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Cell cycle dependent regulation and function of Argonaute 1 in plants [small RNA-seq]

ABSTRACT: The RNA silencing (RNAi) is a biological process, which plays a key role in a wide range of eukaryotes, such as gene expression regulation, transposable elements control and antiviral defense. In human, several studies showed that miRNAs and AGO2 are involved in the regulation of the cell cycle. Indeed, many cell cycle regulators such as cyclins, CDKs, CDK inhibitors, RBR proteins or E2F transcription factors have been identified as miRNA targets. Transcriptional data suggest that these cell cycle miRNAs are differentially accumulated during cell cycle progression and that misregulation of some could lead to proliferative diseases. However, in plants, almost nothing is known about the cell cycle regulation through miRNAs. Unpublished datas from the lab suggest that the Arabidopsis AGO1, which is the most important Argonaute for miRNA-mediated gene regulation, is essential for cell proliferation. In order to investigate the role of AGO1 during cell cycle, we set up a system based on cell cycle synchronization of tobacco BY-2 cell suspensions expressing a GFP-fused AGO1. We sequenced small RNAs from S, G2, M and G1 phases of synchronized cells from total RNA extract and GFP-AGO1 co-purified small RNAs, and investigate whether putative cell cycle specific miRNAs can be highlighted.

ORGANISM(S): Nicotiana tabacum

PROVIDER: GSE102341 | GEO | 2019/06/06

REPOSITORIES: GEO

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Project description:The p53 tumour suppressor is a transcription factor that can regulate the expression of numerous genes encoding either proteins or microRNAs (miRNAs). The predominant outcomes of a typical p53 response are the initiation of apoptotic cascades and the activation of cell cycle checkpoints. HT29-tsp53 cells express a temperature sensitive variant of p53 and in the absence of exogenous DNA damage, these cells preferentially undergo G1 phase cell cycle arrest at the permissive temperature that correlates with increased expression of the cyclin-dependent kinase inhibitor p21WAF1. Recent evidence also suggests that a variety of miRNAs can induce G1 arrest by inhibiting the expression of proteins like CDK4 and CDK6. Here we used oligonucleotide microarrays to identify p53-regulated miRNAs that are induced in these cells undergoing G1 arrest. At the permissive temperature, the expression of several miRNAs was increased through a combination of either transcriptional or post-transcriptional regulation. In particular, miR-34a-5p, miR-143-3p and miR-145-5p were strongly induced and they reached levels comparable to that of reference miRNAs (miR-191 and miR-103). Importantly, miR-34a-5p and miR-145-5p are known to silence the Cdk4 and/or Cdk6 G1 cyclin-dependent kinases (cdks). Surprisingly, there was no p53-dependent decrease in the expression of either of these G1 cdks. To search for other potential targets of p53-regulated miRNAs, p53-downregulated mRNAs were identified through parallel microarray analysis of mRNA expression. Once again, there was no clear effect of p53 on the repression of mRNAs under these conditions despite a remarkable increase in p53-induced mRNA expression. Therefore, despite a strong p53 transcriptional response, there was no clear evidence that p53-responsive miRNA contributed to gene silencing. Taken together, the changes in cell cycle distribution in this cell line at the permissive temperature is likely attributable to transcriptional upregulation of the CDKN1A mRNA and p21WAF1 protein and not to the down regulation of CDK4 or CDK6 by p53-regulated miRNAs. Two independent experiments were performed with 2 samples in each experiment (1 control and 1 treatment condition). In the control sample, RNA was isolated cells maintained at the restrictive temperature (37ËC). The treatment treated sample, was incubated for 16 hours at the permissive temperature (32ËC).

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Cell cycle dependent regulation and function of Argonaute 1 in plants [small RNA-seq]

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