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Proceedings of the National Academy of... Jul 2024The essential role of U4 snRNP in pre-messenger RNA (mRNA) splicing has been well established. In this study, we utilized an antisense morpholino oligonucleotide (AMO)...
The essential role of U4 snRNP in pre-messenger RNA (mRNA) splicing has been well established. In this study, we utilized an antisense morpholino oligonucleotide (AMO) specifically targeting U4 snRNA to achieve functional knockdown of U4 snRNP in HeLa cells. Our results showed that this knockdown resulted in global intronic premature cleavage and polyadenylation (PCPA) events, comparable to the effects observed with U1 AMO treatment, as demonstrated by mRNA 3'-seq analysis. Furthermore, our study suggested that this may be a common phenomenon in both human and mouse cell lines. Additionally, we showed that U4 AMO treatment disrupted transcription elongation, as evidenced by chromatin immunoprecipitation sequencing (ChIP-seq) analysis for RNAPII. Collectively, our results identified a unique role for U4 snRNP in the inhibition of PCPA and indicated a model wherein splicing intrinsically inhibits intronic cleavage and polyadenylation in the context of cotranscriptional mRNA processing.
Topics: Humans; Polyadenylation; RNA Precursors; HeLa Cells; Mice; Animals; RNA Splicing; Ribonucleoprotein, U4-U6 Small Nuclear; RNA, Messenger; Introns
PubMed: 38917004
DOI: 10.1073/pnas.2406710121 -
BioRxiv : the Preprint Server For... Jun 2024Human cone photoreceptors differ from rods and serve as the retinoblastoma cell-of-origin. Here, we used deep full-length single-cell RNA-sequencing to distinguish...
Human cone photoreceptors differ from rods and serve as the retinoblastoma cell-of-origin. Here, we used deep full-length single-cell RNA-sequencing to distinguish post-mitotic cone and rod developmental states and cone-specific features that contribute to retinoblastomagenesis. The analyses revealed early post-mitotic cone- and rod-directed populations characterized by higher THRB or NRL regulon activities, an immature photoreceptor precursor population with concurrent cone and rod gene and regulon expression, and distinct early and late cone and rod maturation states distinguished by maturation-associated declines in RAX regulon activity. Unexpectedly, both L/M cone and rod precursors co-expressed and RNAs, yet they differentially expressed functionally antagonistic isoforms and prematurely terminated transcripts. Early L/M cone precursors exhibited successive expression of lncRNAs along with , which composed the seventh most L/M-cone-specific regulon, and , which contributed to the early cone precursors' proliferative response to loss. These findings reveal previously unrecognized photoreceptor precursor states and a role for early cone-precursor-intrinsic expression in retinoblastoma initiation.
PubMed: 38915659
DOI: 10.1101/2023.02.28.530247 -
International Journal of Biological... Jun 2024The sea cucumber Apostichopus japonicus can expel internal organs under stress and regenerate them subsequently. However, growth is delayed during regeneration,...
The sea cucumber Apostichopus japonicus can expel internal organs under stress and regenerate them subsequently. However, growth is delayed during regeneration, significantly impacting the industry. Circular RNAs (circRNAs) are single-stranded circular RNA molecules produced through alternative splicing of mRNA precursors. They play crucial roles in regulating gene expression via the ceRNA mechanism. In this study, circRNA profiles of control and regenerated intestines were constructed. A total of 15,874 circRNAs were identified, with a length of 300-350 nucleotides (nt) being the most abundant. Sanger sequencing confirmed the circular structure of circRNA398. Compared with the normal intestine, 50 and 83 differentially expressed circRNAs (DE-circRNAs) were identified in the regenerated intestine at 1 and 3 days post evisceration (dpe), respectively. Gene ontology (GO) terms for signal transduction and development regulation were most significantly enriched in 1dpeVScon and 3dpeVScon treatments, respectively. The dual-luciferase assay revealed that circRNA8388 functions as a sponge for miR-2392, participating in the remodeling of the extracellular matrix (ECM). In conclusion, these findings will contribute to the enhancement of the non-coding RNA database for echinoderms and lay the groundwork for future investigations into circRNA regulation during intestinal regeneration.
PubMed: 38909735
DOI: 10.1016/j.ijbiomac.2024.133302 -
Leukemia Jun 2024Malignant growth relies on rapid protein synthesis frequently leading to endoplasmic reticulum (ER) overload and accumulation of unfolded or misfolded protein in this...
Malignant growth relies on rapid protein synthesis frequently leading to endoplasmic reticulum (ER) overload and accumulation of unfolded or misfolded protein in this cellular compartment. In the ER, protein homeostasis is finely regulated by a mechanism called the unfolded protein response (UPR), involving the activation of signalization pathways mediated by three transmembrane proteins, namely PERK, IRE1 and ATF6. IRE1 endoribonuclease activation leads in particular to the splicing of the cytosolic mRNA encoding the key UPR-specific transcription factor XBP1s. Our study shows that sustained activation of XBP1s expression in acute myeloid leukemia (AML) cells induces apoptosis in vitro and in vivo, whereas a moderate XBP1s expression sensitizes cells to chemotherapeutic treatments. ChIP-seq experiments identified specific XBP1s target genes including the MIR22HG lncRNA, the precursor transcript of microRNA-22-3p. miR-22-3p upregulation by XBP1s or forced expression of miR-22 significantly decreases cell's viability and sensitizes leukemic cells to chemotherapy. We found that miR-22-3p intracellular effects result at least partially from the targeting of the mRNA encoding the deacetylase sirtuin-1 (SIRT1), a well-established pro-survival factor. Therefore, this novel XBP1s/miR-22/SIRT1 axis identified could play a pivotal role in the proliferation and chemotherapeutic response of leukemic cells.
PubMed: 38909090
DOI: 10.1038/s41375-024-02321-8 -
Science Advances Jun 2024Neuroblastoma is a childhood developmental cancer; however, its embryonic origins remain poorly understood. Moreover, in-depth studies of early tumor-driving events are...
Neuroblastoma is a childhood developmental cancer; however, its embryonic origins remain poorly understood. Moreover, in-depth studies of early tumor-driving events are limited because of the lack of appropriate models. Herein, we analyzed RNA sequencing data obtained from human neuroblastoma samples and found that loss of expression of trunk neural crest-enriched gene associates with advanced disease and worse outcome. Further, by using single-cell RNA sequencing data of human neuroblastoma cells and fetal adrenal glands and creating in vivo models of zebrafish, chick, and mouse, we show that MOXD1 is a determinate of tumor development. In addition, we found that expression is highly conserved and restricted to mesenchymal neuroblastoma cells and Schwann cell precursors during healthy development. Our findings identify as a lineage-restricted tumor-suppressor gene in neuroblastoma, potentiating further stratification of these tumors and development of novel therapeutic interventions.
Topics: Neuroblastoma; Animals; Humans; Mice; Genes, Tumor Suppressor; Gene Expression Regulation, Neoplastic; Zebrafish; Cell Line, Tumor; Cell Lineage; Neural Crest; Schwann Cells
PubMed: 38905335
DOI: 10.1126/sciadv.ado1583 -
Journal of Insect Physiology Jun 2024Like other lepidopteran insects, males of the tobacco cutworm moth, Spodoptera litura produce two kinds of spermatozoa, eupyrene (nucleate) and apyrene (anucleate)...
A sperm-activating trypsin-like protease from the male reproductive tract of Spodoptera litura: Proteomic identification, sequence characterization, gene expression profile, RNAi and the effects of ionizing radiation.
Like other lepidopteran insects, males of the tobacco cutworm moth, Spodoptera litura produce two kinds of spermatozoa, eupyrene (nucleate) and apyrene (anucleate) sperm. Formed in the testis, both kinds of sperm are released into the male reproductive tract in an immature form and are stored in the duplex region of the tract. Neither type of sperm is motile at this stage. When stored apyrene sperm from the duplex are treated in vitro with an extract of the prostatic region of the male tract, or with mammalian trypsin, they become motile; activation is greater and achieved more rapidly with increasing concentration of extract or enzyme. The activating effect of prostatic extract is blocked by soybean trypsin inhibitor (SBTI), also in a dose-dependent way. These results suggest that the normal sperm-activating process is due to an endogenous trypsin-like protease produced in the prostatic region. Proteomic analysis of S. litura prostatic extracts revealed a Trypsin-Like Serine Protease, TLSP, molecular weight 27 kDa, whose 199-residue amino acid sequence is identical to that of a predicted protein from the S. litura genome and is highly similar to predicted proteins encoded by genes in the genomes of several other noctuid moth species. Surprisingly, TLSP is only distantly related to Serine Protease 2 (initiatorin) of the silkmoth, Bombyx mori, the only identified lepidopteran protein so far shown to activate sperm. TLSP has features typical of secreted proteins, probably being synthesized as an inactive precursor zymogen, which is later activated by proteolytic cleavage. cDNA was synthesized from total RNA extracted from the prostatic region and was used to examine TLSP expression using qPCR. tlsp mRNA was expressed in both the prostatic region and the accessory glands of the male tract. Injection of TLSP-specific dsRNA into adult males caused a significant reduction after 24 h in tlsp mRNA levels in both locations. The number of eggs laid by females mated to adult males that were given TLSP dsRNA in 10 % honey solution, and the fertility (% hatched) of the eggs were reduced. Injecting pupae with TLSP dsRNA caused the later activation of apyrene sperm motility by adult male prostatic extracts to be significantly reduced compared to controls. Exposure of S. litura pupae to ionizing radiation significantly reduced expression of tlsp mRNA in the prostatic part and accessory gland of irradiated males in both the irradiated generation and also in their (unirradiated) F1 progeny. The implications of these findings for the use of the inherited sterility technique for the control of S. litura and other pest Lepidoptera are discussed.
PubMed: 38897288
DOI: 10.1016/j.jinsphys.2024.104664 -
BioRxiv : the Preprint Server For... Jun 2024Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, poses a significant clinical challenge. In particular,...
Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, poses a significant clinical challenge. In particular, unlike adrenergic (ADRN) neuroblastoma cells, mesenchymal (MES) cells are resistant to chemotherapy and retinoid therapy and thereby significantly contribute to relapses and treatment failures. Previous research suggested that overexpression or activation of miR-124, a neurogenic microRNA with tumor suppressor activity, can induce the differentiation of retinoic acid-resistant neuroblastoma cells. Leveraging our established screen for miRNA modulatory small molecules, we validated PP121, a dual inhibitor of tyrosine and phosphoinositide kinases, as a robust inducer of miR-124. A combination of PP121 and miR-132-inducing bufalin synergistically arrests proliferation, induces differentiation, and prolongs the survival of differentiated MES SK-N-AS cells for 8 weeks. RNA- seq and deconvolution analyses revealed a collapse of the ADRN core regulatory circuitry (CRC) and the emergence of novel CRCs associated with chromaffin cells and Schwann cell precursors. Using a similar protocol, we differentiated and maintained other MES neuroblastoma, as well as glioblastoma cells, over 16 weeks. In conclusion, our novel protocol suggests a promising treatment for therapy-resistant cancers of the nervous system. Moreover, these long-lived, differentiated cells provide valuable models for studying mechanisms underlying differentiation, maturation, and senescence.
PubMed: 38895399
DOI: 10.1101/2024.06.05.597584 -
BioRxiv : the Preprint Server For... Jun 2024Vault RNAs (vRNAs) are evolutionarily conserved small non-coding RNAs transcribed by RNA polymerase lll. Initially described as components of the vault particle, they...
Vault RNAs (vRNAs) are evolutionarily conserved small non-coding RNAs transcribed by RNA polymerase lll. Initially described as components of the vault particle, they have since also been described as noncanonical miRNA precursors and as riboregulators of autophagy. As central molecules in these processes, vRNAs have been attributed numerous biological roles including regulation of cell proliferation and survival, response to viral infections, drug resistance, and animal development. Yet, their impact to mammalian physiology remains largely unexplored. To study vault RNAs , we generated a mouse line with a conditional loss of function allele. Because is the sole murine vRNA, this allele enables the characterization of the physiological requirements of this conserved class of small regulatory RNAs in mammals. Using this strain, we show that mice constitutively null for are viable and histologically normal but have a slight reduction in platelet counts pointing to a potential role for vRNAs in hematopoiesis. This work paves the way for further characterizations of this abundant but mysterious RNA molecule. Specifically, it enables the study of the biological consequences of constitutive or lineage-specific deletion and of the physiological requirements for an intact during normal hematopoiesis or in response to cellular stresses such as oncogene expression, viral infection, or drug treatment.
PubMed: 38895289
DOI: 10.1101/2024.06.01.596958 -
International Journal of Molecular... May 2024MicroRNAs (miRNAs) regulate approximately one-third of all human genes. The dysregulation of miRNAs has been implicated in the development of numerous human diseases,...
MicroRNAs (miRNAs) regulate approximately one-third of all human genes. The dysregulation of miRNAs has been implicated in the development of numerous human diseases, including cancers. In our investigation focusing on altering specific miRNA expression in human pancreatic cancer cells, we encountered an interesting finding. While two expression vector designs effectively enhanced miR-708 levels, they were unable to elevate mature forms of miR-29b, -1290, -2467, and -6831 in pancreatic cancer cell lines. This finding was also observed in a panel of other non-pancreatic cancer cell lines, suggesting that miRNA processing efficiency was cell line specific. Using a step-by-step approach in each step of miRNA processing, we ruled out alternative strand selection by the RISC complex and transcriptional interference at the primary miRNA (pri-miRNA) level. DROSHA processing and pri-miRNA export from the nucleus also appeared to be occurring normally. We observed precursor (pre-miRNA) accumulation only in cell lines where mature miRNA expression was not achieved, suggesting that the block was occurring at the pre-miRNA stage. To further confirm this, synthetic pre-miRNA mimics that bypass DICER processing were processed into mature miRNAs in all cases. This study has demonstrated the distinct behaviours of different miRNAs with the same vector in the same cell line, the same miRNA between the two vector designs, and with the same miRNA across different cell lines. We identified a stable vector pre-miRNA processing block. Our findings on the structural and sequence differences between successful and non-successful vector designs could help to inform future chimeric miRNA design strategies and act as a guide to other researchers on the intricate processing dynamics that can impact vector efficiency. Our research confirms the potential of miRNA mimics to surmount some of these complexities.
Topics: MicroRNAs; Humans; Pancreatic Neoplasms; RNA Processing, Post-Transcriptional; Cell Line, Tumor; Ribonuclease III; Gene Expression Regulation, Neoplastic; Transfection; RNA Precursors; Animals
PubMed: 38891854
DOI: 10.3390/ijms25115666 -
Journal of Agricultural and Food... Jun 2024Beyond the key bitter compound kaempferol 3--(2‴--sinapoyl-β-d-sophoroside) previously described in the literature (), eight further bitter and astringent-tasting...
Beyond the key bitter compound kaempferol 3--(2‴--sinapoyl-β-d-sophoroside) previously described in the literature (), eight further bitter and astringent-tasting kaempferol glucosides (-) have been identified in rapeseed protein isolates ( L.). The bitterness and astringency of these taste-active substances have been described with taste threshold concentrations ranging from 3.3 to 531.7 and 0.3 to 66.4 μmol/L, respectively, as determined by human sensory experiments. In this study, the impact of and kaempferol 3--β-d-glucopyranoside () on TAS2R-linked proton secretion by HGT-1 cells was analyzed by quantification of the intracellular proton index. mRNA levels of bitter receptors TAS2R3, 4, 5, 13, 30, 31, 39, 40, 43, 45, 46, 50 and TAS2R8 were increased after treatment with compounds and . Using quantitative UHPLC-MS/MS measurements, the concentrations of - were determined in rapeseed/canola seeds and their corresponding protein isolates. Depending on the sample material, compounds , , and - exceeded dose over threshold (DoT) factors above one for both bitterness and astringency in selected protein isolates. In addition, an increase in the key bitter compound during industrial protein production (apart from enrichment) was observed, allowing the identification of the potential precursor of to be kaempferol 3--(2‴--sinapoyl-β-d-sophoroside)-7--β-d-glucopyranoside (). These results may contribute to the production of less bitter and astringent rapeseed protein isolates through the optimization of breeding and postharvest downstream processing.
PubMed: 38888424
DOI: 10.1021/acs.jafc.4c02342