Arabidopsis Plants Having Defects in Nonsense-mediated mRNA Decay Factors UPF1, UPF2, and UPF3 Show Photoperiod-dependent Phenotypes in Development and Stress Responses

Nonsense-mediated mRNA decay （NMD） is an important mRNA quality surveillance pathway in all eukaryotes that eliminates aberrant mRNAs derived from various sources. Three NMD factor proteins, UPF1, UPF2, and UPF3 are required for the NMD process and were found to be also involved in certain stress responses in mammalian and yeast cells. Using Arabidopsis thaliana mutants of UPF1 and UPF3 and UPF2-silenced lines （irUPF2）, we examined the involvement of UPF1, UPF2, and UPF3 in development and in response to stresses, wounding and infection by Pseudomonas syringae pv. tomato strain DC3000. Under the long （16 h） photoperiod condition, Arabidopsis having a defect in NMD factors exhibited altered morphologies of various organs, disturbed homeostasis of wounding-induced jasmonic acid and pathogen-elicited salicylic acid, and abnormal wounding- and methyl jasmonate-induced changes in the transcript levels of two defense-related genes, LOX2 and VSP2. Importantly, when plants were cultivated under the short （10 h） photoperiod condition, mutants of UPF1 and UPF3 and irUPF2 showed smaller differences from the wild-type plants in growth and stress-induced responses. These data suggest a complex regulatory network, likely composed of light signaling and NMD factor-mediated pathways, in influencing plant development and adaption to environmental stresses.

LncRNA LINC00525 suppresses p21 expression viamRNA decay and triplex-mediated changes in chromatin structure in lung adenocarcinoma

Background:Emerging evidence suggests that long noncoding RNAs(lncRNAs)play crucial roles in various cancers.In the present study,we aim to investigate the function and molecular mechanism of an up-regulated and survivalassociated lncRNA,LINC00525,in lung adenocarcinoma(LUAD).Methods:The expression level of LINC00525 in tissueswas determined by quantitative reverse transcription polymerase chain reaction(RT-qPCR)and in situ hybridization(ISH).The functional role of LINC00525 in LUAD was investigated using gain-and loss-of-function approaches,both in vivo and in vitro.RNA pull-down,RNA immunoprecipitation(RIP),chromatin immunoprecipitation(ChIP),triplex-capture assay,dual-luciferase assay,gene expression microarray,and bioinformatics analysis were used to investigate the potential underlying mechanisms involved.Results:LINC00525 is highly expressed in LUAD cells and tissues.Survival analysis indicated that upregulation of LINC00525 was associated with poor prognosis in patients with LUAD patients.Knockdown of LINC00525 inhibited cell proliferation and cell cycle progression in vitro.In xenograft models,LINC00525 knockdown suppressed tumor growth and tumorigenesis of tumorbearing mice.Mechanistically,LINC00525 epigenetically suppressed p21 transcription by guiding Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit(EZH2)to the p21 promoter through an formation of RNA-DNA triplex with the p21 promoter,leading to increased trimethylation of lysine 27 on histone 3(H3K27me3)of the p21 promoter.In addition,LINC00525 repressed p21 expression post-transcriptionally by enhancing p21mRNA decay.LINC00525 promoted p21mRNAdecay by competitively binding toRNABindingMotif Single Stranded Interacting Protein 2(RBMS2).Conclusion:Our findings demonstrate that LINC00525 promotes the progression of LUAD by reducing the transcription and stability of p21 mRNA in concert with EZH2 and RBMS2,thus suggesting that LINC00525 may be a potential therapeutic target for clinical intervention in LUAD.

Regulation of nonsense-mediated mRNA decay in neural development and disease

Eukaryotes have evolved a variety of mRNA surveillance mechanisms to detect and degrade aberrant mRNAs with potential deleterious outcomes.Among them,nonsense-mediated mRNA decay(NMD)functions not only as a quality control mechanism targeting aberrant mRNAs containing a premature termination codon but also as a posttranscriptional gene regulation mechanism tar-geting numerous physiological mRNAs.Despite its well-characterized molecular basis,the regulatory scope and biological functions of NMD at an organismal level are incompletely understood.In humans,mutations in genes encoding core NMD factors cause specific developmental and neurological syndromes,suggesting a critical role of NMD in the central nervous system.Here,we review the accumulating biochemical and genetic evidence on the developmental regulation and physiological functions of NMD as well as an emerging role of NMD dysregulation in neurodegenerative diseases.

Ghrelin alleviates 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells

Ghrelin is a neuropeptide that has various physiological functions and has been demonstrated to be neuroprotective in a number of neurological disease models.However,the underlying mechanisms of ghrelin in Parkinson’s disease remain largely unexplored.The current study aimed to study the effects of ghrelin in a 6-hydroxydopamine(6-OHDA)-induced Parkinson’s disease model and evaluate the potential underlying mechanisms.In the present study,we treated an SH-SY5 Y cell model with 6-OHDA,and observed that pretreatment with different concentrations of ghrelin(1,10,and 100 nM)for 30 minutes relieved the neurotoxic effects of 6-OHDA,as revealed by Cell Counting Kit-8 and Annexin V/propidium iodide(PI)apoptosis assays.Reverse transcription quantitative polymerase chain reaction and western blot assay results demonstrated that 6-OHDA treatment upregulatedα-synuclein and lincRNA-p21 and downregulated TG-interacting factor 1(TGIF1),which was predicted as a potential transcription regulator of the gene encodingα-synuclein(SNCA).Ghrelin pretreatment was able to reverse the trends caused by 6-OHDA.The Annexin V/PI apoptosis assay results revealed that inhibiting eitherα-synuclein or lincRNA-p21 expression with small interfering RNA(siRNA)relieved 6-OHDA-induced cell apoptosis.Furthermore,inhibiting lincRNA-p21 also partially upregulated TGIF1.By retrieving information from a bioinformatics database and performing both double luciferase and RNA immunoprecipitation assays,we found that lincRNA-p21 and TGIF1 were able to form a double-stranded RNA-binding protein Staufen homolog 1(STAU1)binding site and further activate the STAU1-mediated mRNA decay pathway.In addition,TGIF1 was able to transcriptionally regulateα-synuclein expression by binding to the promoter of SNCA.The Annexin V/PI apoptosis assay results showed that either knockdown of TGIF1 or overexpression of lincRNA-p21 notably abolished the neuroprotective effects of ghrelin against 6-OHDA-induced neurotoxicity.Collectively,these findings suggest that ghrelin exerts neuroprotective effects against 6-OHDA-induced neurotoxicity via the lincRNA-p21/TGIF1/α-synuclein pathway.

Length of the ORF, position of the first AUG and the Kozak motif are important factors in potential dual-coding transcripts

A single mammalian transcript normally encodes one protein, but the transcript of GNAS （G-protein u-subunit） contains two reading frames and produces two structurally unrelated proteins, XLas and ALEX. No other confirmed GNAS-Iike dual-coding transcripts have been reported to date, even though many such candidate genes have been predicted by bioinformatics analysis. In this study, we constructed a series of vectors to test how two protein products were translated from a single transcript in vitro. The length of the ORF （open reading frame）, position of the first AUG and the Kozak motif were found to be important factors. These factors, as well as 55-bp NMD （nonsense-mediated mRNA decay） rule, were used in a bioinformatics search for candidate dual-coding transcripts. A total of 1307, 750 and 474 two-ORF-containing transcripts were found in human, mouse and rat, respectively, of which 170, 89 and 70, respectively, were found to be potential dual-coding transcripts. Most transcripts showed low conservation among species. Interestingly, dual-coding transcripts were significantly enriched for transcripts from the zinc-finger protein family, which are usually DNA-binding proteins involved in regulation of the transcription process.

Environmental Stresses Modulate Abundance and Timing of Alternatively Spliced Circadian Transcripts in Arabidopsis

Environmental stresses profoundly altered accumulation of nonsense mRNAs including intron-retaining （IR） transcripts in Arabidopsis. Temporal patterns of stress-induced IR mRNAs were dissected using both oscillating and non-oscillating transcripts. Broad-range thermal cycles triggered a sharp increase in the long IR CCA1 isoforms and altered their phasing to different times of day. Both abiotic and biotic stresses such as drought or Pseudomonas syringae infection induced a similar increase. Thermal stress induced a time delay in accumulation of CCA1 14Rb transcripts, whereas functional mRNA showed steady oscillations. Our data favor a hypothesis that stress-induced instabilities of the central oscillator can be in part compensated through fluctuations in abundance and out-of-phase oscillations of CCA1 IR transcripts. Taken together, our results support a concept that mRNA abundance can be modulated through altering ratios between functional and nonsense/IR transcripts. SR45 protein specifically bound to the retained CCA1 intron in vitro, suggesting that this sp!icing factor could be involved in regulation of intron retention. Transcriptomes of nonsense-mediated mRNA decay （NMD）-impaired and heat-stressed plants shared a set of retained introns associated with stress- and defense-inducible transcripts. Constitutive activation of certain stress response networks in an NMD mutant could be linked to disequilibrium between functional and nonsense mRNAs.