An R-loop is a DNA:RNA hybrid formed during transcription when a DNA duplex is invaded by a nascent RNA transcript. R-loops accumulate in nucleoli during RNA polymerase I (RNAP I) transcription. Here, we report that mammalian RNase H1 enriches in nucleoli and co-localizes with R-loops in cultured human cells. Co-migration of RNase H1 and R-loops from nucleoli to perinucleolar ring structures was observed upon inhibition of RNAP I transcription. Treatment with camptothecin which transiently stabilized nucleolar R-loops recruited RNase H1 to the nucleoli. It has been reported that the absence of Topoisomerase and RNase H activity in Escherichia coli or Saccharomyces cerevisiae caused R-loop accumulation along rDNA. We found that the distribution of RNase H1 and Top1 along rDNA coincided at sites where R-loops accumulated in mammalian cells. Loss of either RNase H1 or Top1 caused R-loop accumulation, and the accumulation of R-loops was exacerbated when both proteins were depleted. Importantly, we observed that protein levels of Top1 were negatively correlated with the abundance of RNase H1. We conclude that Top1 and RNase H1 are partially functionally redundant in mammalian cells to suppress RNAP I transcription-associate R-loops.

Purpose To report 1-year follow-up of a phase 1/2a clinical trial testing a composite subretinal implant having polarized human embryonic stem cell (hESC)-derived retinal pigment epithelium (RPE) cells on an ultrathin parylene substrate in subjects with advanced non-neovascular age-related macular degeneration (NNAMD) Methods The phase 1/2a clinical trial included 16 subjects in two cohorts. The main endpoint was safety assessed at 365 days using ophthalmic and systemic exams. Pseudophakic subjects with geographic atrophy (GA) and severe vision loss were eligible. Low-dose tacrolimus immunosuppression was utilized for 68 days in the peri-implantation period. The implant was delivered to the worst seeing eye with a custom subretinal insertion device in an outpatient setting. A data safety monitoring committee reviewed all results. Results The treated eyes of all subjects were legally blind with a baseline best-corrected visual acuity (BCVA) of ≤ 20/200. There were no unexpected serious adverse events. Four subjects in cohort 1 had serious ocular adverse events, including retinal hemorrhage, edema, focal retinal detachment, or RPE detachment, which was mitigated in cohort 2 using improved hemostasis during surgery. Although this study was not powered to assess efficacy, treated eyes from four subjects showed an increased BCVA of >5 letters (6–13 letters). A larger proportion of treated eyes experienced a >5-letter gain when compared with the untreated eye (27% vs. 7%; P = not significant) and a larger proportion of nonimplanted eyes demonstrated a >5-letter loss (47% vs. 33%; P = not significant). Conclusions Outpatient delivery of the implant can be performed routinely. At 1 year, the implant is safe and well tolerated in subjects with advanced dry AMD. Translational Relevance This work describes the first clinical trial, to our knowledge, of a novel implant for advanced dry AMD.

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that controls global protein synthesis, in part, by modulating translation initiation, a rate-limiting step for many mRNAs. Previous studies implicate mTOR in regulating stimulant-induced sensitization and antidepressive-like behavior in rodents, as well as drug craving in abstinent heroin addicts. To determine if signaling downstream of mTOR is affected by repeated cocaine administration in reward-associated brain regions, and if inhibition of mTOR alters cocaine-induced behavioral plasticity, C57BL/6J mice received 4 intraperitoneal (IP) injections of 15 mg/kg cocaine and levels of phosphorylated P70S6 kinase and ribosomal S6 protein - two translational regulators directly downstream of mTOR - were analyzed by immunoblotting across several brain regions. Cocaine place-preference and locomotor sensitization were elicited by 4 pairings of cocaine with a distinct environment and the effects of mTOR inhibition were assessed by pretreating the mice with 10 mg/kg rapamycin, 1 hr prior to (a) each saline/cocaine conditioning session, (b) a post-conditioning test or (c) a test for locomotor sensitization conducted at 3 weeks withdrawal. While systemic pretreatment with 10 mg/kg rapamycin during conditioning failed to alter the development of a cocaine place-preference or locomotor sensitization, pretreatment prior to the post-conditioning test attenuated the expression of the place-preference. Additionally, rapamycin pretreatment prior to a cocaine challenge 3 weeks post-conditioning blocked the expression of the sensitized locomotor response. These ndings suggest a role for mTOR activity, and perhaps translational control, in the expression of cocaine-induced place preference and locomotor sensitization.

Antisense oligonucleotides (ASOs) are versatile tools that can regulate multiple steps of RNA biogenesis in cells and living organisms. Significant improvements in delivery, potency, and stability have been achieved through modifications within the oligonucleotide backbone, sugar and heterocycles. However, these modifications can profoundly affect interactions between ASOs and intracellular proteins in ways that are only beginning to be understood. Here, we report that ASOs with specific backbone and sugar modifications can become localized to cytoplasmic ribonucleoprotein granules such as stress granules and those seeded by the aggregation of specific ASO-binding proteins such as FUS/TLS (FUS) and PSF/SFPQ (PSF). Further investigation into the basis for ASO-FUS binding illustrated the importance of ASO backbone and hydrophobic 2′ sugar modifications and revealed that the C-terminal region of FUS is sufficient to retain ASOs in cellular foci. Taken together, the results of this study demonstrate that affinities of various nucleic acid binding domains for ASO depend on chemical modifications and further demonstrate how ASO–protein interactions influence the localization of ASOs.

Macroautophagy is a cellular process whereby the cell sequesters and recycles cytosolic constituents in a lysosome-dependent manner. It has also been implicated in a number of disorders, including cancer and neurodegeneration. Although a previous report that AGS3 over-expression promotes macroautophagy suggests a stimulatory role of AGS3 in this process, we have found that knock-down of AGS3, unexpectedly, also induces macroautophagy, indicating an inhibitory function of endogenous AGS3 in macroautophagy. Interestingly, AGS3 phosphorylation is decreased upon induction of mammalian target of rapamycin (mTOR)-dependent macroautophagy. Moreover, unlike wild-type AGS3, over-expression of an AGS3 mutant lacking this modification fails to enhance macroautophagic activity. These observations imply that AGS3 phosphorylation may participate in the modulation of macroautophagy.

Background: WDR5 is a protein known for its role in the epigenetic control of gene expression. Results: During cytokinesis, WDR5 resides at the midbody in the absence of chromatin and promotes normal abscission. Conclusion: WDR5 is a midbody component and regulates the final steps of cytokinesis. Significance: WDR5 may have additional functions and binding partners outside of the cell nucleus.

Activator of G protein Signaling 3 (AGS3) is a receptor-independent G protein activator that has been implicated in multiple biological events such as brain development, neuroplasticity and addiction, cardiac function, Golgi structure/function, macroautophagy and metabolism. However, how AGS3 is regulated is little known. We demonstrate here that AGS3 interacts with a ubiquitin specific protease USP9x, and this interaction is at least partially mediated through the C-terminal G protein regulatory domain of AGS3. Knockdown of USP9x causes a moderate reduction in the level of AGS3. In contrast, overexpression of either USP9x or its deubiquitinating domain UCH increases the amount of AGS3, whereas expression of the mutant UCH domain that lacks deubiquitinating activity does not have the same effect. As previously observed in AGS3 knockdown cells, the localization of several marker proteins of the late Golgi compartments is disturbed in cells depleted of USP9x. Taken together, our study suggests that USP9x can modulate the level of a subpopulation of AGS3, and this modulation plays a role in regulating the structure of the late Golgi compartments. Finally, we have found that levels of AGS3 and USP9x are co-regulated in the prefrontal cortex of rats withdrawn from repeated cocaine treatment. In conjunction with the above data, this observation indicates a potential role of USP9X in the regulation of the AGS3 level during cocaine-induced neuroplasticity.

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