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1.
Nucleic Acids Res ; 52(10): 6002-6016, 2024 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-38499485

RESUMEN

Alternative splicing (AS) generates multiple RNA isoforms and increases the complexities of transcriptomes and proteomes. However, it remains unclear how RNA structures contribute to AS regulation. Here, we systematically search transcriptomes for secondary structures with concealed branch sites (BSs) in the alternatively spliced introns and predict thousands of them from six organisms, of which many are evolutionarily conserved. Intriguingly, a highly conserved stem-loop structure with concealed BSs is found in animal SF3B3 genes and colocalizes with a downstream poison exon (PE). Destabilization of this structure allows increased usage of the BSs and results in enhanced PE inclusion in human and Drosophila cells, leading to decreased expression of SF3B3. This structure is experimentally validated using an in-cell SHAPE-MaP assay. Through RNA interference screens of 28 RNA-binding proteins, we find that this stem-loop structure is sensitive to U2 factors. Furthermore, we find that SF3B3 also facilitates DNA repair and protects genome stability by enhancing interaction between ERCC6/CSB and arrested RNA polymerase II. Importantly, both Drosophila and human cells with the secondary structure mutated by genome editing exhibit altered DNA repair in vivo. This study provides a novel and common mechanism for AS regulation of PEs and reveals a physiological function of SF3B3 in DNA repair.


Asunto(s)
Empalme Alternativo , Exones , Intrones , Animales , Humanos , Secuencia Conservada , Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Exones/genética , Intrones/genética , Conformación de Ácido Nucleico , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Codón sin Sentido
2.
PLoS Genet ; 19(11): e1011021, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37917726

RESUMEN

Small nuclear RNAs (snRNAs) are structural and functional cores of the spliceosome. In metazoan genomes, each snRNA has multiple copies/variants, up to hundreds in mammals. However, the expressions and functions of each copy/variant in one organism have not been systematically studied. Focus on U1 snRNA genes, we investigated all five copies in Drosophila melanogaster using two series of constructed strains. Analyses of transgenic flies that each have a U1 promoter-driven gfp revealed that U1:21D is the major and ubiquitously expressed copy, and the other four copies have specificities in developmental stages and tissues. Mutant strains that each have a precisely deleted copy of U1-gene exhibited various extents of defects in fly morphology or mobility, especially deletion of U1:82Eb. Interestingly, splicing was changed at limited levels in the deletion strains, while large amounts of differentially-expressed genes and alternative polyadenylation events were identified, showing preferences in the down-regulation of genes with 1-2 introns and selection of proximal sites for 3'-end polyadenylation. In vitro assays suggested that Drosophila U1 variants pulled down fewer SmD2 proteins compared to the canonical U1. This study demonstrates that all five U1-genes in Drosophila have physiological functions in development and play regulatory roles in transcription and 3'-end formation.


Asunto(s)
Drosophila melanogaster , ARN Nuclear Pequeño , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Empalme del ARN/genética , Drosophila/genética , Drosophila/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Mamíferos/genética
3.
Nucleic Acids Res ; 51(10): 5228-5241, 2023 06 09.
Artículo en Inglés | MEDLINE | ID: mdl-37070178

RESUMEN

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.


Asunto(s)
Proteínas de Drosophila , ARN Circular , Proteínas de Unión al ARN , Animales , Femenino , Masculino , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , ARN/genética , ARN/metabolismo , Empalme del ARN/genética , ARN Circular/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo
4.
Plant J ; 113(2): 402-415, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36562774

RESUMEN

Photoperiod plays a critical role in controlling the formation of sexual or vegetative reproductive organs in potato. Although StPHYF-silenced plants overcome day-length limitations to tuberize through a systemic effect on tuberigen StSP6A expression in the stolon, the comprehensive regulatory network of StPHYF remains obscure. Therefore, the present study investigated the transcriptomes of StPHYF-silenced plants and observed that, in addition to known components of the photoperiodic tuberization pathway, florigen StSP3D and other flowering-related genes were activated in StPHYF-silenced plants, exhibiting an early flowering response. Additionally, grafting experiments uncovered the long-distance effect of StPHYF silencing on gene expression in the stolon, including the circadian clock components, flowering-associated MADSs, and tuberization-related regulatory genes. Similar to the AtFT-AtAP1 regulatory module in Arabidopsis, the present study established that the AP1-like StMADS1 functions downstream of the tuberigen activation complex (TAC) and that suppressing StMADS1 inhibits tuberization in vitro and delays tuberization in vivo. Moreover, the expression of StSP6A was downregulated in StMADS1-silenced plants, implying the expression of StSP6A may be feedback-regulated by StMADS1. Overall, these results reveal that the regulatory network of StPHYF controls flowering and tuberization and targets the crucial tuberization factor StMADS1 through TAC, thereby providing a better understanding of StPHYF-mediated day-length perception during potato reproduction.


Asunto(s)
Arabidopsis , Fitocromo , Solanum tuberosum , Fitocromo/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Solanum tuberosum/metabolismo , Transcriptoma , Tubérculos de la Planta/metabolismo , Hojas de la Planta/metabolismo , Fotoperiodo , Arabidopsis/genética , Reproducción , Regulación de la Expresión Génica de las Plantas/genética
5.
PLoS Genet ; 17(11): e1009861, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34723968

RESUMEN

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.


Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Intrones , Mutación , Animales , Sistemas CRISPR-Cas , Drosophila/inmunología
6.
Int J Mol Sci ; 25(4)2024 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-38397102

RESUMEN

The GRAS (GAI\RGA\SCL) gene family encodes plant-specific transcription factors that play crucial roles in plant growth and development, stress tolerance, and hormone network regulation. Plant dwarfing symptom is mainly regulated by DELLA proteins of the GRAS gene subfamily. In this study, the association between the GRAS gene family and Paulownia witches' broom (PaWB) was investigated. A total of 79 PfGRAS genes were identified using bioinformatics methods and categorized into 11 groups based on amino acid sequences. Tandem duplication and fragment duplication were found to be the main modes of amplification of the PfGRAS gene family. Gene structure analysis showed that more than 72.1% of the PfGRASs had no introns. The genes PfGRAS12/18/58 also contained unique DELLA structural domains; only PfGRAS12, which showed significant response to PaWB phytoplasma infection in stems, showed significant tissue specificity and responded to gibberellin (GA3) in PaWB-infected plants. We found that the internodes were significantly elongated under 100 µmol·L-1 GA3 treatment for 30 days. The subcellular localization analysis indicated that PfGRAS12 is located in the nucleus and cell membrane. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays confirmed that PfGRAS12 interacted with PfJAZ3 in the nucleus. Our results will lay a foundation for further research on the functions of the PfGRAS gene family and for genetic improvement and breeding of PaWB-resistant trees.


Asunto(s)
Cytisus , Lamiales , Magnoliopsida , Phytoplasma , Magnoliopsida/genética , Enfermedades de las Plantas/genética , Phytoplasma/genética , Fitomejoramiento , Lamiales/genética
7.
BMC Infect Dis ; 23(1): 843, 2023 Nov 30.
Artículo en Inglés | MEDLINE | ID: mdl-38036959

RESUMEN

BACKGROUND: Whether different anti-hepatitis B virus (HBV) drugs have different effects on COVID-19 is controversial. We aimed to evaluate the incidence of COVID-19 in chronic hepatitis B (CHB) patients receiving anti-HBV treatment, and to compare the impact of entecavir (ETV) and tenofovir disoproxil fumarate (TDF) on the severity of COVID-19. METHODS: CHB outpatients were enrolled from December 2022 to February 2023. Questionnaires were used to collect whether subjects were currently or previously had COVID-19 within the past 2 months, and the information of symptoms, duration, and severity if infected. RESULTS: Six hundred thirty CHB patients were enrolled, 64.3% (405/630) patients were currently or previously had COVID-19. No COVID-19 patient required hospitalization, intensive care unit admission, oxygen support or died. Majority of patients reported mild (32.8% [133/405]) and moderate (48.1% [195/405]) symptoms. After propensity score matching, 400 matched patients were obtained (ETV: 238; TDF: 162), among which the incidences of COVID-19 were comparable between ETV and TDF-treated patients (60.1% [143/238] vs. 64.2% [104/162], p = 0.468). The proportion of patients complicated with any symptom caused by COVID-19 were also similar (ETV vs. TDF: 90.9% [130/143] vs. 91.3% [95/104], p = 1.000). In addition, the severity of overall symptom was comparable between ETV and TDF-treated patients, in terms of proportion of patients complicated with severe symptom (9.8% vs. 8.7%, p = 0.989), symptom duration (4.3 vs. 4.3 days, p = 0.927), and symptom severity score (4.1 vs. 4.0, p = 0.758). Subgroup analysis supported these results. CONCLUSIONS: During the current pandemic, the vast majority of CHB patients experienced non-severe COVID-19, and ETV and TDF did not affect COVID-19 severity differently.


Asunto(s)
COVID-19 , Hepatitis B Crónica , Humanos , Tenofovir/uso terapéutico , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/epidemiología , Antivirales/efectos adversos , Incidencia , Resultado del Tratamiento , COVID-19/epidemiología , Estudios Retrospectivos
8.
Genes Dev ; 29(7): 760-71, 2015 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-25838544

RESUMEN

Unlike typical cis-splicing, trans-splicing joins exons from two separate transcripts to produce chimeric mRNA and has been detected in most eukaryotes. Trans-splicing in trypanosomes and nematodes has been characterized as a spliced leader RNA-facilitated reaction; in contrast, its mechanism in higher eukaryotes remains unclear. Here we investigate mod(mdg4), a classic trans-spliced gene in Drosophila, and report that two critical RNA sequences in the middle of the last 5' intron, TSA and TSB, promote trans-splicing of mod(mdg4). In TSA, a 13-nucleotide (nt) core motif is conserved across Drosophila species and is essential and sufficient for trans-splicing, which binds U1 small nuclear RNP (snRNP) through strong base-pairing with U1 snRNA. In TSB, a conserved secondary structure acts as an enhancer. Deletions of TSA and TSB using the CRISPR/Cas9 system result in developmental defects in flies. Although it is not clear how the 5' intron finds the 3' introns, compensatory changes in U1 snRNA rescue trans-splicing of TSA mutants, demonstrating that U1 recruitment is critical to promote trans-splicing in vivo. Furthermore, TSA core-like motifs are found in many other trans-spliced Drosophila genes, including lola. These findings represent a novel mechanism of trans-splicing, in which RNA motifs in the 5' intron are sufficient to bring separate transcripts into close proximity to promote trans-splicing.


Asunto(s)
Drosophila/genética , Empalme del ARN/genética , ARN Nuclear Pequeño/genética , Trans-Empalme/genética , Secuencias de Aminoácidos , Animales , Secuencia Conservada/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Intrones/genética , Proteínas de Unión al ARN/genética
9.
Nucleic Acids Res ; 48(11): 5799-5813, 2020 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-32399566

RESUMEN

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.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Empalme del ARN , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Alelos , Genes Fúngicos/genética , Genoma Fúngico/genética , Mutación , Fenotipo , Unión Proteica , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por Sustrato , Factores de Transcripción/deficiencia , Factores de Transcripción/genética
10.
Optom Vis Sci ; 99(6): 489-495, 2022 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-35412498

RESUMEN

SIGNIFICANCE: We used an Akeso device to record the visual behavior of children with myopia in two learning modes. We found that online class mode may increase near-viewing time and reduce outdoor time compared with the traditional school mode and may be responsible for accelerating myopia progression. PURPOSE: We aimed to explore the effects of visual behavior in different learning modes on myopia progression among children 9 to 11 years old. METHODS: Forty-nine children were included and requested to use a wearable device to objectively record visual behavior in real time from November 2019 to November 2020; participants took online classes from mid-February to early May 2020 during this period. Data (including glasses-wearing time, outdoor time, and near-viewing time) were collected during two 14-day periods, which included the online class learning mode (March 2 to 15, 2020) and the traditional school mode (May 20 to June 2, 2020). Spherical equivalent refraction and axial length were obtained at baseline, at 6-month intervals, and 1 year later. RESULTS: Outdoor time during online class mode (median, 9.5 minutes; interquartile range, 0.75 to 48 minutes) was significantly lower than during the school mode (median, 29 minutes; interquartile range, 11.50 to 50 minutes; P < .001). The mean ± standard deviation of near-viewing time was significantly different between online class mode (396.58 ± 114.41 minutes) and school mode (376.52 ± 93.99 minutes; P = .007, F = 19.56). In comparison with the baseline examination (-2.33 ± 0.81 D), mean spherical equivalent refraction in oculus dexter corresponding to the 6-month examination was decreased (-2.94 ± 0.83 D, P = .001), indicating a significant increase in myopia during online class mode. CONCLUSIONS: This study provides evidence of the association of learning mode and myopia progression. Accelerated progression of myopia in online class mode may be related to increased near-viewing time and decreased time spent in outdoor activities.


Asunto(s)
Miopía , Niño , Progresión de la Enfermedad , Anteojos , Humanos , Miopía/diagnóstico , Miopía/epidemiología , Refracción Ocular , Encuestas y Cuestionarios , Pruebas de Visión
11.
Ophthalmic Res ; 65(5): 529-539, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35405674

RESUMEN

INTRODUCTION: Scleral hypoxia (HO) is present in myopic eyes, and interleukin (IL)-6 is increased in the aqueous humor of patients with high myopia. The aim of this study was to investigate the effects of IL-6 on scleral fibroblast proliferation, differentiation, and apoptosis under conditions of HO and the possible role of IL-6 in myopic scleral remodeling. METHODS: Primary human scleral fibroblasts (HSFs) were cultured using a tissue mass adherent method. First, cells were cultured under conditions of HO (2% O2) or normoxia (NO, 20% O2) for different times. A quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence were used to detect the expression of IL-6 in HSFs. Next, cells were divided into five groups: NO, HO, HO plus IL-6, HO plus IL-6 receptor inhibitor (IL6RI), and HO plus IL-6 and IL6RI. The groups were treated separately for 72 h. Cell counting kit-8 assay and flow cytometry were used to detect cell proliferation and apoptosis, respectively. Western blotting and qRT-PCR were used to detect the expression of various genes in the transforming growth factor-ß1/Smad2/matrix metalloproteinase-2 pathway; these methods and immunofluorescence were also used to detect transdifferentiation of HSFs. RESULTS: HO resulted in upregulation of IL-6 expression in HSFs. Compared with NO, HO resulted in diminished cell proliferation and increased apoptosis and differentiation in HSFs; the above trend was further enhanced by the addition of IL6RI. Compared with the HO group, the addition of IL-6 led to a decrease in cell proliferation and an increase in apoptosis and differentiation of HSFs; the above trends showed opposite changes after the addition of both IL-6 and IL6RI. Additionally, IL-6 and IL6RI exerted opposite regulatory effects on the transforming growth factor-ß1/Smad2/matrix metalloproteinase-2 pathway under conditions of HO. CONCLUSION: HO caused HSFs to overexpress IL-6. IL-6 has a role in scleral remodeling in myopic eyes through affecting the proliferation, differentiation, and apoptosis of HSFs.


Asunto(s)
Interleucina-6 , Miopía , Apoptosis , Proliferación Celular , Células Cultivadas , Fibroblastos , Humanos , Interleucina-6/metabolismo , Interleucina-6/farmacología , Metaloproteinasa 2 de la Matriz/metabolismo , Miopía/metabolismo , Receptores de Interleucina-6/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo
12.
Nucleic Acids Res ; 47(6): 3142-3157, 2019 04 08.
Artículo en Inglés | MEDLINE | ID: mdl-30649456

RESUMEN

In Drosophila, dosage compensation globally upregulates the expression of genes located on male single X-chromosome. Maleless (MLE) helicase plays an essential role to incorporate the roX lncRNA into the dosage compensation complex (MSL-DCC), and such function is essentially dependent on its dsRNA-binding domains (dsRBDs). Here, we report a 2.90Å crystal structure of tandem dsRBDs of MLE in complex with a 55mer stem-loop of roX2 (R2H1). MLE dsRBDs bind to R2H1 cooperatively and interact with two successive minor grooves and a major groove of R2H1, respectively. The recognition of R2H1 by MLE dsRBDs involves both shape- and sequence-specificity. Moreover, dsRBD2 displays a stronger RNA affinity than dsRBD1, and mutations of key residues in either MLE dsRBD remarkably reduce their affinities for roX2 both in vitro and in vivo. In Drosophila, the structure-based mle mutations generated using the CRISPR/Cas9 system, are partially male-lethal and indicate the inter-regulation among the components of the MSL-DCC at multiple levels. Hence, our research provides structural insights into the interactions between MLE dsRBDs and R2H1 and facilitates a deeper understanding of the mechanism by which MLE tandem dsRBDs play an indispensable role in specific recognition of roX and the assembly of the MSL-DCC in Drosophila dosage compensation.


Asunto(s)
Proteínas Cromosómicas no Histona/química , ADN Helicasas/química , Compensación de Dosificación (Genética) , Proteínas de Drosophila/química , ARN Bicatenario/genética , Factores de Transcripción/química , Animales , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , ARN Bicatenario/química , Factores de Transcripción/genética , Cromosoma X/genética
13.
J Biol Chem ; 293(16): 6011-6021, 2018 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-29500196

RESUMEN

More than 70% of all agricultural pests are insects in the order Lepidoptera, which, unlike other related insect orders, are not very sensitive to RNAi, limiting genetic studies of this insect group. However, the reason for this distinct lepidopteran characteristic is unknown. Previously, using transcriptome analysis of the Asian corn borer Ostrinia furnacalis, we identified a gene, termed up56, that is up-regulated in response to dsRNA. Here we report that this Lepidoptera-specific gene encodes a nuclease that contributes to RNAi insensitivity in this insect order. Its identity was experimentally validated, and sequence analysis indicated that up56 encodes a previously uncharacterized protein with homologous sequences in seven other lepidopteran species. Its computationally predicted three-dimensional structure revealed a high structural similarity to human exonuclease I. Exposure to dsRNA in O. furnacalis strongly up-regulated this gene's expression, and the protein could digest single-stranded RNA (ssRNA), dsRNA, and dsDNA both in vitro and in vivo Of note, we found that this up-regulation of up56 expression is faster than that of the gene encoding the key RNAi-associated nuclease Dicer. up56 knockdown in O. furnacalis significantly enhanced RNAi efficiency. Moreover, up56 overexpression in Drosophila melanogaster suppressed RNAi efficiency. Finally, up56 knockdown significantly increased the amount and diversity of small RNAs. Therefore, we renamed this protein RNAi efficiency-related nuclease (REase). In conclusion, we propose that REase may explain why lepidopterans are refractory to RNAi and that it represents a target for further research of RNAi efficiency in this insect order.


Asunto(s)
Desoxirribonucleasas/genética , Proteínas de Insectos/genética , Lepidópteros/genética , Interferencia de ARN , Ribonucleasas/genética , Secuencia de Aminoácidos , Animales , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Genes de Insecto , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Lepidópteros/química , Lepidópteros/metabolismo , Modelos Moleculares , Filogenia , Estabilidad del ARN , Ribonucleasas/química , Ribonucleasas/metabolismo , Alineación de Secuencia , Transcriptoma
14.
RNA Biol ; 16(6): 809-820, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30836863

RESUMEN

Doublesex is highly conserved and sex-specifically spliced in insect sex-determination pathways, and its alternative splicing (AS) is regulated by Transformer, an exonic splicing activator, in the model system of Drosophila melanogaster. However, due to the lack of a transformer gene, AS regulation of doublesex remains unclear in Lepidoptera, which contain the economically important silkworm Bombyx mori and thousands of agricultural pests. Here, we use yeast three-hybrid system to screen for RNA-binding proteins that recognize sex-specific exons 3 and 4 of silkworm doublesex (Bm-dsx); this approach identified BxRBP1/Lark binding to the exon 3, and BxRBP2/TBPH and BxRBP3/Aret binding to the exon 4. Investigation of tissues shows that BxRBP1 and BxRBP2 have no sex specificity, but BxRBP3 has - three of its four isoforms are expressed with a sex-bias. Using novel sex-specific silkworm cell lines, we find that BxRBP1 and BxRBP3 directly interact with each other, and cooperatively function as splicing repressors. Over-expression of BxRBP1 and BxRBP3 isoforms efficiently inhibits splicing of the exons 3 and 4 in the female-specific cells and generates the male-specific isoform of Bm-dsx. We also demonstrate that the sex-determination upstream gene Masc regulates alternatively transcribed BxRBP3 isoforms. Thus, we identify a new regulatory mechanism of doublesex AS in the silkworm, revealing an evolutionary divergence in insect sex-determination.


Asunto(s)
Empalme Alternativo , Bombyx/genética , Proteínas de Unión al ADN/genética , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Bombyx/metabolismo , Proteínas de Unión al ADN/metabolismo , Exones , Femenino , Genes de Insecto , Proteínas de Insectos/química , Masculino , Señales de Localización Nuclear , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Caracteres Sexuales , Transcripción Genética
15.
RNA ; 22(4): 636-55, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26847261

RESUMEN

Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins.


Asunto(s)
Anticuerpos/química , Proteínas de Drosophila/inmunología , Ribonucleoproteínas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/metabolismo , Antígenos/inmunología , Antígenos/aislamiento & purificación , Western Blotting , Regiones Determinantes de Complementariedad , Proteínas de Drosophila/aislamiento & purificación , Drosophila melanogaster , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Inmunoprecipitación , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Ribonucleoproteínas/aislamiento & purificación
16.
Nucleic Acids Res ; 41(8): 4660-70, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23462954

RESUMEN

Fidelity and efficiency of pre-mRNA splicing are critical for generating functional mRNAs, but how such accuracy in 5' splice site (SS) selection is attained is not fully clear. Through a series of yeast genetic screens, we isolated alleles of prp28 that improve splicing of suboptimal 5'SS substrates, demonstrating that WT-Prp28p proofreads, and consequently rejects, poor 5'SS. Prp28p is thought to facilitate the disruption of 5'SS-U1 snRNA pairing to allow for 5'SS-U6 snRNA pairing in the catalytic spliceosome; unexpectedly, 5'SS proofreading by Prp28p is dependent on competition with the stability of the 5'SS:U6 duplex, but not the 5'SS:U1 duplex. E404K, the strongest prp28 allele containing a mutation located in the linker region between adenosine triphosphatase (ATPase) subdomains, exhibited lower RNA-binding activity and enhanced splicing of suboptimal substrates before first-step catalysis, suggesting that decreased Prp28p activity allows longer time for suboptimal 5'SS substrates to pair with U6 snRNA and thereby reduces splicing fidelity. Residue E404 is critical for providing high splicing activity, demonstrated here in both yeast and Drosophila cells. Thus, the subdomain linker in Prp28p plays important roles both in splicing efficiency across species and in proofreading of 5'SS.


Asunto(s)
ARN Helicasas DEAD-box/genética , Sitios de Empalme de ARN , Empalme del ARN , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Alelos , Animales , Línea Celular , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/metabolismo , Drosophila/genética , Mutación , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
Materials (Basel) ; 17(10)2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38793412

RESUMEN

To mitigate dust pollution generated during various stages of construction activities and reduce the environmental and health hazards posed by airborne dust, this study utilized hydroxyethyl cellulose, glycerol, and isomeric tridecyl alcohol polyoxyethylene ether as raw materials to formulate a composite chemical dust suppressant. The properties of the dust suppressant were characterized through analysis. Employing single-factor experiments, the optimal proportions of the binder, water-retaining agent, and surfactant for the composite dust suppressant were determined. Subsequently, a response surface model was established, and, after analysis and optimization, the optimal mass ratios of each component in the composite dust suppressant were obtained. Under optimal ratios, the physicochemical properties and wind erosion resistance of the composite dust suppressant were analyzed. Finally, the practical application of the suppressant was validated through on-site trials at a construction site. This study revealed that the optimal formulation for the dust suppressant was as follows: 0.2% hydroxyethyl cellulose, 2.097% glycerol, 0.693% isomeric tridecyl alcohol polyoxyethylene ether, and the remainder was pure water. The suppressant is non-toxic, non-corrosive, environmentally friendly, and exhibits excellent moisture retention and bonding properties compared to water. The research findings provide valuable insights for addressing dust pollution issues on construction sites.

18.
Materials (Basel) ; 17(11)2024 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-38893749

RESUMEN

Cross-Laminated Timber (CLT) and concrete composite structures represent an architectural system that integrates the strengths of both materials. In this innovative configuration, the CLT and concrete collaborate synergistically, harnessing their individual merits to achieve enhanced structural performance and functionality. Specifically, the CLT offers a lightweight design, superior bending resistance, and immense engineering plasticity, while concrete boasts exceptional compressive strength and durability. This study investigates the mechanical performance of CLT-concrete composite structures through quasi-static reciprocating loading tests in three full-scale CLT shear wall samples. Designed with varying initial prestressing forces and dimensions of the CLT panel, the prestressed CLT-concrete structures demonstrated a reduced dependence on the steel nodes, resulting in an increase in yield load, yield displacement, and maximum load-carrying capacity. Maximum capacity increased by 39.8% and 33.7% under initial prestressing forces of 23 kN and 46 kN on steel strands. Failure occurred due to localized compressive failure on prestressed steel strands and anchor plates. ABAQUS finite element analysis established three refined models, revealing that the increased initial prestressing force moderately enhanced stiffness but reduced ductility under similar cross-sectional dimensions. Furthermore, under consistent CLT material, dimensions, prestressing force, and loading conditions, prestressed CLT-concrete structures exhibited a higher maximum load-bearing capacity than prestressed CLT-steel composite structures. This study proposes structural design recommendations based on experimental and simulation results, incorporating specific assumptions.

19.
Int Immunopharmacol ; 132: 112000, 2024 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-38583238

RESUMEN

Various studies have been investigated the phenotypic and functional distinctions of craniofacial and long bone cells involved in bone regeneration. However, the process of bone tissue regeneration after bone grafting involves complicated interactions between different cell types at the donor-recipient site. Additionally, differences in alterations of the immune microenvironment at the recipient site remained to be explored. Osteoblasts (OBs) and macrophages (MØ) play essential roles in the bone restoration and regeneration processes in the bone and immune systems, respectively. The modulation of MØ on OBs has been extensively explored in the literature, whereas limited research has been conducted on the influence of OBs on the MØ phenotype and function. In the present study, OBs from the mandible and femur (MOBs and FOBs, respectively) promoted cranial defect regeneration in rats, with better outcomes noted in the MOBs-treated group. After MOBs transplantation, a significant inflammatory response was induced, accompanied by an early increase in IL-10 secretion. And then, there was an upregulation in M2-MØ-related cell markers and inflammatory factor expression. Condition media (CM) of OBs mildly inhibited apoptosis in MØ, enhanced their migration and phagocytic functions, and concurrently increased iNOS and Arg1 expression, with MOB-CM demonstrating more pronounced effects compared to FOB-CM. In conclusion, our investigation showed that MOBs and FOBs have the ability to modulate MØ phenotype and function, with MOBs exhibiting a stronger regulatory potential. These findings provide a new direction for improving therapeutic strategies for bone regeneration in autologous bone grafts from the perspective of the immune microenvironment.


Asunto(s)
Regeneración Ósea , Fémur , Inmunomodulación , Macrófagos , Mandíbula , Osteoblastos , Macrófagos/inmunología , Mandíbula/citología , Mandíbula/inmunología , Fémur/citología , Fémur/inmunología , Osteoblastos/inmunología , Regeneración Ósea/inmunología , Masculino , Animales , Ratas , Ratas Sprague-Dawley , Separación Celular
20.
Front Neurol ; 15: 1421977, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39045431

RESUMEN

Objective: Postoperative deep venous thrombosis (DVT) is commonly observed in patients undergoing craniotomy and is associated with a high incidence of pulmonary embolism and poor clinical outcomes. Herein, we investigated the prophylactic effect of DVT of intraoperative intermittent pneumatic compression (IPC) in patients undergoing craniotomy. Methods: A total of 516 patients who underwent elective craniotomy between December 2021 and December 2022 were enrolled in this study. Patients were randomly assigned to the intervention group (received intraoperative IPC) or control group (without IPC). Lower extremity ultrasound was performed on both legs before and after surgery (1 h, 24 h, and 7 days post-intervention). DVT was defined as the visualization of a thrombus within the vein lumen of the leg. Coagulation and platelet function were measured at the start and end of the craniotomy. Results: A total of 504 patients (251 in the intervention group and 253 in the control group) completed the study. Among these patients, 20.4% (103/504) developed postoperative DVT within the first week after surgery, with 16.7% occurring within 24 h. The incidence of postoperative DVT in the intervention group (9.6%, 24/251) was significantly lower than that in the control group (22.9%, 58/253, p < 0.001). Intraoperative IPC reduced the risk of DVT by 64.6% (0.354, 95% CI, 0.223-0.564, p < 0.001). There was no significant difference in coagulation and platelet function between the two groups (all p > 0.05). Conclusion: DVT may develop within 24 h after the craniotomy. Intraoperative application of IPC reduces the incidence of postoperative DVT.

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