Josephine Galipon

A-to-I editing in the miRNA seed region regulates target mRNA selection and silencing efficiency

Hydrolytic deamination of adenosine to inosine (A-to-I) by adenosine deaminases acting on RNA (ADARs) is a post-transcriptional modification which results in a discrepancy between genomic DNA and the transcribed RNA sequence, thus contributing to the diversity of the transcriptome. Inosine preferentially base pairs with cytidine, meaning that A-to-I modifications in the mRNA sequences may be observed as A-to-G substitutions by the protein-coding machinery. Genome-wide studies have revealed that the majority of editing events occur in non-coding RNA sequences, but little is known about their functional meaning. MiRNAs are small non-coding RNAs that regulate the expression of target mRNAs with complementarities to their seed region. Here, we confirm that A-to-I editing in the miRNA seed duplex globally reassigns their target mRNAs in vivo, and reveal that miRNA containing inosine in the seed region exhibits a different degree of silencing efficiency compared to the corresponding miRNA with guanosine at the same position. The difference in base-pairing stability, deduced by melting temperature measurements, between seed-target duplexes containing either C:G or I:C pairs may account for the observed silencing efficiency. These findings unequivocally show that C:G and I:C pairs are biologically different in terms of gene expression regulation by miRNAs.

Stress‐induced lncRNAs evade nuclear degradation and enter the translational machinery

Long noncoding RNAs (lncRNAs) play important roles in the regulation of gene expression. In fission yeast, glucose starvation triggers a transcriptional cascade of polyadenylated lncRNAs in the upstream region of the fructose‐1,6‐bisphosphatase gene (fbp1+), which is correlated with stepwise chromatin remodeling and necessary for the massive induction of fbp1+ mRNA. Here, we show that these novel metabolic stress‐induced lncRNAs (mlonRNAs) are 5′‐capped, less stable than fbp1+ mRNA and sensitive to a certain extent to the nuclear exosome cofactor Rrp6. However, most mlonRNAs seem to escape nuclear degradation and are exported to the cytoplasm, where they localize to polysomes precisely during glucose starvation‐induced global translation inhibition. It is likely that ribosomes tend to accumulate in the upstream region of mlonRNAs. Although mlonRNAs contain an unusual amount of upstream AUGs (uAUGs) and small open reading frames (uORFs), they escape Upf1‐mediated targeting to the non‐sense‐mediated decay (NMD) pathway. The deletion of Upf1 had no effect on mlonRNA stability, but considerably destabilized fbp1+ mRNA, hinting toward a possible novel role of Upf1. Our findings suggest that the stability of mlonRNAs is distinctly regulated from mRNA and previously described noncoding transcripts.

Malignant pleural mesothelioma cells resist anoikis as quiescent pluricellular aggregates.

Pleural fluid accumulation is a frequent clinical observation in diffuse malignant pleural mesothelioma (MPM). The cytological analysis of pleural fluid often reveals the presence of free spheroid aggregates of malignant cells, giving rise to the question of the ability of non-adherent tumor cells to resist the loss of anchorage-induced apoptosis (termed as anoikis), and to develop new tumor foci in the pleural cavity. Here, we show that MPM cells cultured under non-adherent conditions form well-organized aggregates composed of viable cells, which progressively enter in G0. Although the PI3K/Akt, ERK and SAPK/JNK signaling pathways are activated in adherent MPM cells, loss of anchorage results in the inactivation of these pathways. By comparison, we show that the non-tumoral mesothelial cells MeT-5A enter anoikis in an SAPK/JNK-, Bim- and caspase-9-dependent pathway. The survival of MPM cells can be reversed by activating SAPK/JNK with anisomycin, according to a Bim-dependent mitochondrial pathway. Finally, our findings show that impairment of cell aggregation activates SAPK/JNK and Bim and induces anoikis. Our results underline the importance of intercellular contacts in the anoikis resistance of MPM cells.

Differential Binding of Three Major Human ADAR Isoforms to Coding and Long Non-Coding Transcripts

RNA editing by deamination of adenosine to inosine is an evolutionarily conserved process involved in many cellular pathways, from alternative splicing to miRNA targeting. In humans, it is carried out by no less than three major adenosine deaminases acting on RNA (ADARs): ADAR1-p150, ADAR1-p110, and ADAR2. However, the first two derive from alternative splicing, so that it is currently impossible to delete ADAR1-p110 without also knocking out ADAR1-p150 expression. Furthermore, the expression levels of ADARs varies wildly among cell types, and no study has systematically explored the effect of each of these isoforms on the cell transcriptome. In this study, RNA immunoprecipitation (RIP)-sequencing on overexpressed ADAR isoforms tagged with green fluorescent protein (GFP) shows that each ADAR is associated with a specific set of differentially expressed genes, and that they each bind to distinct set of RNA targets. Our results show a good overlap with known edited transcripts, establishing RIP-seq as a valid method for the investigation of RNA editing biology.

RNA decay systems enhance reciprocal switching of sense and antisense transcripts in response to glucose starvation.

Antisense RNA has emerged as a crucial regulator of opposite‐strand protein‐coding genes in the long noncoding RNA (lncRNA) category, but little is known about their dynamics and decay process in the context of a stress response. Antisense transcripts from the fission yeast fbp1 locus (fbp1‐as) are expressed in glucose‐rich conditions and anticorrelated with transcription of metabolic stress‐induced lncRNA (mlonRNA) and mRNA on the sense strand during glucose starvation. Here, we investigate the localization and decay of antisense RNAs at fbp1 and other loci, and propose a model to explain the rapid switch between antisense and sense mlonRNA/mRNA transcription triggered by glucose starvation. We show that fbp1‐as shares many features with mRNAs, such as a 5′‐cap and poly(A)‐tail, and that its decay partially depends upon Rrp6, a cofactor of the nuclear exosome complex involved in 3′–5′ degradation of RNA. Fluorescence in situ hybridization and polysome fractionation show that the majority of remaining fbp1‐as localizes to the cytoplasm and binds to polyribosomes in glucose‐rich conditions. Furthermore, fbp1‐as and antisense RNA at other stress‐responsive loci are promptly degraded via the cotranslational nonsense‐mediated decay (NMD) pathway. These results suggest NMD may potentiate the swift disappearance of antisense RNAs in response to cellular stress.

Base-pairing probability in the microRNA stem region affects the binding and editing specificity of human A-to-I editing enzymes ADAR1-p110 and ADAR2

ABSTRACT Adenosine deaminases acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I). A-to-I RNA editing targets double-stranded RNA (dsRNA), and increases the complexity of gene regulation by modulating base pairing-dependent processes such as splicing, translation, and microRNA (miRNA)-mediated gene silencing. This study investigates the genome-wide binding preferences of the nuclear constitutive isoforms ADAR1-p110 and ADAR2 on human miRNA species by RNA immunoprecipitation of ADAR-bound small RNAs (RIP-seq). Our results suggest that secondary structure predicted by base-pairing probability in the mainly double-stranded region of a pre-miRNA or mature miRNA duplex may determine ADAR isoform preference for binding distinct subpopulations of miRNAs. Furthermore, we identify 31 unique editing sites with statistical significance, 19 sites of which are novel editing sites. Editing sites are enriched in the seed region responsible for target recognition by miRNAs, and isoform-specific nucleotide motifs in the immediate vicinity and opposite of editing sites are consistent with previous studies, and further reveal that ADAR2 may edit A/C bulges more frequently than ADAR1-p110 in the context of miRNA.