Systematic Analysis of RNA Regulatory Network in Rat Brain after Ischemic Stroke.

Although extensive studies have identified large number of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in ischemic stroke, the RNA regulation network response to focal ischemia remains poorly understood. In this study, we simultaneously interrogate the expression profiles of lncRNAs, miRNAs, and mRNAs changes during focal ischemia induced by transient middle cerebral artery occlusion. A set of 1924 novel lncRNAs were identified and may involve brain injury and DNA repair as revealed by coexpression network analysis. Furthermore, many short interspersed elements (SINE) mediated lncRNA:mRNA duplexes were identified, implying that lncRNAs mediate Staufen1-mediated mRNA decay (SMD) which may play a role during focal ischemia. Moreover, based on the competitive endogenous RNA (ceRNA) hypothesis, a stroke regulatory ceRNA network which reveals functional lncRNA:miRNA:mRNA interactions was revealed in ischemic stroke. In brief, this work reports a large number of novel lncRNAs responding to focal ischemia and constructs a systematic RNA regulation network which highlighted the role of ncRNAs in ischemic stroke.

Downregulation of the Long Non-Coding RNA Meg3 Promotes Angiogenesis After Ischemic Brain Injury by Activating Notch Signaling.

Angiogenesis after ischemic brain injury contributes to the restoration of blood supply in the ischemic zone. Strategies to improve angiogenesis may facilitate the function recovery after stroke. Recent researches have demonstrated that dysfunction of long non-coding RNAs are associated with angiogenesis. We have previously reported that long non-coding RNAs (lncRNAs) are aberrantly expressed in ischemic stroke. However, little is known about long non-coding RNAs and theirs role in angiogenesis after stroke. In this study, we identified a rat lncRNAs, Meg3, and found that Meg3 was significantly decreased after ischemic stroke. Overexpression of Meg3 suppressed functional recovery and decreased capillary density after ischemic stroke. Downregulation of Meg3 ameliorated brain lesion and increased angiogenesis after ischemic stroke. Silencing of Meg3 resulted in a proangiogenic effect evidenced by increased endothelial cell migration, proliferation, sprouting, and tube formation. Mechanistically, we showed that Meg3 negatively regulated notch pathway both in vivo and in vitro. Inhibition of notch signaling in endothelial cells reversed the proangiogenic effect induced by Meg3 downregulation. This study revealed the function of Meg3 in ischemic stroke and elucidated its mechanism in angiogenesis after ischemic stroke.

Conservation and divergence of transcriptional coregulations between box C/D snoRNA and ribosomal protein genes in Ascomycota.

Coordinated assembly of the ribosome is essential for proper translational activity in eukaryotic cells. It is therefore critical to coordinate the expression of components of ribosomal programs with the cell's nutritional status. However, coordinating expression of these components is poorly understood. Here, by combining experimental and computational approaches, we systematically identified box C/D snoRNAs in four fission yeasts and found that the expression of box C/D snoRNA and ribosomal protein (RP) genes were orchestrated by a common Homol-D box, thereby ensuring a constant balance of these two genetic components. Interestingly, such transcriptional coregulations could be observed in most Ascomycota species and were mediated by different cis-regulatory elements. Via the reservation of cis elements, changes in spatial configuration, the substitution of cis elements, and gain or loss of cis elements, the regulatory networks of box C/D snoRNAs evolved to correspond with those of the RP genes, maintaining transcriptional coregulation between box C/D snoRNAs and RP genes. Our results indicate that coregulation via common cis elements is an important mechanism to coordinate expression of the RP and snoRNA genes, which ensures a constant balance of these two components.

[Involvement of intron A into eukaryotic expression vector to improve immunogenicity of mycobacterial heat-shock protein 65 DNA vaccine in mice].

OBJECTIVE: To explore the involvement of intron A into eukaryotic expression vector to improve antigen expression efficiency and enhance immunogenicity of DNA vaccine in mice. METHODS: As model antigen, the coding gene of mycobacterial Hsp65 was cloned into eukaryotic expression vector pCMV4.0 with intron A involved and pVAX1 without intron A involved, respectively. The resulted recombinant expression vectors were transfected into 293T cells and were then injected into BALB/c mice as DNA vaccines. Anti-Hsp65 specific IgG and isotype were detected by ELISA and T cell immune response was analyzed by enzyme-linked immunosorbent spot (ELISPOT) assay and intracellular cytokine staining. RESULTS: Compared with non-intron A pVAX1hsp65, the recombinant plasmid pCMV4.0hsp65 involved with intron A pVAX1hsp65 caused higher expression level of Hsp65 in 293T cells, and enhanced Th1 type immune response, which was defined as higher level of anti-Hsp65 specific total IgG level (3.76 ± 0.23 vs 3.15 ± 0.22, P < 0.01) and IgG2a/IgG1 ratio (4.08 ± 0.04 vs 2.23 ± 0.12, P < 0.01) and more IFN-γ-secreting CD4(+) ((2.0 ± 0.058)% vs (1.5 ± 0.087)%, t = 4.804, P < 0.01) and CD8(+) ((0.6 ± 0.058)% vs (1.0 ± 0.115)%, t = 3.098, P < 0.05) T lymphocytes. The difference showed statistical significance. CONCLUSION: Intron A can improve the expression efficiency of mycobacterial Hsp65 antigen and enhance immunogenicity of DNA vaccine in mice when involved into eukaryotic expression vector.

Incorporation of immunostimulatory motifs in the transcribed region of a plasmid DNA vaccine enhances Th1 immune responses and therapeutic effect against Mycobacterium tuberculosis in mice.

T-helper type 1 (Th1) immune response is involved in the development of protective immunity against Mycobacterium tuberculosis. Thus, an increase in Th1 and cellular immune responses should lead to enhanced anti-mycobacterial activity. In this study, we aimed to improve Th1 immune responses to a DNA vaccine by adding potentially immunostimulatory nucleotide sequences into the transcribed region downstream of the antigen. The Mycobacterium leprae gene for hsp65, codon-optimized for expression in mammalian cells, was inserted into pVAX1 with and without 3'-sequences containing CpG and dsRNA motifs. When the plasmid contained both motifs, transfected murine macrophage-like RAW264.7 cells showed markedly increased levels of mRNA for immune molecules of Th1 (IFN-α, IL-12) and Th17 (IL-17, IL-23 and IL-6) responses and for T cell co-stimulatory molecules (CD80 and CD86) but not for a Th2 response (IL-4 and IL-10). Immunized mice showed substantially increased serum anti-Hsp65 IgG2a antibody levels and IFN-γ production by spleen cells, confirming enhancement of the Th1 response in vivo. Furthermore, when non-vaccinated mice were infected with H37Rv by low-dose aerosol challenge, and then 4 weeks later were treated with plasmids by intramuscular injection, the mice that had been treated with plasmids containing immunostimulatory motifs showed an enhanced reduction in mycobacterial loads in lung and spleen. We conclude that DNA vaccines may be made more highly immunogenic and more effective for treatment by including transcribed stimulatory sequences.

[Mutagenesis, screening and characterization of mutants known as peroxisome biogenesis disorders in yeast].

Peroxisomes are essential for organisms' development and physiology. This fact is underscored by the lethality of a group of genetic disorders collectively known as the peroxisome biogenesis disorders (PBDs), such as Zellweger syndrome, in which peroxisomes fail to assemble properly. Defining the molecular bases of the PBDs has been the impetus behind the identification of the genes controlling peroxisome assembly, the PEX genes, from various modern organisms. Here as a original strain, Yarrowia lipolytica E122 was mutated by diethyl sulfate (DES) treatment and two mutants including temperature sensitive one were obtained. Compared to the initial strain, the two mutants showed more diffuse pattern of fluorescence characteristic of a cytosolic localization in immunofluorescence analysis, and showed no morphologically recognizable peroxisomes in electron micrographs. The two mutants arose from new gene mutation characterized by transformation and will be very useful in identification of new gene controlling peroxisome assembly from yeast.

Molecular identification of Sinopodophyllum hexandrum and Dysosma species using cpDNA sequences and PCR-RFLP markers.

Rhizomes of Sinopodophyllum hexandrum and Dysosma species, which have long been used in the traditional Chinese herbal medicine, have similar morphology and chemical composition. However, the podophyllotoxin content is higher in the rhizomes of S. hexandrum than in those of Dysosma species. The PCR-amplified fragments of trnT -trnL showed length variation between S. hexandrum and Dysosma species, and sequence comparison indicated that the length variation resulted from differential indels. There were species-specific PCR-RFLP markers of the chloroplast trnD -trnT region. Our results suggest that both chloroplast intergenic regions can be used for the identification of S. hexandrum and Dysosma Rhizoma medicines on the market.

Identification and functional analysis of 20 Box H/ACA small nucleolar RNAs (snoRNAs) from Schizosaccharomyces pombe.

Considering all small nucleolar RNAs (snoRNAs) enriched in the nucleolus, we generated a specialized cDNA library of small nuclear RNAs from Schizosaccharomyces pombe and isolated, for the first time, 20 novel box H/ACA snoRNAs. Thirteen of these were characterized as novel guides that were predicted to direct 19 pseudouridylations in 18 S and 25 S rRNAs. The remaining seven snoRNAs were considered as orphan guides that lack sequence complementarity to either rRNAs or snRNAs. We have experimentally demonstrated the function of the 10 novel snoRNAs by gene deletion in the fission yeast. The snoRNAs were shown to be dispensable for the viability of S. pombe, although an impact of snR94 depletion on yeast growth, especially at 23 degrees C, was revealed. A total of 30 pseudouridylation sites were precisely mapped in the S. pombe rRNAs, showing a distinctive pseudouridylation pattern in the budding yeast. Interestingly, the absence of pseudouridylation on U2347 in S. pombe 25 S rRNA pointed out a critical role for Psi2345 in conferring a growth advantage for yeast. In contrast to the intron-encoded box C/D sno-RNAs in yeast, all box H/ACA snoRNAs appeared to be transcribed independently from intergenic regions between two protein-coding genes, except for snR35, which was nested in an open reading frame encoding for a hypothetical protein, although expressed from the opposite strand. Remarkably, snR90 was cotranscribed with an intron-encoded box C/D snoRNA, and this is the first demonstration of a non-coding RNA gene that encodes two different types of snoRNAs by its exon and intron. A detailed comparison of the S. pombe snoRNAs, with their functional homologues in diverse organisms, suggests a mechanism by which the snoRNAs have evolved in coordination with rRNAs to preserve the post-transcriptional modification sites among distant eukaryotes.