Native elongating transcript sequencing reveals global anti-correlation between sense and antisense nascent transcription in fission yeast.

Antisense transcription can regulate sense gene expression. However, previous annotations of antisense transcription units have been based on detection of mature antisense long noncoding (aslnc)RNAs by RNA-seq and/or microarrays, only giving a partial view of the antisense transcription landscape and incomplete molecular bases for antisense-mediated regulation. Here, we used native elongating transcript sequencing to map genome-wide nascent antisense transcription in fission yeast. Strikingly, antisense transcription was detected for most protein-coding genes, correlating with low sense transcription, especially when overlapping the mRNA start site. RNA profiling revealed that the resulting aslncRNAs mainly correspond to cryptic Xrn1/Exo2-sensitive transcripts (XUTs). ChIP-seq analyses showed that antisense (as)XUT's expression is associated with specific histone modification patterns. Finally, we showed that asXUTs are controlled by the histone chaperone Spt6 and respond to meiosis induction, in both cases anti-correlating with levels of the paired-sense mRNAs, supporting physiological significance to antisense-mediated gene attenuation. Our work highlights that antisense transcription is much more extended than anticipated and might constitute an additional nonpromoter determinant of gene regulation complexity.

Neuroprotective Activity of D-HES in Transient Global Cerebral Ischemia in Rats.

We studied the effect of O-((((4-hydroxy-3,5-di(1,7,7-trimethylbicyclo[2.2.1]hept-exo-2-yl) benzyl)oxy)ethyl)-O-(2-hydroxyethyl)-(1→4)-α-D-glucan (D-HES, 80 mg/kg, intravenously) and reference preparation ethylmethylhydroxypyridine succinate (EMHP-S, 50 mg/kg, intravenously) on rat survival and neurological deficit in 24 h after transient global cerebral ischemia in Wistar rats. Intravenous administration of D-HES and EMHP-S significantly increased the number of survivors by 68 and 78%, respectively, in comparison with the control group. In groups treated with D-HES and EMHP-S, the number of animals with severe neurological deficit was significantly lower and the number of animals moderate or mild neurological deficit was significantly higher than in the control group.

Negative Regulation of the Mis17-Mis6 Centromere Complex by mRNA Decay Pathway and EKC/KEOPS Complex in Schizosaccharomyces pombe.

The mitotic kinetochore forms at the centromere for proper chromosome segregation. Deposition of the centromere-specific histone H3 variant, spCENP-A/Cnp1, is vital for the formation of centromere-specific chromatin and the Mis17-Mis6 complex of the fission yeast Schizosaccharomyces pombe is required for this deposition. Here we identified extragenic suppressors for a Mis17-Mis6 complex temperature-sensitive (ts) mutant, mis17-S353P, using whole-genome sequencing. The large and small daughter nuclei phenotype observed in mis17-S353P was greatly rescued by these suppressors. Suppressor mutations in two ribonuclease genes involved in the mRNA decay pathway, exo2 and pan2, may affect Mis17 protein level, as mis17 mutant protein level was recovered in mis17-S353P exo2 double mutant cells. Suppressor mutations in EKC/KEOPS complex genes may not regulate Mis17 protein level, but restored centromeric localization of spCENP-A/Cnp1, Mis6 and Mis15 in mis17-S353P Therefore, the EKC/KEOPS complex may inhibit Mis17-Mis6 complex formation or centromeric localization. Mutational analysis in protein structure indicated that suppressor mutations in the EKC/KEOPS complex may interfere with its kinase activity or complex formation. Our results suggest that the mRNA decay pathway and the EKC/KEOPS complex negatively regulate Mis17-Mis6 complex-mediated centromere formation by distinct and unexpected mechanisms.

Combined targeting of Arf1 and Ras potentiates anticancer activity for prostate cancer therapeutics.

BACKGROUND: Although major improvements have been made in surgical management, chemotherapeutic, and radiotherapeutic of prostate cancer, many prostate cancers remain refractory to treatment with standard agents. Therefore, the identification of new molecular targets in cancer progression and development of novel therapeutic strategies to target them are very necessary for achieving better survival for patients with prostate cancer. Activation of small GTPases such as Ras and Arf1 is a critical component of the signaling pathways for most of the receptors shown to be upregulated in advanced prostate cancer. METHODS: The drug effects on cell proliferation were measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay. The drug effects on cell migration and invasion were determined by Radius™ 24-well and Matrigel-coated Boyden chambers. The drug effects on apoptosis were assessed by FITC Annexin V Apoptosis Detection Kit with 7-AAD and Western blot with antibodies against cleaved PARP and Caspase 3. A NOD/SCID mouse model generated by subcutaneous injection was used to assess the in vivo drug efficacy in tumor growth. ERK activation and tumor cell proliferation in xenografts were examined by immunohistochemistry. RESULTS: We show that Exo2, a small-molecule inhibitor that reduces Arf1 activation, effectively suppresses prostate cancer cell proliferation by blocking ERK1/2 activation. Exo2 also has other effects, inhibiting migration and invasion of PCa cells and inducing apoptosis. The Ras inhibitor salirasib augments Exo2-induced cytotoxicity in prostate cancer cells partially by enhancing the suppression of ERK1/2 phosphorylation. In a xenograft mouse model of prostate cancer, Exo2 reduces prostate tumor burden and inhibits ERK1/2 activation at a dose of 20 mg/kg. Synergistic treatment of salirasib and Exo2 exhibits a superior inhibitory effect on prostate tumor growth compared with either drug alone, which may be attributed to the more efficient inhibition of ERK1/2 phosphorylation. CONCLUSION: This study suggests that simultaneous blockade of Arf1 and Ras activation in prostate cancer cells is a potential targeted therapeutic strategy for preventing prostate cancer development.