Post-transcriptional Gene Expression Research Articles

METTL3/MYCN cooperation drives neural crest differentiation and provides therapeutic vulnerability in neuroblastoma

AbstractNeuroblastoma (NB) is the most common extracranial childhood cancer, caused by the improper differentiation of developing trunk neural crest cells (tNCC) in the sympathetic nervous system. The N6-methyladenosine (m6A) epitranscriptomic modification controls post-transcriptional gene expression but the mechanism by which the m6A methyltransferase complex METTL3/METTL14/WTAP is recruited to specific loci remains to be fully characterized. We explored whether the m6A epitranscriptome could fine-tune gene regulation in migrating/differentiating tNCC. We demonstrate that the m6A modification regulates the expression of HOX genes in tNCC, thereby contributing to their timely differentiation into sympathetic neurons. Furthermore, we show that posterior HOX genes are m6A modified in MYCN-amplified NB with reduced expression. In addition, we provide evidence that sustained overexpression of the MYCN oncogene in tNCC drives METTL3 recruitment to a specific subset of genes including posterior HOX genes creating an undifferentiated state. Moreover, METTL3 depletion/inhibition induces DNA damage and differentiation of MYCN overexpressing cells and increases vulnerability to chemotherapeutic drugs in MYCN-amplified patient-derived xenografts (PDX) in vivo, suggesting METTL3 inhibition could be a potential therapeutic approach for NB.

Genome-wide characterization and evolution analysis of miniature inverted-repeat transposable elements in Barley (Hordeum vulgare)

Miniature inverted-repeat transposable elements (MITEs) constitute a class of class II transposable elements (TEs) that are abundant in plant genomes, playing a crucial role in their evolution and diversity. Barley (Hordeum vulgare), the fourth-most important cereal crop globally, is widely used for brewing, animal feed, and human consumption. However, despite their significance, the mechanisms underlying the insertion or amplification of MITEs and their contributions to barley genome evolution and diversity remain poorly understood. Through our comprehensive analysis, we identified 32,258 full-length MITEs belonging to 2,992 distinct families, accounting for approximately 0.17% of the barley genome. These MITE families can be grouped into four well-known superfamilies (Tc1/Mariner-like, PIF/Harbinger-like, hAT-like, and Mutator-like) and one unidentified superfamily. Notably, we observed two major expansion events in the barley MITE population, occurring approximately 12-13 million years ago (Mya) and 2-3 Mya. Our investigation revealed a strong preference of MITEs for gene-related regions, particularly in promoters, suggesting their potential involvement in regulating host gene expression. Additionally, we discovered that 7.73% miRNAs are derived from MITEs, thereby influencing the origin of certain miRNAs and potentially exerting a significant impact on post-transcriptional gene expression control. Evolutionary analysis demonstrated that MITEs exhibit lower conservation compared to genes, consistent with their dynamic mobility. We also identified a series of MITE insertions or deletions associated with domestication, highlighting these regions as promising targets for crop improvement strategies. These findings significantly advance our understanding of the fundamental characteristics and evolutionary patterns of MITEs in the barley genome. Moreover, they contribute to our knowledge of gene regulatory networks and provide valuable insights for crop improvement endeavors.

RNA Binding Proteins as Potential Therapeutic Targets in Colorectal Cancer

RNA-binding proteins (RBPs) play critical roles in regulating post-transcriptional gene expression, managing processes such as mRNA splicing, stability, and translation. In normal intestine, RBPs maintain the tissue homeostasis, but when dysregulated, they can drive colorectal cancer (CRC) development and progression. Understanding the molecular mechanisms behind CRC is vital for developing novel therapeutic strategies, and RBPs are emerging as key players in this area. This review highlights the roles of several RBPs, including LIN28, IGF2BP1–3, Musashi, HuR, and CELF1, in CRC. These RBPs regulate key oncogenes and tumor suppressor genes by influencing mRNA stability and translation. While targeting RBPs poses challenges due to their complex interactions with mRNAs, recent advances in drug discovery have identified small molecule inhibitors that disrupt these interactions. These inhibitors, which target LIN28, IGF2BPs, Musashi, CELF1, and HuR, have shown promising results in preclinical studies. Their ability to modulate RBP activity presents a new therapeutic avenue for treating CRC. In conclusion, RBPs offer significant potential as therapeutic targets in CRC. Although technical challenges remain, ongoing research into the molecular mechanisms of RBPs and the development of selective, potent, and bioavailable inhibitors should lead to more effective treatments and improved outcomes in CRC.

CEP112 coordinates translational regulation of essential fertility genes during spermiogenesis through phase separation in humans and mice

Spermiogenesis, the complex transformation of haploid spermatids into mature spermatozoa, relies on precise spatiotemporal regulation of gene expression at the post-transcriptional level. The mechanisms underlying this critical process remain incompletely understood. Here, we identify centrosomal protein 112 (CEP112) as an essential regulator of mRNA translation during this critical developmental process. Mutations in CEP112 are discovered in oligoasthenoteratospermic patients, and Cep112-deficient male mice recapitulate key phenotypes of human asthenoteratozoospermia. CEP112 localizes to the neck and atypical centrioles of mature sperm and forms RNA granules during spermiogenesis, enriching target mRNAs such as Fsip2, Cfap61, and Cfap74. Through multi-omics analyses and the TRICK reporter assay, we demonstrate that CEP112 orchestrates the translation of target mRNAs. Co-immunoprecipitation and mass spectrometry identify CEP112’s interactions with translation-related proteins, including hnRNPA2B1, EEF1A1, and EIF4A1. In vitro, CEP112 undergoes liquid-liquid phase separation, forming condensates that recruit essential proteins and mRNAs. Moreover, variants in patient-derived CEP112 disrupt phase separation and impair translation efficiency. Our results suggest that CEP112 mediates the assembly of RNA granules through liquid-liquid phase separation to control the post-transcriptional expression of fertility-related genes. This study not only clarifies CEP112’s role in spermatogenesis but also highlights the role of phase separation in translational regulation, providing insights into male infertility and suggesting potential therapeutic targets.

Interplay of microRNAs and circRNAs in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) with its high death incidence rate is generally detected at advanced stages. During its progression, EOC often develops peritoneal metastasis aggravating the outcomes of EOC patients. Studies on non-coding RNAs (ncRNAs), such as microRNAs (miRNAs) and circular RNAs (circRNAs), have analyzed the impact of miRNAs and circRNAs, along with their interaction among each other, on cancer cells. MiRNAs can act as oncogenes or tumor suppressors modulating post-transcriptional gene expression. There is accumulating evidence that circRNAs apply their stable, covalently closed, continuous circular structures to competitively inhibit miRNA function, and so act as competing endogenous RNAs (ceRNAs). This interplay between both ncRNAs participates in the malignity of a variety of cancer types, including EOC. In the current review, I describe the characteristics of miRNAs and circRNAs, and discuss their interplay with each other in the development, progression, and drug resistance of EOC. Sponging of miRNAs by circRNAs may be used as a biomarker and therapeutic target in EOC.

Abstract PR-11: Altered mRNA splicing mimics chromosome loss and drives pancreatic cancer

Abstract Aiming to identify novel driver mutations that cause pancreatic ductal adenocarcinoma (PDAC), besides p53 mutations, we performed a genetic interaction analysis based on the concept of mutually exclusive mutations, which suggests that mutations exclusive to one another function within the same pathway. Using our unbiased quantitative methods to evaluate the effects of somatic mutations on cancer, we analyzed 3900 human PDAC cases to prioritize mutations among hundreds that appear at low frequencies (5-10%) and are mutually exclusive with p53 mutations. Mutations in RNA splicing factors SF3B1 and RBM10 (∼15% of cases) were among the most significant and mutually exclusive to mutant p53. Thus, we hypothesized that aberrant RNA splicing promoted by mutant SF3B1K700E and truncated RBM10 (RBM10 loss) lead to tumorigenesis and therapy resistance. Through the generation of engineered mouse models to co-express KrasG12D with either Sf3b1K700E or Rbm10 loss in pancreatic cells, we observed that mice harboring KrasG12D and Sf3b1 K700E or Rbm10 loss—like mutant p53—caused PDAC in mice. To identify the RNA splicing mechanism that leads to PDAC formation, we performed deep RNA sequencing and unbiased transcriptome-wide splicing analyses in isolated PDAC cells bearing wild-type SF3B1K700E and RBM10 loss derived from murine and patient PDACs. We established that SF3B1 and RBM10 mutations induce wide splicing changes, primarily affecting exon selection. Since two-thirds of these changes may lead to nonsense-mediated decay (NMD), a mechanism that eliminates aberrant mRNA, we tested whether these changes could trigger this response. Using cycloheximide-dependent NMD inhibition, we confirmed that these mutations cause NMD, resulting in post-transcriptional gene expression loss in mouse chromosomes 2, 7, 11, and 17, also conserved in human models. Intriguingly, recent studies in Nature (2022) have shown that in murine PDACs with loss-of-function p53 mutations, genetic loss of chromosomes 11 and 7 is necessary for PDAC development. These results suggest that mutations in SF3B1 and RBM10, and p53 loss, affect the same chromosomal loci, albeit through different mechanisms, to promote PDAC, supporting the mutual exclusivity findings from patient samples. To determine a personalized therapy for patients with mutant splicing-factor tumors, we found that PDACs with these mutations are more sensitive to Gemcitabine, a component of a PDAC standard-of-care therapy. Moreover, these mutations sensitize cells to small-molecule splicing inhibitor H3B-8800— currently in phase 2 trial for hematologic malignancies. Lastly, we found that combining Gemcitabine and H3B-8800 synergizes to kill mutant splicing cells selectively in vitro and in vivo. We are starting a phase 2 trial for Gemcitabine/Abraxane with escalating doses of H3B-8800 for tumors that have either SF3B1K700E, RBM10 loss, or neomorphic p53, all of which have aberrant RNA splicing. This work has determined novel drivers and mechanisms of PDAC development and a precision therapeutic strategy for treating PDAC patients. Citation Format: Natasha Pinto Medici, Diana Martinez-Saucedo, Danny Lee, Tianyi Chu, Robert Tseng, Vincent Cannataro, Jeffrey Townsend, Christine Iacobuzio-Donahue, Marie Robert, Omar Abdel-Wahab, Steven D. Leach, Luisa Escobar-Hoyos. Altered mRNA splicing mimics chromosome loss and drives pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-11.

Biological Barriers for Drug Delivery and Development of Innovative Therapeutic Approaches in HIV, Pancreatic Cancer, and Hemophilia A/B.

Biological barriers remain a major obstacle for the development of innovative therapeutics. Depending on a disease's pathophysiology, the involved tissues, cell populations, and cellular components, drugs often have to overcome several biological barriers to reach their target cells and become effective in a specific cellular compartment. Human biological barriers are incredibly diverse and include multiple layers of protection and obstruction. Importantly, biological barriers are not only found at the organ/tissue level, but also include cellular structures such as the outer plasma membrane, the endolysosomal machinery, and the nuclear envelope. Nowadays, clinicians have access to a broad arsenal of therapeutics ranging from chemically synthesized small molecules, biologicals including recombinant proteins (such as monoclonal antibodies and hormones), nucleic-acid-based therapeutics, and antibody-drug conjugates (ADCs), to modern viral-vector-mediated gene therapy. In the past decade, the therapeutic landscape has been changing rapidly, giving rise to a multitude of innovative therapy approaches. In 2018, the FDA approval of patisiran paved the way for small interfering RNAs (siRNAs) to become a novel class of nucleic-acid-based therapeutics, which-upon effective drug delivery to their target cells-allow to elegantly regulate the post-transcriptional gene expression. The recent approvals of valoctocogene roxaparvovec and etranacogene dezaparvovec for the treatment of hemophilia A and B, respectively, mark the breakthrough of viral-vector-based gene therapy as a new tool to cure disease. A multitude of highly innovative medicines and drug delivery methods including mRNA-based cancer vaccines and exosome-targeted therapy is on the verge of entering the market and changing the treatment landscape for a broad range of conditions. In this review, we provide insights into three different disease entities, which are clinically, scientifically, and socioeconomically impactful and have given rise to many technological advancements: acquired immunodeficiency syndrome (AIDS) as a predominant infectious disease, pancreatic carcinoma as one of the most lethal solid cancers, and hemophilia A/B as a hereditary genetic disorder. Our primary objective is to highlight the overarching principles of biological barriers that can be identified across different disease areas. Our second goal is to showcase which therapeutic approaches designed to cross disease-specific biological barriers have been promising in effectively treating disease. In this context, we will exemplify how the right selection of the drug category and delivery vehicle, mode of administration, and therapeutic target(s) can help overcome various biological barriers to prevent, treat, and cure disease.

Progress of Exosomal MicroRNAs and Traditional Chinese Medicine Monomers in Neurodegenerative Diseases.

Exosomes, extracellular vesicles secreted by various cells, actively participate in intercellular communication by facilitating the exchange of crucial molecular information such as DNA, RNA, and lipids. Within this intricate network, microRNAs, endogenous non-coding small RNAs, emerge as pivotal regulators of post-transcriptional gene expression, significantly influencing the development of neurodegenerative diseases. The historical prominence of traditional Chinese medicine (TCM) in clinical practice in China underscores its enduring significance. Notably, TCM monomers, serving as active constituents within herbal medicine, assume a critical role in the treatment of neurodegenerative diseases, particularly in mitigating oxidative stress, inhibiting apoptosis, and reducing inflammation. This comprehensive review aims to delineate the specific involvement of exosomal microRNAs in various neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, and amyotrophic lateral sclerosis. Furthermore, the exploration extends to the application of TCM monomers, elucidating their efficacy as therapeutic agents in these conditions. Additionally, the review examines the utilization of exosomes as drug delivery carriers in the context of neurodegenerative diseases, providing a nuanced understanding of the potential synergies between TCM and modern therapeutic approaches. This synthesis of knowledge aims to contribute to the advancement of our comprehension of the intricate molecular mechanisms underlying neurodegeneration and the potential therapeutic avenues offered by TCcom interventions.

MicroRNAs in Genitourinary Malignancies: An Exciting Frontier of Cancer Diagnostics and Therapeutics.

Genitourinary (GU) malignancies, including prostate, urothelial, kidney, testicular, penile, and adrenocortical cancers, comprise a significant burden of cancers worldwide. While many practice-changing advances have been made in the management of GU malignancies in the last decade, there is still significant room for improvement. MicroRNAs (miRNAs) are noncoding RNAs that regulate post-transcription gene expression and which have been implicated in multiple mechanisms of carcinogenesis. Therefore, they have the potential to revolutionize personalized cancer therapy, with several ongoing preclinical and clinical studies underway to investigate their efficacy. In this review, we describe the current landscape of miRNAs as diagnostics, therapeutics, and biomarkers of response for GU malignancies, reflecting a novel frontier in cancer treatment.

RNA-binding proteins potentially regulate alternative splicing of immune/inflammatory-associated genes during the progression of generalized pustular psoriasis.

Generalized pustular psoriasis (GPP) is a rare but severe form of psoriasis. However, the pathogenesis of GPP has not been fully elucidated. Although RNA-binding proteins (RBPs) and the alternative splicing (AS) process are essential for regulating post-transcriptional gene expression, their roles in GPP are still unclear. We aimed to elucidate the regulatory mechanisms to identify potential new therapeutic targets. Here, We analyzed an RNA sequencing (RNA-seq) dataset (GSE200977) of peripheral blood mononuclear cells (PBMCs) of 24 patients with GPP, psoriasis vulgaris (PV), and healthy controls (HCs) from the Gene Expression Omnibus (GEO) database. We found that the abnormal alternative splicing (AS) events associated with GPP were mainly "alt3p/alt5p", and 15 AS genes were differentially expressed. Notably, the proportions of different immune cell types were correlated with the expression levels of regulatory alternatively spliced genes (RASGs): significant differences were observed in expression levels of DTD2, NDUFAF3, NBPF15, and FBLN7 in B cells and ARFIP1, IPO11, and RP11-326L24.9 in neutrophils in the GPP samples. Furthermore, We identified 32 differentially expressed RNA-binding proteins (RBPs) (18 up-regulated and 14 down-regulated). Co-expression networks between 14 pairs of differentially expressed RBPs and RASGs were subsequently constructed, demonstrating that these differentially expressed RBPs may affect the progression of GPP by regulating the AS of downstream immune/inflammatory-related genes such as LINC00989, ENC1 and MMP25-AS1. Our results were innovative in revealing the involvement of inflammation-related RBPs and RASGs in the development of GPP from the perspective of RBP-regulated AS.

RNA Surveillance Factor SMG5 Is Essential for Mouse Embryonic Stem Cell Differentiation.

Nonsense-mediated mRNA decay (NMD) is a highly conserved post-transcriptional gene expression regulatory mechanism in eukaryotic cells. NMD eliminates aberrant mRNAs with premature termination codons to surveil transcriptome integrity. Furthermore, NMD fine-tunes gene expression by destabilizing RNAs with specific NMD features. Thus, by controlling the quality and quantity of the transcriptome, NMD plays a vital role in mammalian development, stress response, and tumorigenesis. Deficiencies of NMD factors result in early embryonic lethality, while the underlying mechanisms are poorly understood. SMG5 is a key NMD factor. In this study, we generated an Smg5 conditional knockout mouse model and found that Smg5-null results in early embryonic lethality before E13.5. Furthermore, we produced multiple lines of Smg5 knockout mouse embryonic stem cells (mESCs) and found that the deletion of Smg5 in mESCs does not compromise cell viability. Smg5-null delays differentiation of mESCs. Mechanistically, our study reveals that the c-MYC protein, but not c-Myc mRNA, is upregulated in SMG5-deficient mESCs. The overproduction of c-MYC protein could be caused by enhanced protein synthesis upon SMG5 loss. Furthermore, SMG5-null results in dysregulation of alternative splicing on multiple stem cell differentiation regulators. Overall, our findings underscore the importance of SMG5-NMD in regulating mESC cell-state transition.

Genome-wide and cell-type-selective profiling of in vivo small noncoding RNA:target RNA interactions by chimeric RNA sequencing

Small noncoding RNAs (sncRNAs) regulate biological processes by impacting post-transcriptional gene expression through repressing the translation and levels of targeted transcripts. Despite the clear biological importance of sncRNAs, approaches to unambiguously define genome-wide sncRNA:target RNA interactions remain challenging and not widely adopted. We present CIMERA-seq, a robust strategy incorporating covalent ligation of sncRNAs to their target RNAs within the RNA-induced silencing complex (RISC) and direct detection of invivo interactions by sequencing of the resulting chimeric RNAs. Modifications are incorporated to increase the capacity for processing low-abundance samples and permit cell-type-selective profiling of sncRNA:target RNA interactions, as demonstrated in mouse brain cortex. CIMERA-seq represents a cohesive and optimized method for unambiguously characterizing the invivo network of sncRNA:target RNA interactions in numerous biological contexts and even subcellular fractions. Genome-wide and cell-type-selective CIMERA-seq enhances researchers' ability to study gene regulation by sncRNAs in diverse model systems and tissue types.

The Role of N6-methyladenosine Modification in Gametogenesis and Embryogenesis: Impact on Fertility.

The most common epigenetic modification of messenger RNAs (mRNAs) is N6-methyladenosine (m6A), which is mainly located near the 3' untranslated region of mRNAs, near the stop codons, and within internal exons. The biological effect of m6A is dynamically modulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers). By controlling post-transcriptional gene expression, m6A has a significant impact on numerous biological functions, including RNA transcription, translation, splicing, transport, and degradation. Hence, m6A influences various physiological and pathological processes, such as spermatogenesis, oogenesis, embryogenesis, placental function, and human reproductive system diseases. During gametogenesis and embryogenesis, genetic material undergoes significant changes, including epigenomic modifications such as m6A. From spermatogenesis and oogenesis to the formation of an oosperm and early embryogenesis, m6A changes occur at every step. m6A abnormalities can lead to gamete abnormalities, developmental delays, impaired fertilization, and maternal-to-zygotic transition blockage. Both mice and humans with abnormal m6A modifications exhibit impaired fertility. In this review, we discuss the dynamic biological effects of m6A and its regulators on gamete and embryonic development and review the possible mechanisms of infertility caused by m6A changes. We also discuss the drugs currently used to manipulate m6A and provide prospects for the prevention and treatment of infertility at the epigenetic level.

Identification and functional prediction of miRNAs that regulate ROS levels in dielectric barrier discharge plasma-treated boar spermatozoa

Dielectric barrier discharge (DBD) plasma regulates the levels of reactive oxygen species (ROS), which are critical for sperm quality. MicroRNAs (miRNAs) are non-coding single-stranded RNA molecules encoded by endogenous genes, which regulate post-transcriptional gene expression in animals. At present, it is unknown whether DBD plasma can regulate sperm ROS levels through miRNAs. To further understand the regulatory mechanism of DBD plasma on sperm ROS levels, miRNAs in fresh boar spermatozoa were detected using Illumina deep sequencing technology. We found that 25 known miRNAs and 50 novel miRNAs were significantly upregulated, and 14 known miRNAs and 74 novel miRNAs were significantly downregulated in DBD plasma-treated spermatozoa. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that target genes of differentially expressed miRNAs were involved in many activities and pathways associated with antioxidants. We verified that DBD plasma significantly increased boar sperm quality and reduced ROS levels. These results suggest that DBD plasma can improve sperm quality by regulating ROS levels via miRNAs. Our findings provide a potential strategy to improve sperm quality through miRNA-targeted regulation of ROS, which helps to increase male reproduction and protect cryopreserved semen in clinical practice.

Biogenesis and Function of miRNAs and Their Role in Cancer

MicroRNA (miRNA) is a small RNA molecule that regulates post-transcriptional gene expression. miRNA combines with the RNA-induced silencing complex (RISC) and carries out its function in controlling translation. The formation of miRNA involves enzymes and proteins that cut it in its processing as well as protein complexes in the cytoplasm that make the miRNA mature. Several changes, for example deletion or overexpression, can occur in miRNAs, causing cancer growth. The aim of this study was to investigate the process of miRNA biogenesis as well as analyze how changes in miRNA expression can affect the biological pathways involved in cancer. This type of research is a literature review. The data that has been collected is then analyzed in three stages, namely data reduction, data presentation and drawing conclusions. The results show that the role of miRNAs is in the post-transcriptional regulation of genes. MiRNAs associate with the RISC complex and interact with mRNA through complementary base pairing thereby halting translation and promoting mRNA degradation. MiRNAs are deregulated in cancer, including over-expression of oncogenic miRNAs or deletion of miRNA characteristics as tumor inhibitors. The results of this research also provide a foundation for further education and advanced research in the field of molecular biology, particularly in genetic regulation and disease mechanisms.

Comparative Analysis of mRNA and lncRNA Expression Profiles in Testicular Tissue of Sexually Immature and Sexually Mature Mongolian Horses.

Testicular development and spermatogenesis are tightly regulated by both coding and non-coding genes, with mRNA and lncRNA playing crucial roles in post-transcriptional gene expression regulation. However, there are significant differences in regulatory mechanisms before and after sexual maturity. Nevertheless, the mRNAs and lncRNAs in the testes of Mongolian horses have not been systematically identified. In this study, we first identified the testicular tissues of sexually immature and sexually mature Mongolian horses at the tissue and protein levels, and comprehensively analyzed the expression profiles of mRNA and lncRNA in the testes of 1-year-old (12 months, n = 3) and 10-year-old (n = 3) Mongolian horses using RNA sequencing technology. Through gene expression analysis, we identified 16,582 mRNAs and 2128 unknown lncRNAs that are commonly expressed in both sexually immature and sexually mature Mongolian horses. Meanwhile, 9217 mRNAs (p < 0.05) and 2191 unknown lncRNAs (p < 0.05) were identified as differentially expressed between the two stages, which were further validated by real-time fluorescent quantitative PCR and analyzed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The analysis results showed that genes in the sexually immature stage were mainly enriched in terms related to cellular infrastructure, while genes in the sexually mature stage were enriched in terms associated with hormones, metabolism, and spermatogenesis. In summary, the findings of this study provide valuable resources for a deeper understanding of the molecular mechanisms underlying testicular development and spermatogenesis in Mongolian horses and offer new perspectives for future related research.

SiRNA therapy in osteoarthritis: targeting cellular pathways for advanced treatment approaches.

Osteoarthritis (OA) is a common joint disorder characterized by the degeneration of cartilage and inflammation, affecting millions worldwide. The disease's complex pathogenesis involves various cell types, such as chondrocytes, synovial cells, osteoblasts, and immune cells, contributing to the intricate interplay of factors leading to tissue degradation and pain. RNA interference (RNAi) therapy, particularly through the use of small interfering RNA (siRNA), emerges as a promising avenue for OA treatment due to its capacity for specific gene silencing. siRNA molecules can modulate post-transcriptional gene expression, targeting key pathways involved in cellular proliferation, apoptosis, senescence, autophagy, biomolecule secretion, inflammation, and bone remodeling. This review delves into the mechanisms by which siRNA targets various cell populations within the OA milieu, offering a comprehensive overview of the potential therapeutic benefits and challenges in clinical application. By summarizing the current advancements in siRNA delivery systems and therapeutic targets, we provide a solid theoretical foundation for the future development of novel siRNA-based strategies for OA diagnosis and treatment, paving the way for innovative and more effective approaches to managing this debilitating disease.

Regulatory roles of long non-coding RNAs in short-term heat stress in adult worker bees

Long non-coding RNAs (lncRNAs) are crucial modulators of post-transcriptional gene expression regulation, cell fate determination, and disease development. However, lncRNA functions during short-term heat stress in adult worker bees are poorly understood. Here, we performed deep sequencing and bioinformatic analyses of honeybee lncRNAs. RNA interference was performed by using siRNA targeting the most highly expressed lncRNA. The silencing effect on lncRNA and the relative expression levels of seven heat shock protein (HSP) genes, were subsequently examined. Overall, 7,842 lncRNAs and 115 differentially expressed lncRNAs (DELs) were identified in adult worker bees following heat stress exposure. Structural analysis revealed that the overall expression abundance, length of transcripts, exon number, and open reading frames of lncRNAs were lower than those of mRNAs. GO analysis revealed that the target genes were mainly involved in “metabolism,” “protein folding,” “response to stress,” and “signal transduction” pathways. KEGG analysis indicated that the “protein processing in endoplasmic reticulum” and “longevity regulating pathway-multiple species” pathways were most enriched. Quantitative real-time polymerase chain reaction (qRT-PCR) detection of the selected DELs confirmed the reliability of the sequencing data. Moreover, the siRNA experiment indicated that feeding siRNA yielded a silencing efficiency of 77.51% for lncRNA MSTRG.9645.5. Upon silencing this lncRNA, the expression levels of three HSP genes were significantly downregulated (p < 0.05), whereas those of three other HSP genes were significantly upregulated (p < 0.05). Our results provide a new perspective for understanding the regulatory mechanisms of lncRNAs in adult worker bees under short-term heat stress.

Inducible deletion of microRNA activity in kidney mesenchymal cells exacerbates renal fibrosis

MicroRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. However, the physiological functions of these non-coding RNAs in renal interstitial mesenchymal cells remain unclear. To conclusively evaluate the role of miRNAs, we generated conditional knockout (cKO) mice with platelet-derived growth factor receptor-β (PDGFR-β)-specific inactivation of the key miRNA pathway gene Dicer. The cKO mice were subjected to unilateral ureteral ligation, and renal interstitial fibrosis was quantitatively evaluated using real-time polymerase chain reaction and immunofluorescence staining. Compared with control mice, cKO mice had exacerbated interstitial fibrosis exhibited by immunofluorescence staining and mRNA expression of PDGFR-β. A microarray analysis showed decreased expressions of miR-9-5p, miR-344g-3p, and miR-7074-3p in cKO mice compared with those in control mice, suggesting an association with the increased expression of PDGFR-β. An analysis of the signaling pathways showed that the major transcriptional changes in cKO mice were related to smooth muscle cell differentiation, regulation of DNA metabolic processes and the actin cytoskeleton, positive regulation of fibroblast proliferation and Ras protein signal transduction, and focal adhesion-PI3K/Akt/mTOR signaling pathways. Depletion of Dicer in mesenchymal cells may downregulate the signaling pathway related to miR-9-5p, miR-344g-3p, and miR-7074-3p, which can lead to the progression of chronic kidney disease. These findings highlight the possibility for future diagnostic or therapeutic developments for renal fibrosis using miR-9-5p, miR-344g-3p, and miR-7074-3p.

Post-transcriptional Regulation of Gene Expression via Unproductive Splicing.

Unproductive splicing is a mechanism of post-transcriptional gene expression control in which premature stop codons are inserted into protein-coding transcripts as a result of regulated alternative splicing, leading to their degradation via the nonsense-mediated decay pathway. This mechanism is especially characteristic of RNA-binding proteins, which regulate each other's expression levels and those of other genes in multiple auto- and cross-regulatory loops. Deregulation of unproductive splicing is a cause of serious human diseases, including cancers, and is increasingly being considered as a prominent therapeutic target. This review discusses the types of unproductive splicing events, the mechanisms of auto- and cross-regulation, nonsense-mediated decay escape, and problems in identifying unproductive splice isoforms. It also provides examples of deregulation of unproductive splicing in human diseases and discusses therapeutic strategies for its correction using antisense oligonucleotides and small molecules.