RNA Transcription Machinery Research Articles

P-745 Follicular fluid exposure may activate gene expression in mature human sperm before fertilization

Abstract Study question Does follicular fluid (FF) exposure shape human sperm RNA content and are these changes acquired through genomic or nongenomic manner? Summary answer FF exposure altered sperm RNA content potentially via activation of sperm RNA transcription machinery and caused selective intake of FF RNA transcripts in sperm. What is known already Mature sperm have been widely recognized as transcriptionally inert. Therefore, sperm RNAs have been regarded as the sole remnants of the gene expression process that occurs during spermatogenesis. Supporting this view, most sperm histones are replaced by protamines in developing spermatids to enable tighter packaging of the genetic material, which should prevent mature sperm cells to express their genes. However, in humans, approximately 15% of the sperm DNA remains associated with histones, raising the possibility that these genome regions could be available for de novo RNA transcription under an appropriate stimulus. Study design, size, duration Fresh, mature sperm were divided into three aliquots. One aliquot was used as an untreated control sample, the second and third aliquots were treated with either PureSperm® Wash (SW) solution or an FF pool of five females for three hours. RNA sequencing (RNA-seq) data was collected from FF and sperm samples. Results were complemented with Chromatin Immunoprecipitation Sequencing (ChIP-seq) data from human mature sperm (GEO: GSE15690 and GSE49624) and nascent RNA capture followed by PCR. Participants/materials, setting, methods All male and female participants were of reproductive age, non-smoking, healthy individuals who reported no history of infertility. Differentially expressed genes (DEG) were identified based on RNA-seq data (adjusted p-value < 0.05, logFC < |0.75|). The histone status of the genomic loci of DEGs was evaluated using ChIP-seq data. DEGs were also subjected to Gene Ontology analysis. Activated RNA transcription was confirmed by the Click-iT™ Nascent RNA Capture kit and PCR. Main results and the role of chance RNA-seq showed 193 DEGs between untreated and FF-treated samples of which 30 genes were upregulated and 163 were downregulated. Furthermore, 18 DEGs were detected between SW- and FF-treated sperm of which 17 genes were upregulated and one downregulated. Finally, 137 DEGs (all downregulated) were found between untreated and SW-treated samples. Among upregulated genes, chemokine-signaling, ion transport, and steroidogenesis-related biological functions were enriched whereas RNA processing, translation, and protein targeting-related biological processes were enriched among downregulated genes (untreated vs. FF-treated). ChIP-seq data revealed that 93% of upregulated DEGs are located within histone-bound chromatin and most of them bear both active and inactive histone markers. Finally, we confirmed that the upregulation of at least two genes (CXCL3 and CXCL8) resulted from active RNA transcription by sperm, whereas the upregulation of IGF2 was due to the selective internalization of mRNA from FF. Additionally, we identified 1396 protein-coding mRNAs present in human FF that were associated with gene expression-related biological processes such as the regulation of transcription, processing of RNA subtypes, and translation. Limitations, reasons for caution Due to the limited sample size, the generalizability of our results should be validated in the future. Furthermore, molecular mechanisms behind observed nascent RNA production needs to be clarified in more detail, including potential FF-mediated remodeling of sperm histone markers involved in the activation of gene expression. Wider implications of the findings Our findings demonstrate that FF exposure changes sperm RNA content, which seems to be partly caused by the active transcription of mature sperm, which has been widely believed to be non-existent. Therefore, our results can have many important implications for a deeper understanding of fertilization, early embryogenesis, and infertility. Trial registration number Not applicaple

Of Keeping and Tipping the Balance: Host Regulation and Viral Modulation of IRF3-Dependent IFNB1 Expression.

The type I interferon (IFN) response is a principal component of our immune system that allows to counter a viral attack immediately upon viral entry into host cells. Upon engagement of aberrantly localised nucleic acids, germline-encoded pattern recognition receptors convey their find via a signalling cascade to prompt kinase-mediated activation of a specific set of five transcription factors. Within the nucleus, the coordinated interaction of these dimeric transcription factors with coactivators and the basal RNA transcription machinery is required to access the gene encoding the type I IFN IFNβ (IFNB1). Virus-induced release of IFNβ then induces the antiviral state of the system and mediates further mechanisms for defence. Due to its key role during the induction of the initial IFN response, the activity of the transcription factor interferon regulatory factor 3 (IRF3) is tightly regulated by the host and fiercely targeted by viral proteins at all conceivable levels. In this review, we will revisit the steps enabling the trans-activating potential of IRF3 after its activation and the subsequent assembly of the multi-protein complex at the IFNβ enhancer that controls gene expression. Further, we will inspect the regulatory mechanisms of these steps imposed by the host cell and present the manifold strategies viruses have evolved to intervene with IFNβ transcription downstream of IRF3 activation in order to secure establishment of a productive infection.

Nucleosome spacing periodically modulates nucleosome chain folding and DNA topology in circular nucleosome arrays

The length of linker DNA that separates nucleosomes is highly variable, but its mechanistic role in modulating chromatin structure and functions remains unknown. Here, we established an experimental system using circular arrays of positioned nucleosomes to investigate whether variations in nucleosome linker length could affect nucleosome folding, self-association, and interactions. We conducted EM, DNA topology, native electrophoretic assays, and Mg2+-dependent self-association assays to study intrinsic folding of linear and circular nucleosome arrays with linker DNA length of 36 bp and 41 bp (3.5 turns and 4 turns of DNA double helix, respectively). These experiments revealed that potential artifacts arising from open DNA ends and full DNA relaxation in the linear arrays do not significantly affect overall chromatin compaction and self-association. We observed that the 0.5 DNA helical turn difference between the two DNA linker lengths significantly affects DNA topology and nucleosome interactions. In particular, the 41-bp linkers promoted interactions between any two nucleosome beads separated by one bead as expected for a zigzag fiber, whereas the 36-bp linkers promoted interactions between two nucleosome beads separated by two other beads and also reduced negative superhelicity. Monte Carlo simulations accurately reproduce periodic modulations of chromatin compaction, DNA topology, and internucleosomal interactions with a 10-bp periodicity. We propose that the nucleosome spacing and associated chromatin structure modulations may play an important role in formation of different chromatin epigenetic states, thus suggesting implications for how chromatin accessibility to DNA-binding factors and the RNA transcription machinery is regulated.

Investigating the Role of DNA Polymerase IV in the Resolution of R‐loops

As the replication fork progresses through the DNA template, it may encounter a number of obstacles, one of which is the RNA transcription machinery. This encounter occurs because both processes utilize the same template and because the rate of replication (~1000 nt/s) is much faster than the rate of transcription (40–80 nt/s). The encounter of the two machineries, in either a co‐directional or a head‐on orientation, results in replication‐transcription collisions (RTCs). Failure to resolve these collisions leads to replication fork stalling and eventually cell death. One of the events required to resolve RTCs is the removal of RNA: DNA hybrids, known as R‐loops. In E. coli, RNase H1, coded by rnhA, is a member of a family of highly conserved endonucleases mediating resolution of R‐loops by cleaving the RNA strand on RNA: DNA hybrids. If not resolved, R‐loops cause genomic instability in the form of double‐stranded breaks through the collapse of the replication fork and accumulation of DNA lesions.In E. coli, both replication stalling and accumulation of DNA lesions leads to induction of the SOS response. Among the first genes to be upregulated is dinB, encoding DNA polymerase IV (DinB), becoming the most abundant DNA polymerase in the cell (~2500 nM). DinB main role is to synthesize DNA over template lesions. There is evidence to support the notion that DinB is involved in recombination, a mechanism that is used to repair double‐stranded breaks. In stressful conditions, DinB, due to its abundance, outcompetes other polymerases for the recombination intermediate and synthesizes DNA from it. Because of its abundance during SOS induction and its role in recombination, DinB may also be involved in the resolution of R‐loops. Here, we investigate the possible role of DinB in R‐loop resolution in a rnhA‐deletion strain. Loss of rnhA results in slow growth that is rescued by expression of DinB from a low copy plasmid. This data suggest DinB is multicopy suppressor of the rnhA deletion and that DinB plays a role in the resolution of R‐loops.Support or Funding InformationNational Institute of General Medical Sciences (RO1GM088230 to VG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The distinctive cellular responses to DNA strand breaks caused by a DNA topoisomerase I poison in conjunction with DNA replication and RNA transcription.

Camptothecin (CPT) inhibits DNA topoisomerase I (Top1) through a non-catalytic mechanism that stabilizes the Top1-DNA cleavage complex (Top1cc) and blocks the DNA re-ligation step, resulting in the accumulation in the genome of DNA single-strand breaks (SSBs), which are converted to secondary strand breaks when they collide with the DNA replication and RNA transcription machinery. DNA strand breaks mediated by replication, which have one DNA end, are distinct in repair from the DNA double-strand breaks (DSBs) that have two ends and are caused by ionizing radiation and other agents. In contrast to two-ended DSBs, such one-ended DSBs are preferentially repaired through the homologous recombination pathway. Conversely, the repair of one-ended DSBs by the non-homologous end-joining pathway is harmful for cells and leads to cell death. The choice of repair pathway has a crucial impact on cell fate and influences the efficacy of anticancer drugs such as CPT derivatives. In addition to replication-mediated one-ended DSBs, transcription also generates DNA strand breaks upon collision with the Top1cc. Some reports suggest that transcription-mediated DNA strand breaks correlate with neurodegenerative diseases. However, the details of the repair mechanisms of, and cellular responses to, transcription-mediated DNA strand breaks still remain unclear. In this review, combining our recent results and those of previous reports, we introduce and discuss the responses to CPT-induced DNA damage mediated by DNA replication and RNA transcription.

Chemogene treatment consisting of recombinant adenoviral transfection of p16cDNA (SVN-22/3), and docetaxel eradicates chemoresistant aneuploid pancreatic adenosquamous ca characterised by overexpression of k-ras and hypermethylation of CPG islands of P16

IntroductionAdenosquamous Ca is an aggressive and highly metastatic variant of adenocarcinoma with both glandular and squamous differentiation. Usually, it occurs in chemoradiated patients.MethodsTumour cells were obtained from a resected pancreatic...

Naturally occurring antisense: Transcriptional leakage or real overlap?

Naturally occurring antisense transcription is associated with the regulation of gene expression through a variety of biological mechanisms. Several recent genome-wide studies reported the identification of potential antisense transcripts for thousands of mammalian genes, many of them resulting from alternatively polyadenylated transcripts or heterogeneous transcription start sites. However, it is not clear whether this transcriptional plasticity is intentional, leading to regulated overlap between the transcripts, or, alternatively, represents a "leakage" of the RNA transcription machinery. To address this question through an evolutionary approach, we compared the genomic organization of genes, with or without antisense, between human, mouse, and the pufferfish Fugu rubripes. Our hypothesis was that if two neighboring genes overlap and have a sense-antisense relationship, we would expect negative selection acting on the evolutionary separation between them. We found that antisense gene pairs are twice as likely to preserve their genomic organization throughout vertebrates' evolution compared to nonantisense pairs, implying an overlap existence in the ancestral genome. In addition, we show that increasing the genomic distance between pairs of genes having a sense-antisense relationship is selected against. These findings indicate that, at least in part, the abundance of antisense transcripts observed in expressed data represents real overlap rather than transcriptional leakage. Moreover, our results imply that natural antisense transcription has considerably affected vertebrate genome evolution.

An RNA cap (nucleoside-2'-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization.

Viruses represent an attractive system with which to study the molecular basis of mRNA capping and its relation to the RNA transcription machinery. The RNA-dependent RNA polymerase NS5 of flaviviruses presents a characteristic motif of S-adenosyl-L-methionine-dependent methyltransferases at its N-terminus, and polymerase motifs at its C-terminus. The crystal structure of an N-terminal fragment of Dengue virus type 2 NS5 is reported at 2.4 A resolution. We show that this NS5 domain includes a typical methyltransferase core and exhibits a (nucleoside-2'-O-)-methyltransferase activity on capped RNA. The structure of a ternary complex comprising S-adenosyl-L-homocysteine and a guanosine triphosphate (GTP) analogue shows that 54 amino acids N-terminal to the core provide a novel GTP-binding site that selects guanine using a previously unreported mechanism. Binding studies using GTP- and RNA cap-analogues, as well as the spatial arrangement of the methyltransferase active site relative to the GTP-binding site, suggest that the latter is a specific cap-binding site. As RNA capping is an essential viral function, these results provide a structural basis for the rational design of drugs against the emerging flaviviruses.

Decoupling of DNA excision repair and RNA transcription in translocation breaksite regions of plasmacytoma-susceptible BALB/cAnPt mice.

Preferential repair of pyrimidine dimers in rodent cells is thought to be directly coupled to the RNA transcription machinery. The most compelling evidence for this notion is the finding that excision repair occurs more rapidly in the template strand of DNA of transcribed genes than in the non-template strand. A thorough test of this coupling concept by careful comparison of the rate of repair to the rate of transcription of a gene and its regulatory region has not been reported. In the present study, we used nuclear run-on as a measure of transcription in the c-myc and Pvt1 genes in normal B-lymphoblasts from plasmacytoma-susceptible (BALB/cAnPt) and plasmacytoma-resistant (DBA/2N) strains of mice. Previous studies have shown that these loci, but not c-abl or Dhfr are repaired differently in mouse strains: poorly in BALB/cAnPt but efficiently in DBA/2N. The results presented here indicate that in DBA/2N cells, run-on transcription from both DNA strands can be readily detected in the regions of c-myc and Pvt1 that were efficiently repaired. Unexpectedly, however, in BALB/cAnPt lymphoblasts, transcription was equivalent to that of DBA/2N, despite a dramatic reduction in efficiency of excision repair. This finding indicates that, in BALB/cAnPt lymphoblasts, DNA repair 5' to c-myc and in Pvt1 is decoupled from the RNA transcription machinery. We postulate that this dissociation of repair and transcription represents a BALB/cAnPt-specific defect in a component of the transcription/repair complex that specifically compromises repair activity but not transcription. This defect may be responsible for the inability of normal BALB/cAnPt lymphoblasts to repair DNA sequences in the c-myc 5' flank and the Pvt1 gene, inducing gene-specific instability that predisposes these loci to genetic accidents, including chromosomal translocation, retroviral integration and other mutations.

The effects of glucocorticoids on human eosinophils

It is readily apparent that eosinophils are important targets of steroid effects. Disruption of the cytokine network by glucocorticoids can indirectly diminish eosinophil generation, survival, and function. In addition, glucocorticoids appear to have direct effects on the survival, and perhaps on the function, of these cells. Rapid advances in our knowledge of the biochemistry of signal transduction, as well as the interaction between cell surface receptors and RNA transcription machinery, will allow detailed analysis of the mechanisms by which the function and life cycle of eosinophils are influenced by glucocorticoids.

Metabolic processing of cyclobutyl pyrimidine dimers and (6-4) photoproducts in UV-treated human cells. Evidence for distinct excision-repair pathways.

A new nuclease digestion assay was developed to elucidate the human excision-repair system operating on cyclobutyl pyrimidine dimers and (6-4) photoproducts. We analyzed lesions that accumulated in excised oligonucleotide fragments during incubation of UV-treated cultured fibroblasts. (6-4) photoproducts were removed intact, whereas excised cyclobutyl dimers often contained ruptured interpyrimidine phosphodiester bonds, raising the possibility that the intradimer backbone-cleavage reaction may help promote the bypass of unexcised dimers by the DNA replication or RNA transcription machinery. Cell strains representing eight different inherited forms of the cancer-prone skin disease xeroderma pigmentosum (XP) were generally found to exhibit characteristic abilities to excise the two classes of photolesions, ranging from total deficiency in groups A and G to normal proficiency in the variant. The capacity of any given XP group to act on one class of photoproducts in no way predicted its ability to act on the other. Finally, in those XP strains displaying significant levels of dimer removal, the ratio of intact-versus-modified dimers was normal, implying that rupture of the intradimer backbone linkage occurs independently of subsequent excision-repair reactions. Our data indicate that cyclobutyl dimers and (6-4) photoproducts are processed by distinct nucleotide-excision-repair pathways in human cells.