Small RNAs reveal two target sites of the RNA-maturation factor Mbb1 in the chloroplast of Chlamydomonas

Many chloroplast transcripts are protected against exonucleolytic degradation by RNA-binding proteins. Such interactions can lead to the accumulation of short RNAs (sRNAs) that represent footprints of the protein partner. By mining existing data sets of Chlamydomonas reinhardtii small RNAs, we identify chloroplast sRNAs. Two of these correspond to the 5′-ends of the mature psbB and psbH messenger RNAs (mRNAs), which are both stabilized by the nucleus-encoded protein Mbb1, a member of the tetratricopeptide repeat family. Accordingly, we find that the two sRNAs are absent from the mbb1 mutant. Using chloroplast transformation and site-directed mutagenesis to survey the psbB 5′ UTR, we identify a cis-acting element that is essential for mRNA accumulation. This sequence is also found in the 5′ UTR of psbH, where it plays a role in RNA processing. The two sRNAs are centered on these cis-acting elements. Furthermore, RNA binding assays in vitro show that Mbb1 associates with the two elements specifically. Taken together, our data identify a conserved cis-acting element at the extremity of the psbH and psbB 5′ UTRs that plays a role in the processing and stability of the respective mRNAs through interactions with the tetratricopeptide repeat protein Mbb1 and leads to the accumulation of protected sRNA

Maternal exposure to life events during pregnancy and congenital heart disease in offspring: a case-control study in a Chinese population

Background: Previous studies have suggested that maternal stress could increase the risk of some adverse pregnancy outcomes, but evidence on congenital heart disease (CHD) is limited. We aimed to explore the association between maternal exposure to life events during pregnancy and CHD in offspring. Methods: The data was based on an unmatched case-control study about CHD conducted in Shaanxi province of China from 2014 to 2016. We included 2280 subjects, 699 in the case group and 1581 in the control group. The cases were infants or fetuses diagnosed with CHD, and the controls were infants without any birth defects. The life events were assessed by the Life Events Scale for Pregnant Women, and were divided into positive and negative events for synchronous analysis. A directed acyclic graph was drawn to screen the confounders. Logistic regression was employed to estimate the odds ratio and 95% confidence interval for the effects of life events on CHD. Results: After controlling for the potential confounders, the pregnant women experiencing the positive events during pregnancy had lower risk of CHD in offspring than those without positive events (OR = 0.38, 95%CI: 0.30 ~ 0.48). The risk of CHD in offspring could increase by 62% among the pregnant women experiencing the negative events compared to those without (OR = 1.62, 95%CI: 1.29 ~ 2.03). Both effects showed a certain dose-response association. Besides, the positive events could weaken the risk impact of negative events on CHD. Conclusion: It may suggest that maternal exposure to negative life events could increase the risk of CHD in offspring, while experiencing positive events could play a potential protective role

Ectopic Transplastomic Expression of a Synthetic MatK Gene Leads to Cotyledon-Specific Leaf Variegation

Chloroplasts (and other plastids) harbor their own genetic material, with a bacterial-like gene-expression systems. Chloroplast RNA metabolism is complex and is predominantly mediated by nuclear-encoded RNA-binding proteins. In addition to these nuclear factors, the chloroplast-encoded intron maturase MatK has been suggested to perform as a splicing factor for a subset of chloroplast introns. MatK is essential for plant cell survival in tobacco, and thus null mutants have not yet been isolated. We therefore attempted to over-express MatK from a neutral site in the chloroplast, placing it under the control of a theophylline-inducible riboswitch. This ectopic insertion of MatK lead to a variegated cotyledons phenotype. The addition of the inducer theophylline exacerbated the phenotype in a concentration-dependent manner. The extent of variegation was further modulated by light, sucrose and spectinomycin, suggesting that the function of MatK is intertwined with photosynthesis and plastid translation. Inhibiting translation in the transplastomic lines has a profound effect on the accumulation of several chloroplast mRNAs, including the accumulation of an RNA antisense to rpl33, a gene coding for an essential chloroplast ribosomal protein. Our study further supports the idea that MatK expression needs to be tightly regulated to prevent detrimental effects and establishes another link between leaf variegation and chloroplast translation

Analysis of targets and functions of the chloroplast intron maturase MatK

In Chloroplasten durchlaufen primäre Transkripte eine großen Anzahl von bzw. Reifungsprozesse. Diese Ereignisse spielen eine wichtige Rolle bei der Regulation der Genexpression und sind im Wesentlichen durch Proteinfaktoren, insbesondere RNA-Bindeproteine, reguliert. Der plastidäre Spleißfaktor MatK zählt zu den prokaryotischen Gruppe-II-Intron. MatK aus Nicotiana tabacum interagiert mit seinem Heimatintron trnK und sechs weiteren Gruppe IIA Introns. In dieser Untersuchung, MatK-Bindestellen konnten unterschiedlichen Regionen der Gruppe-II-Introns zugewiesen werden mit RIP-seq in Nicotiana tabacum. Die vorliegenden Ergebnisse zeigen, dass MatK im Vergleich zu seinen bakteriellen Vorfahren an Vielseitigkeit in der RNA-Erkennung gewonnen hat. MatK zeigt somit beispielhaft, wie eine Maturase die Fähigkeit erworben haben könnte, in trans auf mehrere Introns zu wirken. Quantitative Untersuchung und mathematische Modellierung der Expression von MatK und dessen Zielen offenbart ein komplexes Muster möglicher regulatorischer Feedback-Mechanismen. In dieser Studie konnte ein möglicher Feedback- Mechanismus durch Analyse von polysomal gebundenen Transkripten ausgeschlossen werden. Stabile Bindung von Proteinen an spezifische RNA-Bindestellen und anschließender Abbau der ungeschützten RNA kann zu Akkumulation von kleinen RNAs (sRNAs) führen. Solche Footprints von RNA-Bindeproteinen wurden durch die Untersuchung von Datensätzen kleiner RNAs in Chlamydomonas reinhardtii identifiziert. Zwei der sRNAs entsprechen den 5'' Enden der reifen psbB und psbH mRNAs. Beide sRNAs sind abhängig von Mbb1, einem TPR (Tetratrico-peptide repeat) Protein. Die beiden sRNAs besitzen eine hohe Ähnlichkeit in ihrer Primärsequenz und fehlen in der mbb1 Mutante. Dies legt nahe, dass auch andere der hier identifizierten sRNAs an 5'' Enden plastidärer mRNAs Protein-Bindestellen repräsentieren, die für die korrekte RNA-Prozessierung und RNA-Stabilisierung in Chlamydomonas Chloroplasten erforderlich sind.In chloroplasts, primary transcripts are subjected to a number of processing events. These events play important roles in the regulation of gene expression and are extensively controlled by protein factors, especially by RNA-binding proteins. Chloroplast splicing factor MatK is related to prokaryotic group II intron maturases. Nicotiana tabacum MatK interacts with its home intron trnK and six additional group IIA introns. In this study, binding sites of MatK were narrowed down to varying regions of its group II targets by RIP-seq in Nicotiana tabacum. The results obtained demonstrate that MatK has gained versatility in RNA recognition relative to its bacterial ancestors. MatK thus exemplifies how a maturase could have gained the ability to act in trans on multiple introns during the dispersion of the group II introns through the eukaryotic genome early in the eukaryote evolution. Quantitative investigation and mathematical modeling of the expression of MatK and its targets revealed a complex pattern of possible feedback regulatory interactions. In this study, one possible feedback regulation mechanism was ruled out by the analysis of polysome associated transcripts. Stable binding of proteins to specific RNA sites and subsequent degradation of the unprotected RNA regions can result in small RNA, footprint of the RNA binding protein. Such footprints were identified by examining small RNA datasets of Chlamydomonas reinhardtii. Two of the sRNAs correspond to the 5’ ends of mature psbB and psbH mRNAs. Both sRNAs are dependent on Mbb1, a nuclear-encoded TPR (Tetratrico-peptide repeat) protein. The two sRNAs have high similarity in primary sequence, and both are absent in the mbb1 mutant. This suggests that sRNAs at the 5’ ends of chloroplast mRNAs identified here generally represent the binding sites of proteins, which function in RNA processing and RNA stabilization in Chlamydomonas chloroplast

Factors Controlling Shale Reservoirs and Development Potential Evaluation: A Case Study

The shale of the Lower Silurian Longmaxi Formation is an important gas-producing layer for shale gas development in southern China. This set of shale reservoir characteristics and shale gas development potential provide an important foundation for shale gas development. This study takes wellblock XN111 in the Sichuan Basin, China, as an example and uses X-ray diffraction (XRD), scanning electron microscopy (SEM), isothermal adsorption, and other techniques to analyze the shale reservoir characteristics of the Lower Silurian Longmaxi Formation. The results show that the Lower Silurian Longmaxi Formation was deposited in a deep-water shelf environment. During this period, carbonaceous shale and siliceous shale characterized by a high brittle mineral content (quartz>40 wt.%, carbonate mineral>10 wt.%) and a low clay mineral content (<30 wt.%, mainly illite) were widely deposited throughout the region. The total organic carbon (TOC) content reaches up to 6.07 wt.%, with an average of 2.66 wt.%. The vitrinite reflectance is 1.6–2.28%, with an average of 2.05%. The methane adsorption capacity is 0.84–4.69 m3/t, with an average of 2.92 m3/t. Pores and fractures are developed in the shale reservoirs. The main reservoir space is composed of connected mesopores with an average porosity of 4.78%. The characteristics and development potential of the shale reservoirs in the Lower Silurian Longmaxi Formation are controlled by the following factors: (1) the widespread deep-water shelf deposition in wellblock XN111 was a favorable environment for the development of high-quality shale reservoirs with a cumulative thickness of up to 50 m; (2) the high TOC content enabled the shale reservoir to have a high free gas content and a high adsorptive gas storage capacity; and (3) the shale’s high maturity or over maturity is conducive to the development of pores and fractures in the organic matter, which effectively improves the storage capacity of the shale reservoirs. The reservoir characteristic index was constructed using the high-quality shale’s thickness, gas content, TOC, fracture density, and clay content. Using production data from shale gas wells in adjacent blocks, a mathematical relationship was established between the Estimated Ultimate Recovery (EUR) of a single well and the Reservoir Characteristics Index (Rci). The EUR of a single well in wellblock XN111 was estimated

A Nucleus-Encoded Chloroplast Phosphoprotein Governs Expression of the Photosystem I Subunit PsaC in Chlamydomonas reinhardtii

The nucleo-cytoplasmic compartment exerts anterograde control on chloroplast gene expression through numerous proteins that intervene at posttranscriptional steps. Here, we show that the maturation of psaC mutant (mac1) of Chlamydomonas reinhardtii is defective in photosystem I and fails to accumulate psaC mRNA. The MAC1 locus encodes a member of the Half-A-Tetratricopeptide (HAT) family of super-helical repeat proteins, some of which are involved in RNA transactions. The Mac1 protein localizes to the chloroplast in the soluble fraction. MAC1 acts through the 5' untranslated region of psaC transcripts and is required for their stability. Small RNAs that map to the 5'end of psaC RNA in the wild type but not in the mac1 mutant are inferred to represent footprints of MAC1-dependent protein binding, and Mac1 expressed in bacteria binds RNA in vitro. A coordinate response to iron deficiency, which leads to dismantling of the photosynthetic electron transfer chain and in particular of photosystem I, also causes a decrease of Mac1. Overexpression of Mac1 leads to a parallel increase in psaC mRNA but not in PsaC protein, suggesting that Mac1 may be limiting for psaC mRNA accumulation but that other processes regulate protein accumulation. Furthermore, Mac 1 is differentially phosphorylated in response to iron availability and to conditions that alter the redox balance of the electron transfer chain

Extracellular Metabolites of Heterotrophic Auxenochlorella protothecoides: A New Source of Bio-Stimulants for Higher Plants

The biodiversity of microalgal species is enormous, and their versatile metabolism produces a wide diversity of compounds that can be used in food, healthcare, and other applications. Microalgae are also a potential source of bio-stimulants that enhance nutrition efficiency, abiotic stress tolerance, and/or crop quality traits. In this study, the extracellular metabolites of Auxenochlorella protothecoides (EAp) were prepared using three different culture strategies, and their effects on plant growth were examined. Furthermore, the composition of EAp was analyzed by GC-MS. The elongation of lateral roots and the cold-tolerance of Arabidopsis thaliana and Nicotiana benthamiana were promoted by EAp. Moreover, EAp from high-cell-density fermentation stimulated the growth of the leafy vegetables Brassica rapa and Lactuca sativa at dilutions as high as 500- and 1000-fold. Three major groups of compounds were identified by GC-MS, including organic acids or organic acid esters, phenols, and saccharides. Some of these compounds have known plant&ndash;stimulating effects, while the rest requires further investigation in the future. Our study demonstrates that EAp is a potential bio-stimulant, while also providing an environmentally friendly and economical microalgae fermentation process

Case of cryptic TNIP1::PDGFRB rearrangement presenting with myelodysplastic syndrome achieved hematologic and cytogenetic remission with low-dose imatinib plus decitabine therapy

For a long time, FIP1L1::PDGFRA fusion seems to be the only cryptic rearrangement of myeloid/lymphoid neoplasm with tyrosine kinase gene fusions. Recently, with the wide application of RNA sequencing, more cryptic rearrangements of other TK genes have been identified, especially the PDGFRB. Here we report a case of myelodysplastic syndrome with severe thrombocytopenia. Conventional karyotype analysis revealed a t (5;19) (q33; p13.2) but no PDGFRB rearrangement was detected by the PDGFRB break-apart probe. The TNIP1::PDGFRB fusion was eventually found by RNA sequencing, leading us to treat with low-dose imatinib plus decitabine, and the patient achieved hematologic improvement and cytogenetic remission