SPEN is required for Xist upregulation during initiation of X chromosome inactivation

At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen−/− ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in initiation of the XCI process.

Antagonistic cotranscriptional regulation through ARGONAUTE1 and the THO/TREX complex orchestrates FLC transcriptional output

Quantitative transcriptional control is essential for physiological and developmental processes in many organisms. Transcriptional output is influenced by cotranscriptional processes interconnected to chromatin regulation, but how the functions of different cotranscriptional regulators are integrated is poorly understood. The Arabidopsis floral repressor locus FLOWERING LOCUS C (FLC) is cotranscriptionally repressed by alternative processing of the antisense transcript COOLAIR. Proximal 3′-end processing of COOLAIR resolves a cotranscriptionally formed R-loop, and this process physically links to a histone-modifying complex FLD/SDG26/LD. This induces a chromatin environment locally that determines low transcription initiation and a slow elongation rate to both sense and antisense strands. Here, we show that ARGONAUTE1 (AGO1) genetically functions in this cotranscriptional repression mechanism. AGO1 associates with COOLAIR and influences COOLAIR splicing dynamics to promote proximal COOLAIR, R-loop resolution, and chromatin silencing. Proteomic analyses revealed physical associations between AGO1, subunits of RNA Polymerase II (Pol II), the splicing-related proteins—the spliceosome NineTeen Complex (NTC) and related proteins (NTR)—and the THO/TREX complex. We connect these activities by demonstrating that the THO/TREX complex activates FLC expression acting antagonistically to AGO1 in COOLAIR processing. Together these data reveal that antagonistic cotranscriptional regulation through AGO1 or THO/TREX influences COOLAIR processing to deliver a local chromatin environment that determines FLC transcriptional output. The involvement of these conserved cotranscriptional regulators suggests similar mechanisms may underpin quantitative transcriptional regulation generally.

Regulation of GSTu1-mediated insecticide resistance in Plutella xylostella by miRNA and lncRNA

The evolution of resistance to insecticides is well known to be closely associated with the overexpression of detoxifying enzymes. Although the role of glutathione S-transferase (GST) genes in insecticide resistance has been widely reported, the underlying regulatory mechanisms are poorly understood. Here, one GST gene (GSTu1) and its antisense transcript (lnc-GSTu1-AS) were identified and cloned, and both of them were upregulated in several chlorantraniliprole-resistant Plutella xylostella populations. GSTu1 was confirmed to be involved in chlorantraniliprole resistance by direct degradation of this insecticide. Furthermore, we demonstrated that lnc-GSTu1-AS interacted with GSTu1 by forming an RNA duplex, which masked the binding site of miR-8525-5p at the GSTu1-3′UTR. In summary, we revealed that lnc-GSTu1-AS maintained the mRNA stability of GSTu1 by preventing its degradation that could have been induced by miR-8525-5p and thus increased the resistance of P. xylostella to chlorantraniliprole. Our findings reveal a new noncoding RNA-mediated pathway that regulates the expression of detoxifying enzymes in insecticide-resistant insects and offer opportunities for the further understanding of the mechanisms of insecticide and drug resistance.

Vernalization-triggered expression of the antisense transcript COOLAIR is mediated by CBF genes

Many plants undergo vernalization, a long-term winter-triggered acceleration of flowering, to align their flowering time with spring. In Arabidopsis thaliana, this is achieved by silencing a floral repressor, FLOWERING LOCUS C (FLC). COOLAIR, an antisense noncoding RNA expressed at the FLC locus, is induced during the early phase of vernalization, preceding FLC suppression. However, the mechanism by which long-term cold induces COOLAIR is not well understood. Here, we showed that C-repeat (CRT)/dehydration-responsive elements (DREs) at the 3′-end of FLC and CRT/DRE-binding factors (CBFs) are required for vernalization-induced COOLAIR activation. The CBFs bind to CRT/DREs at the 3′-end of FLC, both in vitro and in vivo, and the CBFs levels increased gradually during vernalization. Additionally, vernalization-induced COOLAIR expression was highly suppressed in the cbfs mutant, in which all CBFs were knocked-out. Contrastingly, CBF-overexpressing plants showed COOLAIR upregulation, even at warm temperatures. We propose that COOLAIR is induced by CBFs in the early phase of vernalization but is downregulated as CBFs are evicted from closed FLC chromatin during the late vernalization phase. We also demonstrated that cbfs and COOLAIR mutants have a normal vernalization response, although they show defects in vernalization-induced COOLAIR activation, indicating that COOLAIR is not necessary for this process.

RNAa-Mediated Epigenetic Attenuation of The Cell Senescence via Locus Specific Induction of Endogenous SIRT1

SIRT1, a known regulator of cellular senescence, is a therapeutic target for age related disorders and its upregulation is a strategy to improve the cell therapeutic potentials of human mesenchymal stem cell (MSCs). Knockdown of natural antisense transcripts via small activating RNAs (RNAa) is an emerging approach for safe and locus specific gene regulation. We have recently identified a natural antisense transcript at human SIRT1 locus (SIRT1-NAT), the expression of which shows a negative correlation with that of SIRT1. To test the hypothetic upregulation of SIRT1 via knockdown of SIRT1-NAT, in this study we designed a single stranded oligonucleotide (SIRT1-antagoNAT) against the antisense transcript, transfection of which efficiently knocked down the SIRT1-NAT and induced SIRT1 transcription in human MSCs. In addition, activation of SIRT1 transfection via knockdown of SIRT1-NAT in human MSCs enhanced their proliferation and differentiation potentials, reduced senescence associated β-galactosidase activity and reversed the senescence associated molecular alterations. Our findings introduce an RNAa mediated approach for epigenetic induction of endogenous SIRT1 and the consequent attenuation of senescence. Further studies should evaluate the therapeutic potentials of this approach against various age related disorders.

The fate of notch-1 transcript is linked to cell cycle dynamics by activity of a natural antisense transcript

A core imprint of metazoan life is that perturbations of cell cycle are offset by compensatory changes in successive cellular generations. This trait enhances robustness of multicellular growth and requires transmission of signaling cues within a cell lineage. Notably, the identity and mode of activity of transgenerational signals remain largely unknown. Here we report the discovery of a natural antisense transcript encoded in exon 25 of notch-1 locus (nAS25) by which mother cells control the fate of notch-1 transcript in daughter cells to buffer against perturbations of cell cycle. The antisense transcript is transcribed at G1 phase of cell cycle from a bi-directional E2F1-dependent promoter in the mother cell where the titer of nAS25 is calibrated to the length of G1. Transmission of the antisense transcript from mother to daughter cells stabilizes notch-1 sense transcript in G0 phase of daughter cells by masking it from RNA editing and resultant nonsense-mediated degradation. In consequence, nAS25-mediated amplification of notch-1 signaling reprograms G1 phase in daughter cells to compensate for the altered dynamics of the mother cell. The function of nAS25/notch-1 in integrating G1 phase history of the mother cell into that of daughter cells is compatible with the predicted activity of a molecular oscillator, slower than cyclins, that coordinates cell cycle within cell lineage.

LncRNA Nqo1-AS1 Attenuates Cigarette Smoke-Induced Oxidative Stress by Upregulating its Natural Antisense Transcript Nqo1

Evidence of the involvement of long noncoding RNAs (lncRNAs) in the pathogenesis of chronic obstructive pulmonary disease (COPD) is growing but still largely unknown. This study aims to explore the expression, functions and molecular mechanisms of Fantom3_F830212L20, a lncRNA that transcribes in an antisense orientation to Nqo1.We name this lncRNA as Nqo1 antisense transcript 1 (Nqo1-AS1). The distribution, expression level and protein coding potential of Nqo1-AS1 were determined. The effects of Nqo1-AS1 on cigarette smoke (CS)-induced oxidative stress were also evaluated. The results showed that Nqo1-AS1 were mainly located in the cytoplasm of mouse alveolar epithelium and had a very low protein coding potential. Nqo1-AS1 (or its human homologue) was increased with the increase of CS exposure. Nqo1-AS1 overexpression enhanced the mRNA and protein levels of Nqo1 and Serpina1 mRNA expression, and attenuated CS-induced oxidative stress, whereas knockdown of Nqo1-AS1 significantly decreased Nqo1 and Serpina1 mRNA expressions, and aggravated CS-induced oxidative stress. Nqo1-AS1 increased Nqo1 mRNA stability and upregulated Nqo1 expression through antisense pairing with Nqo1 3′UTR. In conclusion, these results suggest that Nqo1-AS1 attenuates CS-induced oxidative stress by increasing Nqo1 mRNA stability and upregulating Nqo1 expression, which might serve as a novel approach for the treatment of COPD.