MiRNA-Mediated Control of HLA-G Expression and Function

HLA-G is a non-classical HLA class-Ib molecule expressed mainly by the extravillous cytotrophoblasts (EVT) of the placenta. The expression of HLA-G on these fetal cells protects the EVT cells from immune rejection and is therefore important for a healthy pregnancy. The mechanisms controlling HLA-G expression are largely unknown. Here we demonstrate that miR-148a and miR-152 down-regulate HLA-G expression by binding its 3′UTR and that this down-regulation of HLA-G affects LILRB1 recognition and consequently, abolishes the LILRB1-mediated inhibition of NK cell killing. We further demonstrate that the C/G polymorphism at position +3142 of HLA-G 3′UTR has no effect on the miRNA targeting of HLA-G. We show that in the placenta both miR-148a and miR-152 miRNAs are expressed at relatively low levels, compared to other healthy tissues, and that the mRNA levels of HLA-G are particularly high and we therefore suggest that this might enable the tissue specific expression of HLA-G

Epigenetic loss of RNA‑methyltransferase NSUN5 in glioma targets ribosomes to drive stress adaptive translational program

Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease

Small Nucleolar (Sno)RNA: Therapy Lays in Translation

The ribosome is one of the largest complexes in the cell. Adding to its complexity are more than 200 RNA modification sites present on ribosomal RNAs (rRNAs) in a single human ribosome. These modifications occur in functionally important regions of the rRNA molecule, and they are vital for ribosome function and proper gene expression. Until recent technological advancements, the study of rRNA modifications and their profiles has been extremely laborious, leaving many questions unanswered. Small nucleolar RNAs (snoRNAs) are non-coding RNAs that facilitate and dictate the specificity of rRNA modification deposition, making them an attractive target for ribosome modulation. Here, we propose that through the mapping of rRNA modification profiles, we can identify cell-specific modifications with high therapeutic potential. We also describe the challenges of achieving the targeting specificity needed to implement snoRNAs as therapeutic targets in cancers

Expression of miR-148a, miR-152 and HLA-G mRNA in various human tissues.

<p>(A–C) Quantitative real-time PCR analysis of miR-148a (A), miR-152 (B) and the mRNA of HLA-G (C) in different healthy tissues, presented relative to miR-16 (A and B) or hUBC (C). (D–E) Ratio of HLA-G mRNA levels divided by the miR-148a levels (D) or by miR-152 levels (E). One out of three representative experiments is shown.</p

MiR-148a and miR-152 down-regulate HLA-G expression and reduce LILRB1 binding.

<p>721.221/HLA-G (G variant, A, or C variant, B) were transduced with the control miRNA, miR-148a or miR-152. The various cells were then stained with anti HLA-G mAb and analyzed by FACS. One out of three representative experiments is shown. (C) FACS histograms re HLA-G staining of 721.221/HLA-G cells not expressing the 3′UTR of HLA-G. One out of three representative experiments is shown. (D–E) LILRB1-Ig staining of 721.221/HLA-G (G variant, D, or C variant, E) and of 21.221/HLA-G cells not expressing the 3′UTR of HLA-G (F) transduced with control miRNA, with miR-148a or with miR-152. Black histogram: cells transduced with a control miRNA. Dark grey histogram: cells transduced with miR-148a. Light grey histogram: cells transduced with miR-152. One out of three representative experiments is shown. (G) Quantitative real-time PCR analysis of HLA-G mRNA levels in 721.221/HLA-G cells (G variant), presented relative to hUBC. One out of three representative experiments is shown.</p

Expression of miR-148a, miR-152 and HLA-G mRNA in choriocarcinoma cell lines.

<p>(A) Quantitative real-time PCR analysis for miR-148a (black columns) and miR-152 (white columns) in BEWO, JAR and JEG-3 cell lines, presented relative to U6. (B) Quantitative real-time PCR analysis of HLA-G mRNA in BEWO, JAR and JEG-3 cell lines, presented relative to hUBC. One out of three representative experiments is shown.</p

MiR-148a and miR-152 specifically target the 3′UTR of HLA-G.

<p>(A) Luciferase activity in RKO cells transduced with control miRNA (black columns), miR-148a (white columns) or miR-152 (grey columns). Results are presented relative to control reporter activity. G/C variant, reporter containing the WT sequence of the 3′UTR of HLA-G, which contains guanine or cytosine at position +3142, as indicated in the figure. Mut, reporter mutated at the seed sequence of HLA-G 3′UTR. Values are mean ± s.d. of triplicate samples. * <i>P</i><0.05 (two-tailed Student's <i>t</i>-test). One out of three representative experiments is shown. (B) FACS histogram showing GFP levels (indicative for miRNA expression) of the miRNA-infected 721.221 cells. (C–D) Quantitative real-time PCR analysis of miR-148a (C) or miR-152 (D) in 721.221/HLA-G cells expressing the G variant transduced with the relevant miRNA or a control miRNA, as indicated in the figure. Results presented relative to U6. One out of three representative experiments is shown.</p

The miRNA-mediate reduction of HLA-G expression enhances NK cell cytotoxicity.

<p>(A–B) 721.221 and 721.221/HLA-G cells, expressing either the G variant (A) or the C variant (B), transduced with control miRNA, miR-148a or miR-152, were used as target cells in killing assays against NK clones expressing LILRB1. The effector: target (E∶T) ratio was 1∶1. Values are mean ± s.d. for triplicate samples. * <i>P</i><0.02 (two-tailed Student's <i>t</i>-test). One out of three representative experiments is shown.</p

MiR-148a and miR-152 potentially target the 3′UTR of HLA-G.

<p>(A) Immunohistochemisty performed on first trimester placental tissue sections. Right panel: negative control. Left panel: HLA-G expression is detected in the trophoblast cell columns and extravillous trophoblasts. (B) Alignment of the 3′UTR of HLA-G (G variant) with the predicted miRNAs: miR-152 left panel, miR-148a right panel. (C) Alignment of the 3′UTR of HLA-G (C variant) with the predicted miRNAs: miR-152 left panel, miR-148a right panel.</p