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Conversely to canonical splicing, back-splicing connects the upstream 3' splice site (SS) with a downstream 5'SS and generates exonic circular RNAs (circRNAs) that are widely identified and have regulatory functions in eukaryotic gene expression. However, sex-specific back-splicing in Drosophila has not been investigated and its regulation remains unclear. Here, we performed multiple RNA analyses of a variety sex-specific Drosophila samples and identified over ten thousand circular RNAs, in which hundreds are sex-differentially and -specifically back-spliced. Intriguingly, we found that expression of SXL, an RNA-binding protein encoded by Sex-lethal (Sxl), the master Drosophila sex-determination gene that is only spliced into functional proteins in females, promoted back-splicing of many female-differential circRNAs in the male S2 cells, whereas expression of a SXL mutant (SXLRRM) did not promote those events. Using a monoclonal antibody, we further obtained the transcriptome-wide RNA-binding sites of SXL through PAR-CLIP. After splicing assay of mini-genes with mutations in the SXL-binding sites, we revealed that SXL-binding on flanking exons and introns of pre-mRNAs facilitates back-splicing, whereas SXL-binding on the circRNA exons inhibits back-splicing. This study provides strong evidence that SXL has a regulatory role in back-splicing to generate sex-specific and -differential circRNAs, as well as in the initiation of sex-determination cascade through canonical forward-splicing.

A flame-retardant polylactic acid composite added with piperazine pyrophosphate and melamine cyanurate was developed. Piperazine pyrophosphate and melamine cyanurate showed synergistic effect in the polylactic acid composites. When the weight ratio of piperazine pyrophosphate to melamine cyanurate was 4:1 and the loading of the piperazine pyrophosphate/melamine cyanurate flame retardant was 15 wt.%, the prepared polylactic acid composites passed V-0 rating in the UL-94 test and achieved the high limit oxygen index value of 34.9%. The cone calorimeter test result confirmed that the addition of piperazine pyrophosphate/melamine cyanurate flame retardant reduced the total heat release value of polylactic acid. The effect of polylactic acid biodegradation on the flame retardancy of polylactic acid composite was further discussed. The biodegraded polylactic acid, which was exposed to the air for 8 months, showed high flame retardancy and even passed V-0 rating in the UL-94 test without the addition of piperazine pyrophosphate/melamine cyanurate flame retardant, which proved that biodegradation could affect the flame retardancy of polylactic acid.

Catalyzed by spliceosomes in the nucleus, RNA splicing removes intronic sequences from precursor RNAs in eukaryotes to generate mature RNA, which also significantly increases proteome complexity and fine-tunes gene expression. Most metazoans have two coexisting spliceosomes; the major spliceosome, which removes99.5% of introns, and the minor spliceosome, which removes far fewer introns (only 770 at present have been predicted in the human genome). Both spliceosomes are large and dynamic machineries, each consisting of five small nuclear RNAs (snRNAs) and more than 100 proteins. However, the dynamic assembly, catalysis, and protein composition of the minor spliceosome are still poorly understood. With different splicing signals, minor introns are rare and usually distributed alone and flanked by major introns in genes, raising questions of how they are recognized by the minor spliceosome and how their processing deals with the splicing of neighboring major introns. Due to large numbers of introns and close similarities between the two machinery, cooperative, and competitive recognition by the two spliceosomes has been investigated. Functionally, many minor-intron-containing genes are evolutionarily conserved and essential. Mutations in the minor spliceosome exhibit a variety of developmental defects in plants and animals and are linked to numerous human diseases. Here, we review recent progress in the understanding of minor splicing, compare currently known components of the two spliceosomes, survey minor introns in a wide range of organisms, discuss cooperation and competition of the two spliceosomes in splicing of minor-intron-containing genes, and contributions of minor splicing mutations in development and diseases. This article is categorized under: RNA ProcessingProcessing of Small RNAs RNA ProcessingSplicing Mechanisms RNA Structure and DynamicsRNA Structure, Dynamics and Chemistry.

G-quadruplexes (G4s) have been shown to be involved in the regulation of multiple cellular processes. Exploring putative G4-forming sequences (PQSs) in heat-responsive genes of rice and their folding structures under different conditions will help to understand the mechanism in response to heat stress. In this work, we discovered a prevalence of PQSs in nuclease hypersensitive sites within the promoters of heat-responsive genes. Moreover, 50 % of the searched G

Transcription and pre-mRNA splicing are coupled to promote gene expression and regulation. However, mechanisms by which transcription and splicing influence each other are still under investigation. The ATPase Prp5p is required for pre-spliceosome assembly and splicing proofreading at the branch-point region. From an open UV mutagenesis screen for genetic suppressors of prp5 defects and subsequent targeted testing, we identify components of the TBP-binding module of the Spt–Ada–Gcn5 Acetyltransferase (SAGA) complex, Spt8p and Spt3p. Spt8Δ and spt3Δ rescue the cold-sensitivity of prp5-GAR allele, and prp5 mutants restore growth of spt8Δ and spt3Δ strains on 6-azauracil. By chromatin immunoprecipitation (ChIP), we find that prp5 alleles decrease recruitment of RNA polymerase II (Pol II) to an intron-containing gene, which is rescued by spt8Δ. Further ChIP-seq reveals that global effects on Pol II-binding are mutually rescued by prp5-GAR and spt8Δ. Inhibited splicing caused by prp5-GAR is also restored by spt8Δ. In vitro assays indicate that Prp5p directly interacts with Spt8p, but not Spt3p. We demonstrate that Prp5p's splicing proofreading is modulated by Spt8p and Spt3p. Therefore, this study reveals that interactions between the TBP-binding module of SAGA and the spliceosomal ATPase Prp5p mediate a balance between transcription initiation/elongation and pre-spliceosome assembly.

Phytophthora effector PSR1 suppresses small RNA (sRNA)-mediated immunity in plants, but the underlying mechanism remains unknown. Here, we show that Phytophthora suppressor of RNA silencing 1 (PSR1) contributes to the pathogenicity of Phytophthora sojae and specifically binds to three conserved C-terminal domains of the eukaryotic PSR1-Interacting Protein 1 (PINP1). PINP1 encodes PRP16, a core pre-mRNA splicing factor that unwinds RNA duplexes and binds to primary microRNA transcripts and general RNAs. Intriguingly, PSR1 decreased both RNA helicase and RNA-binding activity of PINP1, thereby dampening sRNA biogenesis and RNA metabolism. The PSR1–PINP1 interaction caused global changes in alternative splicing (AS). A total of 5,135 genes simultaneously exhibited mis-splicing in both PSR1-overexpressing and PINP1-silenced plants. AS upregulated many mRNA transcripts that had their introns retained. The high occurrence of intron retention in AS-induced transcripts significantly promoted Phytophthora pathogen infection in Nicotiana benthamiana, and this might be caused by the production of truncated proteins. Taken together, our findings reveal a key role for PINP1 in regulating sRNA biogenesis and plant immunity.

SF3B1 mutations occur in many cancers, and the highly conserved His662 residue is one of the hotspot mutation sites. To address effects on splicing and development, we constructed strains carrying point mutations at the corresponding residue His698 in Drosophila using the CRISPR-Cas9 technique. Two mutations, H698D and H698R, were selected due to their frequent presence in patients and notable opposite charges. Both the sf3b1-H698D and–H698R mutant flies exhibit developmental defects, including less egg-laying, decreased hatching rates, delayed morphogenesis and shorter lifespans. Interestingly, the H698D mutant has decreased resistance to fungal infection, while the H698R mutant shows impaired climbing ability. Consistent with these phenotypes, further analysis of RNA-seq data finds altered expression of immune response genes and changed alternative splicing of muscle and neural-related genes in the two mutants, respectively. Expression of Mef2-RB, an isoform of Mef2 gene that was downregulated due to splicing changes caused by H698R, partly rescues the climbing defects of the sf3b1-H698R mutant. Lariat sequencing reveals that the two sf3b1-H698 mutations cause aberrant selection of multiple intronic branch sites, with the H698R mutant using far upstream branch sites in the changed alternative splicing events. This study provides in vivo evidence from Drosophila that elucidates how these SF3B1 hotspot mutations alter splicing and their consequences in development and in the immune system., Author summary In the past decade, one of the important findings in the RNA splicing field has been that somatic SF3B1 mutations widely occur in many cancers. Including R625, H662, K666, K700 and E902, there are five hotspot mutation sites in the highly conserved HEAT repeats of SF3B1. Several kinds of H662 mutations have been found widely in MDS, AML, CLL and breast cancers; however, it remains unclear how these H662 mutations alter splicing and whether they have in vivo effects on development. To address these questions, in this manuscript, we first summarized the H662 mutations in human diseases and constructed two corresponding Drosophila mutant strains, sf3b1-H698D and -H698R using CRISPR-Cas9. Analyses of these two fly strains find that the two oppositely charged Sf3b1-H698 mutants are defective in development. In addition, one mutant has decreased climbing ability, whereas the other mutant has impaired immune response. Further RNA-seq allows us to find responsible genes in each mutant strain, and lariat sequencing reveals that both mutations cause aberrant selection of the intronic branch sites. Our findings provide the first in vivo evidence that Sf3b1 mutations result in defective development, and also reveal a molecular mechanism of these hotspot histidine mutations that enhance the use of cryptic branch sites to alter splicing. Importantly, we demonstrate that the H698R mutant prefers to use far upstream branch sites.

In this study, we investigate the smithsonite surface sulfidation by zinc sulfide precipitation adsorption. Smithsonite solubility experiments indicated that the CZnT in Na2S solution (1.0 × 10−3 mol/L) was 1–2 orders of magnitude lower compared with the corresponding experiment results in deionized water. Na2S addition benefited the dissolution rate of smithsonite in deionized water. Visual MINTEQ model results confirmed that the zinc sulfide precipitation formed after the main sulfidation reaction between Zn(OH)2(aq) and HS− ions at pH 10, compared with the conversion from ZnS(aq), Zn2S32− ions, and Zn4S64− ions to sphalerite. Moreover, X-ray diffraction and X-ray photo-electron spectroscopy (XPS) analysis showed that the average crystallite sizes of zinc sulfide precipitation particle obtained by co-precipitation technique were approximately 2.32 nm in size. These particles were composed of zinc monosulfide and zinc polysulfide. Time-of-flight secondary ion mass spectrometry and XPS analysis provided strong evidence for zinc sulfide precipitation adsorption on the smithsonite surfaces. Furthermore, more mass signals of S−, ZnS−, S2−, and Zn2S− were observed from the zinc sulfide precipitation than from natural sphalerite, and the monosulfide in the zinc sulfide precipitation was easily adsorbed on the smithsonite surfaces. Results elucidate the smithsonite surface sulfidation behavior during zinc sulfide precipitation adsorption.

Many post-transcriptional mRNA processing steps play crucial roles in tumorigenesis and the progression of cancers, such as N6-methyladenosine (m6A) modification and alternative splicing. Upregulation of methyltransferase-like 3 (METTL3), the catalytic core of the m6A methyltransferase complex, increases m6A levels and results in significant effects on the progression of hepatocellular carcinoma (HCC). However, alternative splicing of METTL3 has not been fully investigated, and the functions of its splice variants remain unclear. Here, we analyzed both our and online transcriptomic data, obtaining 13 splice variants of METTL3 in addition to canonical full-length METTL3-A in HCC cell lines and tissues. Validated by RT–qPCR and Western blotting, we found that METTL3-D, one of the splice variants expressing a truncated METTL3 protein, exhibits higher levels than METTL3-A in normal human livers but lower levels than METTL3-A in HCC tumor tissues and cell lines. Further functional assays demonstrated that METTL3-D expression decreased cellular m6A modification, inhibited the proliferation, migration, and invasion of HCC cells, and was negatively associated with the malignancy of patient tumors, exhibiting functions opposite to those of full-length METTL3-A. This study demonstrates that the METTL3-D splice variant is a tumor suppressor that could potentially be used as a target for HCC therapy.

The minor spliceosome is evolutionarily conserved in higher eukaryotes, but its biological significance remains poorly understood. Here, by precise CRISPR/Cas9-mediated disruption of the U12 and U6atac snRNAs, we report that a defective minor spliceosome is responsible for spinal muscular atrophy (SMA) associated phenotypes in Drosophila. Using a newly developed bioinformatic approach, we identified a large set of minor spliceosome-sensitive splicing events and demonstrate that three sensitive intron-containing neural genes, Pcyt2, Zmynd10, and Fas3, directly contribute to disease development as evidenced by the ability of their cDNAs to rescue the SMA-associated phenotypes in muscle development, neuromuscular junctions, and locomotion. Interestingly, many splice sites in sensitive introns are recognizable by both minor and major spliceosomes, suggesting a new mechanism of splicing regulation through competition between minor and major spliceosomes. These findings reveal a vital contribution of the minor spliceosome to SMA and to regulated splicing in animals., Spinal muscular atrophy (SMA) is associated with minor splicing-related defects. Here the authors develop Drosophila models with minor spliceosomal-snRNA deletions, and demonstrate SMA-like phenotypes.

Hydroxymethyl carbon black (HCB) was prepared as an asphalt modifier with a high oxygen content and active surface chemical properties. The microstructure of HCB was analyzed by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and Fourier-transform infrared spectroscopy. The improvement effect of HCB on asphalt&rsquo, s physical, dynamic shear, rheological, and aging properties was evaluated. To analyze the dynamic properties of the HCB-modified asphalt mixtures, a simple performance test (SPT) was conducted, and then the change laws of the dynamic modulus and phase angle for the HCB mixtures were clarified. The results showed that the surface of HCB is smooth and that the oxygen content increases with the generation of hydroxyl functional groups. Polar oxygen-containing functional groups and hydrogen bonds are helpful in improving the resistance to cracking and aging. The surface activity of HCB is susceptible to temperature and frequency, causing a slight influence of HCB on the viscoelasticity of asphalt mixtures at high and low frequencies. At low temperatures and high frequencies, the HCB enhanced the elasticity characteristics and weakened the viscosity characteristics of asphalt mixtures.

The preparation of crumb rubber modified asphalt (RA) shall effectively recycle waste rubber and abate environmental contamination. To ascertain the composition and the factors affecting the viscosity of RA, some samples were prepared in the case of different conditions of mixing temperature and processing time for different rubber dosage and rubber size. Brookfield viscosity tests were conducted on RA samples, and the impact exerted by these factors on viscosity of RA was anatomized. The visco-flow activation energy (Eη) of RA was acquired through adopting Arrhenius equation in line with the viscosity values of asphalt in the case of different temperatures. The changing regularity of Eη was anatomized. The viscosity of rubber asphalt can be split into three parts: the contribution of base asphalt (BE), rubber and asphalt interaction effect (IE) and crumb rubber particle effect (PE), and additionally the viscosity definition is proposed as η R A = η 0 ( 1 + IE + PE ) . To isolate the PE, the sieving test was designed. Rotary viscosity tests were conducted on RA samples and asphalt samples separated from crumb rubber through adopting Brookfield viscosity meter. The impact exerted by these factors on the viscosity composition parameters IE and PE of rubber asphalt was anatomized. In line with the viscosity definition, the viscosity prediction model of rubber asphalt was established. It is indicated from the results and analysis that the viscosity values of RA increase by the exponential growth model continuously with the increase of rubber content. There is a certain equivalence for rubber asphalt viscosity between raising mixing temperature and extending the processing time. But too high temperature or excessive time may result in excessive degradation of rubber molecules, which shall cause the decrease of RA viscosity. With the rising of the dosage of crumb rubber, Eη of RA for origin or aged samples both goes up first and thereupon drops down. And peak Eη exists in the range of rubber content from 10% to 15%. The Eη value of aged RA outstrips that of original sample for the same rubber dosage. For the rubber asphalt samples prepared through adopting the same type and size crumb rubber, the IE values show linear relation with the dosage of crumb rubber, while PE values increase by the exponential growth model continuously with the dosage of crumb rubber. Within a certain temperature range, IE and PE values both show linear relation with the mixing temperature, and show parabola relationship with the processing time. The proposed prediction model can manifest the viscosity composition of rubber asphalt and has an expected fitting precision.

Numerous attempts have been carried out to develop potential substitutes for petroleum asphalt binders from various biomass resources. This research aims to investigate the effectiveness of partially replacing asphalt binders with thermochemical liquefied products (LPs) of wheat straw. The LPs were obtained by acid-catalyzed liquefaction in organic solvents, based on which nine kinds of bio-asphalt binders distinguished by the type and content of LPs were prepared by mechanical stirring. To evaluate the physicochemical properties of the LPs, the Fourier transform infrared (FTIR) spectroscopy test, gel permeation chromatography (GPC) test, thermogravimetric (TG) test and rotational viscosity (RV) test were conducted. The potential advantages of bio-asphalt binders were explored by the RV test, dynamic shear rheometer (DSR) test, scanning electronic microscopy (SEM) test and FTIR test. The results demonstrate that LPs consist of 79%–88% bio-oils with low molecular weight and a small amount of solid residues. Compared with asphalt binders, more polar groups (e.g. hydroxyl, carbonyl and alcohol, etc.) with low boiling points are found in LPs, resulting in a lower volatilization and degradation temperature. There is a high chance for the fusion of LPs and asphalt binders to be physical integration. Further, the non-Newtonian flow behavior of asphalt binders is deepened by LPs. Nevertheless, better low-temperature cracking resistance and high-temperature performance are found from different types of bio-asphalt binders. It should be noted that the relationship between performances of bio-asphalt binders and the content of LPs needs more attention in further researches.

The efficient recovery of micro-fine particle ilmenite using froth flotation remains a challenge. In this study, a novel method for the activation flotation of micro-fine particle ilmenite was performed. Micro-flotation experiments, solution chemistry, X-ray photoelectron spectroscopy, reaction thermodynamic and Time-of-flight secondary ion mass spectrometry analysis were conducted to investigate the activation mechanism. The results indicated that the flotation recovery was increased by ~ 18% with the addition of H2SO4-H2O2 and [Cu(NH3)4]2+ ions at pH value 5.5 using sodium oleate. The dominant RCOOH (l) species and RCOO− ions could interact with Fe and Cu active sites on ilmenite surfaces via chemical adsorption. The treatment of H2SO4-H2O2 promoted the valence transition of iron from Fe (II) to Fe (III). Eventually, the proportion of Fe (III) increased from 44% to 66%. This activity reinforced the formation of Fe(OL)3 complex under a low-acidic condition and it was well consist with the corresponding results of reaction thermodynamic. [Cu(NH3)4]2+ ions adsorbed onto ilmenite surfaces and further improved the surface activity of ilmenite. Accordingly, the normalized peak intensities of C18H33O2− increased from 1.42 × 10−2 to 1.80 × 10−2. Insights into copper-ammonia ions activation for the micro-fine particle ilmenite was mainly attributed to the dual oxidization of H2SO4 and H2O2, as well as the enhanced adsorptions of molecule/colloid oleate and oleate compounds of Fe and Cu. Results enriched basic theories of ilmenite flotation.