snRNA Research Articles

B cell selection sites in the nurse shark spleen may represent evolutionary precursors of mammalian germinal centers

Abstract Sharks are members of the oldest extant taxonomic group (Chondrichthyes) to have immunoglobulin (Ig)-based adaptive immunity. While research has demonstrated that sharks can produce antigen-specific antibody responses, affinity mature these antibodies, and establish immunological memory, histological examination has so far failed to find evidence of germinal centers (GCs). To explore this apparent discrepancy, we immunized several nurse sharks (Ginglymostoma cirratum) with phycoerythrin and visualized the subsequent splenic response using a combination of single nuclei (sn)RNA sequencing and fluorescent microscopy. Our snRNA data reveal the presence of both centrocyte-like and centroblast-like B cells in sharks, as well as a T cell subset expressing several markers found in mammalian T follicular helper cells. RNA fluorescent in situ hybridization experiments show distinct regions of AID expression, polarization of CXCR4 and CXCR5 expression, and association of T cell aggregates with splenic B cell follicles. Fluorescent microscopy clearly demonstrates presentation of nondegraded antigen in the center of B cell follicles. Finally, analysis of Ig variable regions amplified from such B cell follicles reveals evidence of positive selection of clonotypes. Together, our data supports the presence of B cell selection sites in the cartilaginous fish spleen that functionally support the selection of antigen-specific B cells and affinity maturation of the shark Ig repertoire. Further, these sites potentially represent the primordial structures that gave rise to mammalian GCs. Supported by grants from NIH T32 AI095190, F31 AI147532

Coilin Modulates Nuclear Organization by Promoting Protein SUMOylation

Coilin was first identified as the marker protein for Cajal Bodies (CBs). However, CBs are only abundant in neuronal, embryonic, and cancer cells, while primary cells have few CBs. Even in cells with CBs, 70% of coilin is nucleoplasmic, not found in CBs. CBs are subnuclear domains that function in small nuclear ribonucleoprotein (snRNP) biogenesis as well as production of telomerase RNP, expression and 3’end processing of small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and small CB‐associated RNAs (scaRNAs). The function of nucleoplasmic coilin is not well understood. We have previously shown that coilin promotes miRNA biogenesis by promoting phosphorylation of the Microprocessor component, DGCR8, at its S377 residue in both cells with and without CBs. We have since identified 7 additional serine residues of DGCR8 with decreased phosphorylation in coilin knockdown through phosphoproteomic analysis of HeLa cells. In addition to phosphorylation, the addition of a small ubiquitin‐like modifier (SUMO), or SUMOylation, of DGCR8 also increases its stability. Because of coilin’s role in the promotion of one post‐translational modification, here we aimed to test the hypothesis that coilin knockdown alters protein SUMOylation and results in altered nuclear organization. We conducted a series of siRNA knockdowns, Western blots, and immunoprecipitation experiments. Here we show, for the first time, that coilin knockdown results in global decrease of protein SUMOylation in cells with (HeLa) and without CBs (WI‐38), suggesting that regulation of protein SUMOylation may be a function of nucleoplasmic coilin. Specifically, we found that coilin knockdown results in decreased SUMOylation of DGCR8 and Speckled protein 100kDa (Sp100), a major component of the promyelocytic leukemia nuclear body (PML‐NB). Sp100 is vital to PML‐NB formation, and decreased SUMOylation of Sp100 decreases its stability. This information led us to the second part of our hypothesis. We used immunofluorescence and found that coilin knockdown results in decreased numbers of PML‐NBs per cell. To summarize, we have identified coilin as a regulator of protein phosphorylation, SUMOylation, and nuclear organization.

QRNAstruct: a method for extracting secondary structural features of RNA via regression with biological activity.

Recent technological advances have enabled the generation of large amounts of data consisting of RNA sequences and their functional activity. Here, we propose a method for extracting secondary structure features that affect the functional activity of RNA from sequence–activity data. Given pairs of RNA sequences and their corresponding bioactivity values, our method calculates position-specific structural features of the input RNA sequences, considering every possible secondary structure of each RNA. A Ridge regression model is trained using the structural features as feature vectors and the bioactivity values as response variables. Optimized model parameters indicate how secondary structure features affect bioactivity. We used our method to extract intramolecular structural features of bacterial translation initiation sites and self-cleaving ribozymes, and the intermolecular features between rRNAs and Shine–Dalgarno sequences and between U1 RNAs and splicing sites. We not only identified known structural features but also revealed more detailed insights into structure–activity relationships than previously reported. Importantly, the datasets we analyzed here were obtained from different experimental systems and differed in size, sequence length and similarity, and number of RNA molecules involved, demonstrating that our method is applicable to various types of data consisting of RNA sequences and bioactivity values.

Karyotypic analysis and isolation of four DNA markers of the scleractinian coral Favitespentagona (Esper, 1795) (Scleractinia, Anthozoa, Cnidaria).

We performed conventional and molecular cytogenetic studies on the Favitespentagona Esper, 1795, a scleractinian coral mostly found along the west coast of Japan. Karyotype analysis of F.pentagona by G-banding revealed a karyogram containing a homogenously staining region (HSR) on chromosome 10 in more than 50% of the examined metaphase spreads. This HSR consisted of sequences from 18S ribosomal RNA (rRNA) genes, as demonstrated by fluorescence in situ hybridization (FISH) and DNA sequencing. We highlighted the development of four chromosomal FISH markers from repetitive genes such as U2 small nuclear RNA linked to 5S rRNA sequence (U2 snRNA-5S), 18S rRNA, histone H3, and uncharacterized gene FP-9X. The chromosomal locations of the U2 snRNA-5S and 18S RNA were on the terminal end of long arm of chromosomes 2 and 10, respectively, while the histone H3 and the uncharacterized gene were located near the centromeres of chromosomes 1 and 9, respectively. These FISH markers will improve the karyotyping of F.pentagona from mitotic preparations which helps in widening our understanding of coral genetic structure and chromosome organization. In addition, these improvements in karyotyping will provide the basis in constructing of chromosome-level genome assembly for F.pentagona.

Exploring the expanding universe of small RNAs.

The world of small noncoding RNAs (sncRNAs) is ever-expanding, from small interfering RNA, microRNA and Piwi-interacting RNA to the recently emerging non-canonical sncRNAs derived from longer structured RNAs (for example, transfer, ribosomal, Y, small nucleolar, small nuclear and vault RNAs), showing distinct biogenesis and functional principles. Here we discuss recent tools for sncRNA identification, caveats in sncRNA expression analysis and emerging methods for direct sequencing of sncRNAs and systematic mapping of RNA modifications that are integral to their function.

U2 small nuclear RNA auxiliary factor 2, transcriptionally activated by the transcription factor Dp-1/E2F transcription factor 1 complex, enhances the growth and aerobic glycolysis of leiomyosarcoma cells

ABSTRACT The dysregulation of U2 Small Nuclear RNA Auxiliary Factor 2 (U2AF2) is associated with malignant behaviors of multiple types of tumors. In this study, we explored the association between U2AF2 dysregulation and the survival of patients with primary leiomyosarcoma, the regulatory effect of U2AF2 on cell growth/aerobic glycolysis, and the mechanisms of U2AF2 dysregulation at the transcriptional level. Gene expression and survival time of patients with primary leiomyosarcoma were extracted from TCGA-Sarcoma (SARC). Leiomyosarcoma cell lines SK-LMS-1 and SK-UT-1 were utilized to construct in vitro and in vivo models. Results showed that the higher U2AF2 expression group had significantly shorter progression-free survival (HR: 2.049, 95%CI: 1.136–3.697, p = 0.011) and disease-specific survival (4.656, 95%CI: 2.141–10.13, p < 0.001) compared to the lower U2AF2 expression group. U2AF2 knockdown suppressed leiomyosarcoma cell growth and aerobic glycolysis (decreased glucose uptake, lactate production, and extracellular acidification rate) in vitro. Tumors derived from SK-LMS-1 cells with U2AF2 knockdown grew significantly slower, with lower GLUT1, PGK1, and PGAM1 protein expression than the control groups. TFDP1 and E2F1 could interact with each other in leiomyosarcoma cells. Both TFDP1 and E2F1 could bind to the promoter of U2AF2 and exert a synergistic activating effect on U2AF2 transcription. In conclusion, this study revealed that U2AF2 upregulation is associated with poor survival of leiomyosarcoma. Its upregulation enhances proliferation and aerobic glycolysis of leiomyosarcoma cells in vitro and in vivo. TFDP1 and E2F1 can form a complex, which binds to the U2AF2 gene promoter and synergistically activates its transcription.

GRAPHENE OXIDE AFFECT THE EXPRESSION OF PROLIFERATION RELATED GENES AND microRNA IN NORMAL HUMAN ASTROCYTES

Aim. In this study we investigate the impact of low doses of graphene oxide on the expression of key regulatory genes which control cell proliferation as well as microRNAs in normal human astrocytes. Methods. The expression level of genes related to cell proliferation was studied by real-time qPCR in normal human astrocytes line NHA/TS (Cambrex Bio Science, Walkersville, MD, USA) using SYBRGreen Mix and specific for each mRNA forward and reverse primers. These astrocytes were treated with graphene oxide (1 and 4 ng/ml of medium) for 24 hrs. Graphene oxide (2 mg/ml, dispersion in water) was received from Sigma-Aldrich Chemie GmbH, Germany. Total RNA was extracted using TRIZOL reagent. For reverse transcription of mRNAs we used Thermo Scientific Verso cDNA Synthesis Kit (Germany). The values of mRNA expressions were normalized to the level of ACTB mRNA and represented as percent of control (100 %). For polyadenylation and reverse transcription of miRNAs we used Mir-X miRNA First-Strand Synthesis Kit (Takara, Japan). The expression level of microRNAs was studied by real-time qPCR using SYBRGreen Mix and specific for each miRNA forward primers and universal reverse primer. For normalization of microRNA expressions the level of U6 RNA expression was used. Results. It was shown that the expression level of TOB1, HSPA5, EDEM1, MYBL1, and MYBL2 significantly increased in normal human astrocytes line NHA/TS, which were treated with graphene oxide (1 and 4 ng/ml of medium) for 24 hrs. Up-regulation of these genes expression was dose-dependent: bigger dose of graphene oxide (4 ng/ml of medium) introduced more significant changes in the expression of all these genes. Furthermore, bioinformatics analysis of 3′-untranslated regions of mRNA allowed identifying binding sites of microRNA: miR-19a for MYBL1, miR-143 for MYBL2 and miR-182 for TOB1. It was also shown that the expression of all these microRNA significantly down-regulated by graphene oxide, supporting the idea of both post-transcriptional and transcriptional regulation of MYBL1, MYBL2 and TOB1 gene expressions. Conclusions. Graphene oxide significantly disturbs genome stability by up-regulation of the expression of key regulatory genes and down-regulation of microRNA.

Small Non-Coding RNAs in the Human Placenta: Regulatory Roles and Clinical Utility.

The placenta is a vital organ formed during pregnancy, and being the interface between the mother and fetus, it is paramount that placental functioning is strictly controlled. Gene expression in the placenta is finely tuned—with aberrant expression causing placental pathologies and inducing stress on both mother and fetus. Gene regulation is brought upon by several mechanisms, and small non-coding RNAs (sncRNAs) have recently been appreciated for their contribution in gene repression. Their dysregulation has been implicated in a range of somatic and inherited disorders, highlighting their importance in maintaining healthy organ function. Their specific roles within the placenta, however, are not well understood, and require further exploration. To this end, we summarize the mechanisms of microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and transfer RNAs (tRNAs), their known contributions to human placental health and disease, the relevance of sncRNAs as promising biomarkers throughout pregnancy, and the current challenges faced by placental sncRNA studies.

SNHG14 Upregulation Was a Molecular Mechanism Underlying MPP+ Neurotoxicity in Dopaminergic SK-N-SH Cells via SNHG14-miR-519a-3p-ATG10 ceRNA Pathway.

Long non-coding RNA small nuclear RNA host gene 14 (SNHG14) is a novel contributor of dopaminergic neuronal injury in Parkinson's disease. We further explored its role in 1-methyl-4-phenylpyridinium (MPP+)-damaged dopaminergic neurons (DAn) and the possible mechanism involving SNHG14, microRNA (miR)-519a-3p, and autophagy-related 10 (ATG10). MPP+ cytotoxicity was measured by MTS cell viability assay, flow cytometry, and a series of assay kits for detecting apoptosis and oxidative stress. Molecule expression was examined by qPCR and Western blotting, and RNA interaction was predicted by starBase2.0 of ENCORI platform and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. SNHG14 and ATG10 expression was increased, and miR-519a-3p was decreased in MPP+-treated SK-N-SH cells, and SNHG14 knockdown alleviated MPP+-induced SK-N-SH cell damage by regulating cell viability, cell cycle arrest, apoptosis, and oxidative stress. Additionally, antisense RNA of miR-519a-3p abated the suppressive role of SNHG14 knockdown, and ectopic expression of ATG10 counteracted the protective role of miR-519a-3p against MPP+ neurotoxicity. Mechanistically, SNHG14 and ATG10 were competitive endogenous RNAs (ceRNAs) for miR-519a-3p, and ATG10 expression could be positively modulated by SNHG14 via sponging miR-519a-3p. Target silencing SNHG14 and restoring miR-519a-3p could prevent DAn from MPP+ toxicity via regulation of ATG10.

Major and minor U small nuclear RNAs genes characterization in a neotropical fish genome: Chromosomal remodeling and repeat units dispersion in Parodontidae

In association with many proteins, small nuclear RNAs (snRNAs) organize the spliceosomes that play a significant role in processing precursor mRNAs during gene expression. According to snRNAs genic arrangements, two kinds of spliceosomes (major and minor) can be organized into eukaryotic cells. Although in situ localization of U1 and U2 snDNAs have been performed in fish karyotypes, studies with genomic characterization and functionality of U snRNAs integrated into chromosomal changes on Teleostei are still scarce. This study aimed to achieve a genomic characterization of the U snRNAs genes in Apareiodon sp. (2n = 54, ZZ/ZW), apply these data to recognize functional/defective copies, and map chromosomal changes involving snDNAs in Parodontidae species karyotype diversification. Nine snRNA multigene families (U1, U2, U4, U5, U6, U11, U12, U4atac and U6atac) arranged in putatively functional copies in the genome were analyzed. Proximal Sequence Elements (PSE) and TATA-box promoters occurrence, besides an entire transcribed region and conserved secondary structures, qualify them for spliceosome activity. In addition, several defective copies or pseudogenes were identified for the snRNAs that make up the major spliceosome. In situ localization of snDNAs in five species of Parodontidae demonstrated that U1, U2, and U4 snDNAs were involved in chromosomal location changes or units dispersion. The U snRNAs defective/pseudogenes units dispersion could be favored by the probable occurrence of active retrotransposition enzymes in the Apareiodon genome. The U2 and U4 snDNAs sites were involved in independent events in the differentiation of sex chromosomes among Parodontidae lineages. The study characterized U snRNA genes that compose major and minor spliceosomes in the Apareiodon sp. genome and proposes that their defective copies trigger chromosome differentiation and diversification events in Parodontidae.

The Arabidopsis cyclophilin CYP18-1 facilitates PRP18 dephosphorylation and the splicing of introns retained under heat stress.

In plants, heat stress induces changes in alternative splicing, including intron retention; these events can rapidly alter proteins or downregulate protein activity, producing nonfunctional isoforms or inducing nonsense-mediated decay of messenger RNA (mRNA). Nuclear cyclophilins (CYPs) are accessory proteins in the spliceosome complexes of multicellular eukaryotes. However, whether plant CYPs are involved in pre-mRNA splicing remain unknown. Here, we found that Arabidopsis thaliana CYP18-1 is necessary for the efficient removal of introns that are retained in response to heat stress during germination. CYP18-1 interacts with Step II splicing factors (PRP18a, PRP22, and SWELLMAP1) and associates with the U2 and U5 small nuclear RNAs in response to heat stress. CYP18-1 binds to phospho-PRP18a, and increasing concentrations of CYP18-1 are associated with increasing dephosphorylation of PRP18a. Furthermore, interaction and protoplast transfection assays revealed that CYP18-1 and the PP2A-type phosphatase PP2A B'η co-regulate PRP18a dephosphorylation. RNA-seq and RT-qPCR analysis confirmed that CYP18-1 is essential for splicing introns that are retained under heat stress. Overall, we reveal the mechanism of action by which CYP18-1 activates the dephosphorylation of PRP18 and show that CYP18-1 is crucial for the efficient splicing of retained introns and rapid responses to heat stress in plants.

Species diversity, systematic revision and molecular phylogeny of Ganodermataceae (Polyporales, Basidiomycota) with an emphasis on Chinese collections.

Ganodermataceae is one of the main families of macrofungi since species in the family are both ecologically and economically important. The double-walled basidiospores with ornamented endospore walls are the characteristic features of Ganodermataceae. It is a large and complex family; although many studies have focused on Ganodermataceae, the global diversity, geographic distribution, taxonomy and molecular phylogeny of Ganodermataceae still remained incompletely understood. In this work, taxonomic and phylogenetic studies on worldwide species of Ganodermataceae were carried out by morphological examination and molecular phylogenetic analyses inferred from six gene loci including the internal transcribed spacer regions (ITS), the large subunit of nuclear ribosomal RNA gene (nLSU), the second largest subunit of RNA polymerase II gene (rpb2), the translation elongation factor 1-α gene (tef1), the small subunit mitochondrial rRNA gene (mtSSU) and the small subunit nuclear ribosomal RNA gene (nSSU). A total of 1 382 sequences were used in the phylogenetic analyses, of which 817 were newly generated, including 132 sequences of ITS, 139 sequences of nLSU, 83 sequences of rpb2, 124 sequences of tef1, 150 sequences of mtSSU and 189 sequences of nSSU. The combined six-gene dataset included sequences from 391 specimens representing 146 taxa from Ganodermataceae. Based on morphological and phylogenetic analyses, 14 genera were confirmed in Ganodermataceae: Amauroderma, Amaurodermellus, Cristataspora, Foraminispora, Furtadoella, Ganoderma, Haddowia, Humphreya, Magoderna, Neoganoderma, Sanguinoderma, Sinoganoderma, Tomophagus and Trachydermella. Among these genera, Neoganoderma gen. nov. is proposed for Ganoderma neurosporum; Sinoganoderma gen. nov. is proposed for Ganoderma shandongense; Furtadoella gen. nov. is proposed to include taxa previously belonging to Furtadoa since Furtadoa is a homonym of a plant genus in the Araceae; Trachydermella gen. nov. is proposed to include Trachyderma tsunodae since Trachyderma is a homonym of a lichen genus in the Pannariaceae. Twenty-three new species, viz., Ganoderma acaciicola, G. acontextum, G. alpinum, G. bubalinomarginatum, G. castaneum, G. chuxiongense, G. cocoicola, G. fallax, G. guangxiense, G. puerense, G. subangustisporum, G. subellipsoideum, G. subflexipes, G. sublobatum, G. tongshanense, G. yunlingense, Haddowia macropora, Sanguinoderma guangdongense, Sa. infundibulare, Sa. longistipitum, Sa. melanocarpum, Sa. microsporum and Sa. tricolor are described. In addition, another 33 known species are also described in detail for comparison. Scanning electron micrographs of basidiospores of 10 genera in Ganodermataceae are provided. A key to the accepted genera of Ganodermataceae and keys to the accepted species of Ganoderma, Haddowia, Humphreya, Magoderna, Sanguinoderma and Tomophagus are also provided. In total, 278 species are accepted as members of Ganodermataceae including 59 species distributed in China. Taxonomic novelties: New genera: Furtadoella B.K. Cui & Y.F. Sun, Neoganoderma B.K. Cui & Y.F. Sun, Sinoganoderma B.K. Cui, J.H. Xing & Y.F. Sun and Trachydermella B.K. Cui & Y.F. Sun; New species: Ganoderma acaciicola B.K. Cui, J.H. Xing & Y.F. Sun, G. acontextum B.K. Cui, J.H. Xing & Vlasák, G. alpinum B.K. Cui, J.H. Xing & Y.F. Sun, G. bubalinomarginatum B.K. Cui, J.H. Xing & Y.F. Sun, G. castaneum B.K. Cui, J.H. Xing & Y.F. Sun, G. chuxiongense B.K. Cui, J.H. Xing & Y.F. Sun, G. cocoicola B.K. Cui, J.H. Xing & Y.F. Sun, G. fallax B.K. Cui, J.H. Xing & Vlasák, G. guangxiense B.K. Cui, J.H. Xing & Y.F. Sun, G. puerense B.K. Cui, J.H. Xing & Y.F. Sun, G. subangustisporum B.K. Cui, J.H. Xing & Y.F. Sun, G. subellipsoideum B.K. Cui, J.H. Xing & Y.F. Sun, G. subflexipes B.K. Cui, J.H. Xing & Y.F. Sun, G. sublobatum B.K. Cui, J.H. Xing & Y.F. Sun, G. tongshanense B.K. Cui, J.H. Xing & Y.F. Sun, G. yunlingense B.K. Cui, J.H. Xing & Y.F. Sun, Haddowia macropora B.K. Cui, Vlasák & Y.F. Sun, Sanguinoderma guangdongense B.K. Cui & Y.F. Sun, Sa. infundibulare B.K. Cui & Y.F. Sun, Sa. longistipitum B.K. Cui & Y.F. Sun, Sa. melanocarpum B.K. Cui & Y.F. Sun, Sa. microsporum B.K. Cui & Y.F. Sun and Sa. tricolor B.K. Cui & Y.F. Sun; New combinations: Furtadoella biseptata (Costa-Rezende et al.) B.K. Cui & Y.F. Sun, Fu. brasiliensis (Singer) B.K. Cui & Y.F. Sun, Fu. corneri (Gulaid & Ryvarden) B.K. Cui & Y.F. Sun, Neoganoderma neurosporum (J.S. Furtado) B.K. Cui & Y.F. Sun, Sinoganoderma shandongense (J.D. Zhao & L.W. Xu) B.K. Cui, J.H. Xing & Y.F. Sun and Trachydermella tsunodae (Yasuda ex Lloyd) B.K. Cui & Y.F. Sun. Citation: Sun Y-F, Xing J-H, He X-L, Wu D-M, Song C-G, Liu S, Vlasák J, Gates G, Gibertoni TB, Cui B-K (2022). Species diversity, systematic revision and molecular phylogeny of Ganodermataceae (Polyporales, Basidiomycota) with an emphasis on Chinese collections. Studies in Mycology 101: 287-415. doi: 10.3114/sim.2022.101.05.

An Efficient Genetic Transformation and CRISPR/Cas9-Based Genome Editing System for Moso Bamboo (Phyllostachys edulis).

Moso bamboo (Phyllostachys edulis) is the most important monopodial bamboo species worldwide. Without a genetic transformation system, it is difficult to verify the functions of genes controlling important traits and conduct molecular breeding in moso bamboo. Here, we established a plant regeneration system from immature embryos. Calli were induced on MS medium added 4–6 mg⋅L–1 2,4-dichlorophenoxyacetic acid (2,4-D) with high efficiency (>60%). A plant growth regulator combination of 0.5 mg⋅L–1 1-naphthylacetic acid (NAA), 2.0 mg⋅L–1 6-benzylaminopurine (BAP), and 3.0 mg⋅L–1 zeatin (ZT) was suitable for shoot differentiation, and the shoot induction frequency was increased to 43% after 0.5 mg⋅L–1 abscisic acid (ABA) pretreatment. An effective antibiotic screening concentration was determined by hygromycin sensitivity test. We further optimized the Agrobacterium concentration and added vacuum infiltration for infection, which improves the transient expression efficiency. A genetic transformation system was established for the first time in moso bamboo, with the transformation efficiency of approximately 5%. To optimize genome editing, two endogenous U3 small nuclear RNA (snRNA) promoters were isolated and used to drive small guide RNA (sgRNA) expression. The results showed that the PeU3.1 promoter exhibited higher efficiency, and it was used for subsequent genome editing. Finally, homozygous pds1pds2 mutants were obtained by an efficient CRISPR/Cas9 genome-editing system. These technical systems will be conducive to gene functional validation and accelerate the molecular breeding process of moso bamboo.

PiggyBac-based transgenic RNAi of serine protease 2 results in male sterility in Hyphantria cunea

Fall webworm, Hyphantria cunea, is a global invasive forest pest that causes serious damage to the economy and ecosystem of agriculture and forestry. Due to the extent of the problem and the difficulty of conventional chemical control, new technologies must be pursued, such as genetic-based inheritable insect sterile technology (gSIT), which exhibits promise for pest control. In the present study, we established a piggyBac-based transgenic system in fall webworm and generated a dominant male-sterile strain by targeting the seminal fluid protein serine protease 2 (Hcser2), displaying an outstanding trait of gSIT. First, an RNA polymerase type III (Pol III) promoter, the HcU62 small nuclear RNA (snRNA) gene promoter, was identified and characterized through direct injection of RNAi plasmids in vivo. Quantitative real-time PCR revealed that HcU62 had the greatest knockdown efficiency of the Hcyellow gene among five short hairpin RNA (shRNA) plasmids tested, designated HcU61–HcU65. Second, subsequent application of piggyBac-based transgenic RNAi (HcU62: shHcyellow, Ysh2) significantly reduced the expression level of the Hcyellow gene, resulting in a stable yellow observable phenotype from the larval to pupal stages in Ysh2 transgenic mutants. Finally, an HcU62-driven transgenic RNAi strain targeting the Hcser2 gene was obtained, resulting in a dominant male-sterile phenotype. Significantly, this process did not affect the growth, development, mating behavior or egg laying of the mutants, and the dominant sterile trait could be inherited in the next generation through female Hcser2 mutants. Furthermore, CRISPR/Cas9-mediated disruption of the Hcser2 gene further confirmed the dominant sterile phenotype, supporting it as a generalized target for genetic control of H. cunea. This study reports the first piggyBac-mediated transgenic system in H. cunea, providing a promising genetic method for controlling this pest by targeting Hcser2 gene.

Humanization and characterization of the S. cerevisiae splicing factor Prp8 for drug screening

To remove non-coding regions (introns) from pre-mRNA and splice together exons, eukaryotes rely on the spliceosome, which itself is made up of five small nuclear RNAs and dozens of proteins. RNA splicing is essential for eukaryotic gene expression and it has recently emerged as an attractive drug target. One essential splicing factor is pre-mRNA processing protein 8 (Prp8), which helps to scaffold the active site during both catalytic steps of splicing. The human homolog, PRPF8, shares 60% sequence homology to yeast Prp8.

The potential role of U2AF1L4 cDNA 3’ stretch mutations in oral carcinogenesis

BackgroundU2 small nuclear RNA auxiliary factor 1-like protein 4 (U2AF1L4) plays a crucial role in the physiological alternative splicing of pre-mRNA pools. Material and methodsWe analyzed the mutations within U2AF1L4 cDNA 3’ stretch, by Q PCR-Tm calling in cancer and pre-cancer oral biopsies. The conservation of U2AF1L4 across mammals and the binding sites on human U2AF1L4 for carcinogens and therapeutants were scrutinized followed by String mediated protein interactome prediction. ResultsIn Q PCR-Tm calling, the U2AF1L4 cDNA 3’ stretch showed amplicons with multiple Tm values in diseased (pre-cancer and cancer lesions) cDNA pools varying from the control, indicating mutation in the U2AF1L4 cDNA 3’ stretch oral cancer and pre-cancer lesions of addicted cases. The evolutionary analyses showed that human U2AF1L4 is a highly conserved protein. The molecular docking revealed stable interactions between carcinogens and therapeutants with human U2AF1L4 respectively. The U2AF1L4 interactome comprised of key splice factors like SRSF indicating the plausible broad impact of 3’cDNA mutations in altering the global splicing processes of the oral mucosa. ConclusionsThe U2AF1L4 cDNA 3’ stretch comprising exons 6, 7, and 8 imbibes mutations mediated by tobacco, areca nut, and alcohol chronic addiction; playing a crucial role in oral precancer-cancer pathophysiologies.

Correlation between circulating microRNA and lipid indicesin patients with ischemic heart disease and type 2 diabetes mellitus

The aim of the study was to investigate circulating miRNAs-27a, -221 levels and their correlations with lipid indices in patients with ischemic heart disease (IHD) and type 2 diabetes mellitus. Materials and methods. The study included 58 patients with stable IHD and type 2 diabetes mellitus, 22 IHD patients without diabetes and 19 healthy controls. MicroRNAs-27a-3р and 221-3р were determined in blood plasma by real time polymerase chain reaction. U6 small nuclear RNA was used for normalization. Results. The lowest levels of circulating miRNAs-27a and -221 were in IHD patients with diabetes. The decrease in the levels of both microRNAs in patients with diabetes was significant not only in comparison with the control group (P = 0.024, P = 0.006), but also with patients without diabetes (P = 0.011, P = 0.001). In IHD patients without diabetes, microRNAs-27a and -221 levels did not change significantly. In IHD patients with diabetes, microRNA-221 showed a significant positive correlation with high-density lipoprotein cholesterol (HDL-C) (R = 0.382, P = 0.003). MicroRNA-27a was correlated negatively with triglycerides (TG) (R = -0.284, P = 0.051) and positively – with HDL-C (R = 0.257, P = 0.078) at a borderline significance level. The lower level of microRNA-27a (the 1st tertile) was associated with significantly increased TG level in comparison with the 3rd tertile (P = 0.004), and significantly decreased HDL-C level in comparison with the 2nd (P = 0.001) and the 3rd (P = 0.023) tertiles. Patients with lower microRNA-221 level (the 1st tertile) had significantly reduced HDL-C level in comparison with the 3rd tertile(P = 0.007). Conclusions. The results have demonstrated the significant decrease in circulating microRNAs-27a and -221 levels in IHD patients with type 2 diabetes mellitus and no change in both microRNAs in patients without diabetes. In IHD patients with diabetes, the lower expression of microRNA-27a was associated with the significant elevation of TG and the significant decrease in HDL-C. The lower microRNA-221 level was associated only with the significant decrease in HDL-C.

Mechanisms of lncRNA biogenesis as revealed by nascent transcriptomics.

Mammalian genomes express two principal gene categories through RNA polymerase II-mediated transcription: protein-coding transcription units and non-coding RNA transcription units. Non-coding RNAs are further divided into relatively abundant structural RNAs, such as small nuclear RNAs, and into a myriad of long non-coding RNAs (lncRNAs) of often low abundance and low stability. Although at least some lncRNA synthesis may reflect transcriptional 'noise', recent studies define unique functions for either specific lncRNAs or for the process of lncRNA synthesis. Notably, the transcription, processing and metabolism of lncRNAs are regulated differently from protein-coding genes. In this Review, we provide insight into the regulation of lncRNA transcription and processing gleaned from the application of recently devised nascent transcriptomics technology. We first compare and contrast different methodologies for studying nascent transcription. We then discuss the molecular mechanisms regulating lncRNA transcription, especially transcription initiation and termination, which emphasize fundamental differences in their expression as compared with protein-coding genes. When perturbed, lncRNA misregulation leads to genomic stress such as transcription-replication conflict and R-loop-mediated DNA damage. We discuss many unresolved but important questions about the synthesis and potential functions of lncRNAs.

Trimethylguanosine synthase 1 is a novel regulator of pancreatic beta-cell mass and function

Type 2 diabetes is a metabolic disorder associated with abnormal glucose homeostasis and is characterized by intrinsic defects in β-cell function and mass. Trimethylguanosine synthase 1 (TGS1) is an evolutionarily conserved enzyme that methylates small nuclear and nucleolar RNAs and that is involved in pre-mRNA splicing, transcription, and ribosome production. However, the role of TGS1 in β-cells and glucose homeostasis had not been explored. Here, we show that TGS1 is upregulated by insulin and upregulated in islets of Langerhans from mice exposed to a high-fat diet and in human β-cells from type 2 diabetes donors. Using mice with conditional (βTGS1KO) and inducible (MIP-CreERT-TGS1KO) TGS1 deletion, we determined that TGS1 regulates β-cell mass and function. Using unbiased approaches, we identified a link between TGS1 and endoplasmic reticulum stress and cell cycle arrest, as well as and how TGS1 regulates β-cell apoptosis. We also found that deletion of TGS1 results in an increase in the unfolded protein response by increasing XBP-1, ATF-4, and the phosphorylation of eIF2α, in addition to promoting several changes in cell cycle inhibitors and activators such as p27 and Cyclin D2. This study establishes TGS1 as a key player regulating β-cell mass and function. We propose that these observations can be used as a stepping-stone for the design of novel strategies focused on TGS1 as a therapeutic target for the treatment of diabetes.

Non-Coding Ribonucleic Acids as Regulators of Ferroptosis in Cancer

Non-coding ribonucleic acids were thought to be non-functional transcription products decades ago. But recently they have been found to play an essential role in programmed regulation in a variety of diseases, including cancer. They include micro ribonucleic acids and their upstream long non-coding ribonucleic acids and circular ribonucleic acids. Long non-coding ribonucleic acids or circular ribonucleic acids can form a regulatory network through sponging micro ribonucleic acids. The network regulates biological functions of various tumors such as proliferation, invasion and drug resistance. In addition, other non-coding ribonucleic acids such as piwi-interacting ribonucleic acids, transfer ribonucleic acids, ribosomal ribonucleic acids, small nuclear ribonucleic acids and small nucleolar ribonucleic acids can also act as regulators of the biological characteristics of tumor cells. Ferroptosis is a programmed cell death, which is distinct from the classical caspase splicing and has received increasing attention in recent years with regard to the tumorigenesis and treatment of cancer. Interestingly, non-coding ribonucleic acids have been found to regulate ferroptosis in tumors and to maintain the biological function of tumor cells. Herein, we summarize the related research and emphasize that the regulation of ferroptosis in tumor cells by non-coding ribonucleic acids may provide a basis for future targeted cancer therapy.