we demonstrate that NEXT substrates arise from heterogenous and predominantly pA- 3' ends often covering kilobase-wide genomic regions. In contrast, PAXT targets harbor well-defined pA+ 3' ends defined by canonical pA site use. Irrespective of this clear division, NEXT and PAXT act redundantly in two ways: (1) regional redundancy, where the majority of exosome-targeted transcription units produce NEXT- and PAXT-sensitive RNA isoforms, and (2) isoform redundancy, where the PAXT connection ensures fail-safe decay of post-transcriptionally polyadenylated NEXT targets. In conjunction, this provides a two-layered targeting mechanism for efficient nuclear sorting of the human transcriptome.

NRDE2 negatively regulates exosome functions by inhibiting MTR4 recruitment and exosome interaction.

The exosome functions in the degradation of diverse RNA species, yet how it is negatively regulated remains largely unknown. Here, we show that NRDE2 forms a 1:1 complex with MTR4, a nuclear exosome cofactor critical for exosome recruitment, via a conserved MTR4-interacting domain (MID). Unexpectedly, NRDE2 mainly localizes in nuclear speckles, where it inhibits MTR4 recruitment and RNA degradation, and thereby ensures efficient mRNA nuclear export. Structural and biochemical data revealed that NRDE2 interacts with MTR4's key residues, locks MTR4 in a closed conformation, and inhibits MTR4 interaction with the exosome as well as proteins important for MTR4 recruitment, such as the cap-binding complex (CBC) and ZFC3H1. Functionally, MID deletion results in the loss of self-renewal of mouse embryonic stem cells. Together, our data pinpoint NRDE2 as a nuclear exosome negative regulator that ensures mRNA stability and nuclear export.

Exosome cofactor hMTR4 competes with export adaptor ALYREF to ensure balanced nuclear RNA pools for degradation and export.

The exosome is a key RNA machine that functions in the degradation of unwanted RNAs. Here, we found that significant fractions of precursors and mature forms of mRNAs and long noncoding RNAs are degraded by the nuclear exosome in normal human cells. Exosome-mediated degradation of these RNAs requires its cofactor hMTR4. Significantly, hMTR4 plays a key role in specifically recruiting the exosome to its targets. Furthermore, we provide several lines of evidence indicating that hMTR4 executes this role by directly competing with the mRNA export adaptor ALYREF for associating with ARS2, a component of the cap-binding complex (CBC), and this competition is critical for determining whether an RNA is degraded or exported to the cytoplasm. Together, our results indicate that the competition between hMTR4 and ALYREF determines exosome recruitment and functions in creating balanced nuclear RNA pools for degradation and export.

High-density genetic map construction and QTLs identification for plant height in white jute (Corchorus capsularis L.) using specific locus amplified fragment (SLAF) sequencing.

BACKGROUND: Genetic mapping and quantitative trait locus (QTL) detection are powerful methodologies in plant improvement and breeding. White jute (Corchorus capsularis L.) is an important industrial raw material fiber crop because of its elite characteristics. However, construction of a high-density genetic map and identification of QTLs has been limited in white jute due to a lack of sufficient molecular markers. The specific locus amplified fragment sequencing (SLAF-seq) strategy combines locus-specific amplification and high-throughput sequencing to carry out de novo single nuclear polymorphism (SNP) discovery and large-scale genotyping. In this study, SLAF-seq was employed to obtain sufficient markers to construct a high-density genetic map for white jute. Moreover, with the development of abundant markers, genetic dissection of fiber yield traits such as plant height was also possible. Here, we present QTLs associated with plant height that were identified using our newly constructed genetic linkage groups. RESULTS: An F8 population consisting of 100 lines was developed. In total, 69,446 high-quality SLAFs were detected of which 5,074 SLAFs were polymorphic; 913 polymorphic markers were used for the construction of a genetic map. The average coverage for each SLAF marker was 43-fold in the parents, and 9.8-fold in each F8 individual. A linkage map was constructed that contained 913 SLAFs on 11 linkage groups (LGs) covering 1621.4 cM with an average density of 1.61 cM per locus. Among the 11 LGs, LG1 was the largest with 210 markers, a length of 406.34 cM, and an average distance of 1.93 cM between adjacent markers. LG11 was the smallest with only 25 markers, a length of 29.66 cM, and an average distance of 1.19 cM between adjacent markers. 'SNP_only' markers accounted for 85.54% and were the predominant markers on the map. QTL mapping based on the F8 phenotypes detected 11 plant height QTLs including one major effect QTL across two cultivation locations, with each QTL accounting for 4.14-15.63% of the phenotypic variance. CONCLUSIONS: To our knowledge, the linkage map constructed here is the densest one available to date for white jute. This analysis also identified the first QTL in white jute. The results will provide an important platform for gene/QTL mapping, sequence assembly, genome comparisons, and marker-assisted selection breeding for white jute.

Aly and THO are required for assembly of the human TREX complex and association of TREX components with the spliced mRNA.

The mRNA export complex TREX (TREX) is known to contain Aly, UAP56, Tex1 and the THO complex, among which UAP56 is required for TREX assembly. Here, we systematically investigated the role of each human TREX component in TREX assembly and its association with the mRNA. We found that Tex1 is essentially a subunit of the THO complex. Aly, THO and UAP56 are all required for assembly of TREX, in which Aly directly interacts with THO subunits Thoc2 and Thoc5. Both Aly and THO function in linking UAP56 to the cap-binding protein CBP80. Interestingly, association of UAP56 with the spliced mRNA, but not with the pre-mRNA, requires Aly and THO. Unexpectedly, we found that Aly and THO require each other to associate with the spliced mRNA. Consistent with these biochemical results, similar to Aly and UAP56, THO plays critical roles in mRNA export. Together, we propose that Aly, THO and UAP56 form a highly integrated unit to associate with the spliced mRNA and function in mRNA export.