Auswahl der wissenschaftlichen Literatur zum Thema „SncRNA silencing“

microRNAs (miRNAs) are small non-coding RNAs (sncRNAs) that play an important role in the life cycle of human viruses. We sought to characterize human immunodeficiency virus 1 (HIV-1)-encoded miRNAs and determine their role in viral replication. Initially, a bioinformatic analysis was used to predict HIV-1-encoded miRNAs. Next, a representative number of these predicted sequences were verified using a miRNA microarray chip, reverse transcription PCR (RT-PCR), and the deep sequencing of RNA extracted from HIV-1-infected cells. Eight HIV-1-encoded sncRNA sequences conforming to the criteria that define miRNAs were identified in HIV-1-infected immortalized T cells and human primary CD4+ lymphocytes; five of the eight sequences have not been previously reported. Deep sequencing validated the presence of these virus-encoded miRNA sequences and uncovered large numbers of atypical sncRNA sequences, lacking characteristics of conventional miRNAs. We named these sequences small RNAs (smRNAs). The overexpression of four candidate HIV-1-encoded miRNAs and silencing of two smRNAs significantly increased HIV-1 viral replication. Our study uncovered novel HIV-1-encoded sncRNAs that, upon deregulated expression, alter viral titers in HIV-1-infected cells, suggesting that miRNAs and smRNAs play an important role in regulating viral replication. Future studies may reveal the function of HIV-1-encoded sncRNAs and their possible implications for diagnosis and treatment.

MicroRNA is a small non-coding RNA (sncRNA) involved in gene silencing and regulating post-transcriptional gene expression. miRNAs play an essential role in the pathogenesis of numerous diseases, including diabetes, cardiovascular diseases, viral diseases and cancer. Diffuse large B-cell lymphoma (DLBCL) is an aggressive non-Hodgkin’s lymphoma (NHL), arising from different stages of B-cell differentiation whose pathogenesis involves miRNAs. Various viral and non-viral vectors are used as a delivery vehicle for introducing specific miRNA inside the cell. Adenoviruses are linear, double-stranded DNA viruses with 35 kb genome size and are extensively used in gene therapy. Meanwhile, Adeno-associated viruses accommodate up to 4.8 kb foreign genetic material and are favorable for transferring miRNA due to small size of miRNA. The genetic material is integrated into the DNA of the host cell by retroviruses so that only dividing cells are infected and stable expression of miRNA is achieved. Over the years, remarkable progress was made to understand DLBCL biology using advanced genomics and epigenomics technologies enabling oncologists to uncover multiple genetic mutations in DLBCL patients. These genetic mutations are involved in epigenetic modification, ability to escape immunosurveillance, impaired BCL6 and NF-κβ signaling pathways and blocking terminal differentiation. These pathways have since been identified and used as therapeutic targets for the treatment of DLBCL. Recently miRNAs were also identified to act either as oncogenes or tumor suppressors in DLBCL pathology by altering the expression levels of some of the known DLBCL related oncogenes. i.e., miR-155, miR-17-92 and miR-21 act as oncogenes by altering the expression levels of MYC, SHIP and FOXO1, respectively, conversely; miR-34a, mir-144 and miR-181a act as tumor suppressors by altering the expression levels of SIRT1, BCL6 and CARD11, respectively. Hundreds of miRNAs have already been identified as biomarkers in the prognosis and diagnosis of DLBCL because of their significant roles in DLBCL pathogenesis. In conclusion, miRNAs in addition to their role as biomarkers of prognosis and diagnosis could also serve as potential therapeutic targets for treating DLBCL.

Abstract In mammalian cells, small non-coding RNAs (sncRNAs) negatively regulate gene expression in a pathway known as RNA interference (RNAi). RNAi can be categorized into post-transcriptional gene silencing (PTGS), which involves the cleavage of target messenger RNA (mRNA) or inhibition of translation in the cytoplasm, and transcriptional gene silencing (TGS), which is mediated by the establishment of repressive epigenetic marks at target loci. Transfer RNAs (tRNAs), which are essential for translation, can be processed into small ncRNAs, termed tRNA-derived small RNAs (tsRNAs). The biogenesis of tsRNAs and their role in gene expression regulation has not yet been fully understood. Here, we show that Dicer dependent tsRNAs promote gene silencing through a mechanism distinct from PTGS and TGS. tsRNAs can lead to downregulation of target genes by targeting introns via nascent RNA silencing (NRS) in nuclei. Furthermore, we show that Ago2 slicer activity is required for this mechanism. Synthetic tsRNAs can significantly reduce expression of a target gene at both RNA and protein levels. Target genes regulated by NRS are associated with various diseases, which further underpins its biological significance. Finally, we show that NRS is evolutionarily conserved and has the potential to be explored as a novel synthetic sRNA based therapeutic.

PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs (sncRNAs) that perform crucial biological functions in metazoans and defend against transposable elements (TEs) in germ lines. Recently, ubiquitously expressed piRNAs were discovered in soma and germ lines using small RNA sequencing (sRNA-seq) in humans and animals, providing new insights into the diverse functions of piRNAs. However, the role of piRNAs has not yet been fully elucidated, and sRNA-seq studies continue to reveal different piRNA activities in the genome. In this review, we summarize a set of simplified processes for piRNA analysis in order to provide a useful guide for researchers to perform piRNA research suitable for their study objectives. These processes can help expand the functional research on piRNAs from previously reported sRNA-seq results in metazoans. Ubiquitously expressed piRNAs have been discovered in the soma and germ lines in Annelida, Cnidaria, Echinodermata, Crustacea, Arthropoda, and Mollusca, but they are limited to germ lines in Chordata. The roles of piRNAs in TE silencing, gene expression regulation, epigenetic regulation, embryonic development, immune response, and associated diseases will continue to be discovered via sRNA-seq.

Background: Parkinson’s disease (PD) is seriously threatening the health and life quality of the elderly, who have a high incidence and high disability rate. Resveratrol (RES) was reported to play a protective role in PD. However, the functions and potential mechanism of RES in PD remain unclear, which need to be further explored. Methods: Human neuroblastoma cells (SH-SY5Y and SK-N-SH) were subjected to 1-Methyl-4-phenylpyridium (MPP+) induction to construct a cell model of PD. Cell viability was evaluated using CCK-8. The gene expression was evaluated using qRT-PCR and western blot. Luciferase activity assay and RIP were performed to validate interactions among SNHG1, miR-128-3p and SNCA. Results: Our results exhibited that RES reduced SNHG1 and SNCA expression but elevated miR-128-3p expression in human neuroblastoma cells upon MPP+ induction. Functionally, RES resulted in the promotion of cell autophagy in MPP+-induced human neuroblastoma cells, while these influences were abolished by SNHG1 overexpression. Mechanistically, SNHG1 could indirectly elevate SNCA expression via sponging miR-128-3p. Moreover, SNCA overexpression reversed SNHG1 silencing-induced cell autophagy in MPP+-induced human neuroblastoma cells upon RES pre-incubation. Conclusions: RES prevented MPP+-induced repression of cell autophagy through inhibiting the SNHG1/miR-128-3p/SNCA axis, suggesting that RES might play a preventive effect on PD progression.

AbstractObjectives:Lewy body dementia (LBD) is the second most prevalent neurodegenerative dementia and it causes more morbidity and mortality than Alzheimer’s disease. Several genetic associations of LBD have been reported and their functional implications remain uncertain. Hence, we aimed to do a systematic review of all gene expression studies that investigated people with LBD for improving our understanding of LBD molecular pathology and for facilitating discovery of novel biomarkers and therapeutic targets for LBD.Methods:We systematically reviewed five online databases (PROSPERO protocol: CRD42017080647) and assessed the functional implications of all reported differentially expressed genes (DEGs) using Ingenuity Pathway Analyses.Results:We screened 3,809 articles and identified 31 eligible studies. In that, 1,242 statistically significant (p < 0.05) DEGs including 70 microRNAs have been reported in people with LBD. Expression levels of alternatively spliced transcripts of SNCA, SNCB, PRKN, APP, RELA, and ATXN2 significantly differ in LBD. Several mitochondrial genes and genes involved in ubiquitin proteasome system and autophagy–lysosomal pathway were significantly downregulated in LBD. Evidence supporting chronic neuroinflammation in LBD was inconsistent. Our functional analyses highlighted the importance of ribonucleic acid (RNA)-mediated gene silencing, neuregulin signalling, and neurotrophic factors in the molecular pathology of LBD.Conclusions:α-synuclein aggregation, mitochondrial dysfunction, defects in molecular networks clearing misfolded proteins, and RNA-mediated gene silencing contribute to neurodegeneration in LBD. Larger longitudinal transcriptomic studies investigating biological fluids of people living with LBD are needed for molecular subtyping and staging of LBD. Diagnostic biomarker potential and therapeutic promise of identified DEGs warrant further research.

Alpha-synuclein (α-syn) is a protein considered to be detrimental in a number of degenerative disorders (synucleinopathies) of which α-syn aggregates are considered a pathological hallmark. The clearance of α-syn strongly depends on autophagy, which can be stimulated by inhibiting the mechanistic target of rapamycin (mTOR). Thus, the overexpression of mTOR and severe autophagy suppression may produce α-syn accumulation, including the proteinase K-resistant protein isoform. Glioblastoma multiforme (GBM) is a lethal brain tumor that features mTOR overexpression and severe autophagy inhibition. Cell pathology in GBM is reminiscent of a fast, progressive degenerative disorder. Therefore, the present work questions whether, as is analogous to neurons during degenerative disorders, an overexpression of α-syn occurs within GBM cells. A high amount of α-syn was documented in GBM cells via real-time PCR (RT-PCR), Western blotting, immunohistochemistry, immuno-fluorescence, and ultrastructural stoichiometry, compared with the amount of β- and γ-synucleins and compared with the amount of α-syn counted within astrocytes. The present study indicates that (i) α-syn is overexpressed in GBM cells, (ii) α-syn expression includes a proteinase-K resistant isoform, (iii) α-syn is dispersed from autophagy-like vacuoles to the cytosol, (iv) α-syn overexpression and cytosol dispersion are mitigated by rapamycin, and (v) the α-syn-related GBM-like phenotype is mitigated by silencing the SNCA gene.

Abstract Genetic elements encoded in nuclear DNA determine the sex of an individual in many animals. In certain bivalve lineages that possess doubly uniparental inheritance (DUI), mitochondrial DNA (mtDNA) has been hypothesized to contribute to sex determination. In these cases, females transmit a female mtDNA to all offspring, while male mtDNA (M mtDNA) is transmitted only from fathers to sons. Because M mtDNA is inherited in the same way as Y chromosomes, it has been hypothesized that mtDNA may be responsible for sex determination. However, the role of mitochondrial and nuclear genes in sex determination has yet to be validated in DUI bivalves. In this study, we used DNA, RNA, and mitochondrial short non-coding RNA (sncRNA) sequencing to explore the role of mitochondrial and nuclear elements in the sexual development pathway of the freshwater mussel Potamilus streckersoni (Bivalvia: Unionida). We found that the M mtDNA shed a sncRNA partially within a male-specific mitochondrial gene that targets a pathway hypothesized to be involved in female development and mitophagy. RNA-seq confirmed the gene target was significantly upregulated in females, supporting a direct role of mitochondrial sncRNAs in gene silencing. These findings support the hypothesis that M mtDNA inhibits female development. Genome-wide patterns of genetic differentiation and heterozygosity did not support a nuclear sex determining region, although we cannot reject that nuclear factors are involved with sex determination. Our results provide further evidence that mitochondrial loci contribute to diverse, non-respiratory functions and additional insights into an unorthodox sex determining system.

Abstract Small non-coding RNAs (sncRNA) are involved in many steps of the gene expression cascade and regulate processing and expression of mRNAs by the formation of ribonucleoprotein complexes (RNP) such as the RNA-induced silencing complex (RISC). By analyzing small RNA Seq data sets, we identified a sncRNA annotated as piR-hsa-1254, which is likely derived from the 3′-end of 7SL RNA2 (RN7SL2), herein referred to as snc7SL RNA. The 7SL RNA is an abundant long non-coding RNA polymerase III transcript and serves as structural component of the cytoplasmic signal recognition particle (SRP). To evaluate a potential functional role of snc7SL RNA, we aimed to define its cellular localization by live cell imaging. Therefore, a Molecular Beacon (MB)-based method was established to compare the subcellular localization of snc7SL RNA with its precursor 7SL RNA. We designed and characterized several MBs in vitro and tested those by live cell fluorescence microscopy. Using a multiplex approach, we show that 7SL RNA localizes mainly to the endoplasmic reticulum (ER), as expected for the SRP, whereas snc7SL RNA predominately localizes to the nucleus. This finding suggests a fundamentally different function of 7SL RNA and its derivate snc7SL RNA.

Exosomes, a subset of small extracellular vesicles, carry various nucleic acids, proteins, lipids, amino acids and metabolites. They function as a mode of intercellular communication and molecular transfer. Exosome cargo molecules, including small non-coding RNAs (sncRNAs), are involved in the immune response in various organisms. However, the role of exosome-derived sncRNAs in immune responses in molluscs remains unclear. Here, we aimed to reveal the sncRNAs involved in the immune response during grafting transplantation by the pearl oyster Pinctada fucata . Exosomes were successfully extracted from the P. fucata haemolymph during graft transplantation. Abundant microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) were simultaneously discovered in P. fucata exosomes by small RNA sequencing. The expression patterns of the miRNAs and piRNAs at the grafting and initial stages were not substantially different, but varied significantly between the initial and later stages. Target prediction and functional analysis indicate that these miRNAs and piRNAs are related to immune response upon grafting transplantation, whereas piRNAs may also be associated with transposon silencing by targeting with genome transposon elements. This work provides the basis for a functional understanding of exosome-derived sncRNAs and helps to gain further insight into the PIWI/piRNA pathway function outside of germline cells in molluscs.

La 2’-O-methylation (Nm) peut affecter les ARNs de multiples façons et les enzymes qui la déposent sont évolutivement conservées. Leurs mutations sont souvent liées à des cancers et des maladies du cerveau. FTSJ1, une ARNt-methyltransferase (Nm-MTase), associée à la déficience intellectuelle (DI) chez l’Homme et conservée chez la levure, est un excellent exemple. Au cours de mon travail doctoral j’ai démontré la fonction moléculaire de ses homologues prédits de Drosophila (Trm7_32 et Trm7_34) en utilisant la technique de pointe RiboMethSeq, ainsi que le MALDI-TOF. J'ai également découvert de nouvelles cibles ARNt de FTSJ1 au sein des lignées cellulaires dérivées du sang de patients atteint de DI. Une analyse de séquençage des petits ARN (ARNseq) a révélé une dérégulation de la population des micro-ARN (miARN) dans les cellules portant une mutation perte de fonction de FTSJ1, tandis que chez la Drosophila, nos senseurs génétiques ont montré des dysfonctionnements dans les voies de silencing des petits ARN non-codants en l'absence des orthologues de FTSJ1. Une analyse par northern blot a détecté l'accumulation de fragments d'ARNt spécifiques (tRF) dérivés de tRNAPhe (une cible majeure des enzymes en question). Aujourd'hui, nous examinons le potentiel de biomarqueurs de ces tRF et testons leurs conservation dans les lignées cellulaires de patients souffrants de DI. Enfin, nous avons utilisé le RiboMethSeq pour évaluer tout changement dans les profils Nm de la population d'ARNm dans le contexte mutant FTSJ1. Les résultats de ces études fournissent des informations supplémentaires sur les voies moléculaires qui régissent la pathogenèse DI dépendante de FTSJ1
2’-O-methylation (Nm) can affect RNAs in multiple ways and Nm-modifying enzymes are highly conserved and their dysfunctions are often linked to the development of cancers and brain diseases. An excellent example is the human FTSJ1 - a tRNA Nm-methyltransferase (Nm-MTase) that is conserved in yeast and is associated with Intellectual Disability (ID) in humans and mice. During my PhD work, I contributed on extending the evolutionary portfolio of this enzyme by demonstrating the molecular function of its predicted Drosophila homologs (Trm7_32 and Trm7_34) by using the cutting edge RiboMethSeq technique and the more classical MALDI-TOF. I also unraveled novel tRNA and even potential mRNA targets of human FTSJ1 in cell lines derived from ID patients’ blood carrying various mutations in FTSJ1. A small RNAseq analysis revealed a deregulation of the miRNAs population in FTSJ1 loss of function mutant cells, while in flies, our genetic sensors showed dysfunctional piRNA and Ago2-dependent miRNA silencing pathways when lacking the FTSJ1 orthologs. Northern blot analysis detected the accumulation of specific tRNA fragments (tRFs) derived from tRNAPhe (a major target of the enzymes). Today, we are considering the biomarker potential of these tRFs and testing their conservation in the ID patients’ cell lines. Finally, we used RiboMethseq to evaluate any changes in the Nm-profiles of the mRNA population in FTSJ1 mutant context, thus, opening new possibilities for biological significance of this enzyme. The results from these studies provide further insights in the molecular pathways that govern FTSJ1-dependent ID pathogenesis