The lncRNA Malat1 Inhibits miR-15/16 to Enhance Cytotoxic T Cell Activation and Memory Cell Formation
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eLife assessment
This is an important study that revealed a new noncoding RNA regulatory circuit involved in T cell function. The authors provide compelling evidence, that is more rigorous than the state-of-the-art, using genetically engineered mice and cell-based experiments. The interpretation of the results should be tempered due to the small effect size observed.
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Abstract
Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, many intracellular bacteria and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor miRNA family important for cell proliferation and survival. miR-15/16 also play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and T cell memory. Comparative Argonaute-2 high throughput sequencing of crosslinking immunoprecipitation (Ago2 HITS-CLIP, or AHC) combined with gene expression profiling in normal and miR-15/16-deficient T cells revealed a large network of several hundred direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, the long non-coding RNA Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak in T cells. This binding site was also among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16 binding site in Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of IL-2 and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of long noncoding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed.
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eLife assessment
This is an important study that revealed a new noncoding RNA regulatory circuit involved in T cell function. The authors provide compelling evidence, that is more rigorous than the state-of-the-art, using genetically engineered mice and cell-based experiments. The interpretation of the results should be tempered due to the small effect size observed.
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Reviewer #1 (Public Review):
Wheeler et al. have discovered a new RNA circuit that regulates T-cell function. They found that the long non-coding RNA Malat1 sponges miR-15/16, which controls many genes related to T cell activation, survival, and memory. This suggests that Malat1 indirectly regulates T-cell function. They used CRISPR to mutate the miR-15/16 binding site in Malat1 and observed that this disrupted the RNA circuit and impaired cytotoxic T-cell responses. While this study presents a novel molecular mechanism of T-cell regulation by Malat1-miR-15/16, the effects of Malat1 are weaker compared to miR-15/16. This could be due to several reasons, including higher levels of miR-15/16 compared to Malat1 or Malat1 expression being mostly restricted to the nucleus. Although the role of miR15/16 in T-cell activation has been …
Reviewer #1 (Public Review):
Wheeler et al. have discovered a new RNA circuit that regulates T-cell function. They found that the long non-coding RNA Malat1 sponges miR-15/16, which controls many genes related to T cell activation, survival, and memory. This suggests that Malat1 indirectly regulates T-cell function. They used CRISPR to mutate the miR-15/16 binding site in Malat1 and observed that this disrupted the RNA circuit and impaired cytotoxic T-cell responses. While this study presents a novel molecular mechanism of T-cell regulation by Malat1-miR-15/16, the effects of Malat1 are weaker compared to miR-15/16. This could be due to several reasons, including higher levels of miR-15/16 compared to Malat1 or Malat1 expression being mostly restricted to the nucleus. Although the role of miR15/16 in T-cell activation has been previously published, if the authors can demonstrate that miR15/16 and/or Malat1 affect the clearance of Listeria or LCMV, this will significantly add to the current findings and provide physiological context to the study.
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Reviewer #2 (Public Review):
This study connects prior findings on MicroRNA15/16 and Malat1 to demonstrate a functional interaction that is consequential for T cell activation and cell fate.
The study uses mice (Malat1scr/scr) with a precise genetic modification of Malat1 to specifically excise the sites of interaction with the microRNA, but sparing all other sequences, and mice with T-cell specific deletion of miR-15/16. The effects of genetic modification on in vivo T-cell responses are detected using specific mutations and shown to be T-cell intrinsic.
It is not known where in the cell the consequential interactions between MicroRNA15/16 and Malat1 take place. The authors depict in the graphical abstract Malat1 to be a nuclear lncRNA. Malat 1 is very abundant, but it is unclear if it can shuttle between the nucleus and cytoplasm. As …
Reviewer #2 (Public Review):
This study connects prior findings on MicroRNA15/16 and Malat1 to demonstrate a functional interaction that is consequential for T cell activation and cell fate.
The study uses mice (Malat1scr/scr) with a precise genetic modification of Malat1 to specifically excise the sites of interaction with the microRNA, but sparing all other sequences, and mice with T-cell specific deletion of miR-15/16. The effects of genetic modification on in vivo T-cell responses are detected using specific mutations and shown to be T-cell intrinsic.
It is not known where in the cell the consequential interactions between MicroRNA15/16 and Malat1 take place. The authors depict in the graphical abstract Malat1 to be a nuclear lncRNA. Malat 1 is very abundant, but it is unclear if it can shuttle between the nucleus and cytoplasm. As the authors discuss future work defining where in the cell the relevant interactions take place will be important.
In addition to showing physiological phenotypic effects, the mouse models prove to be very helpful when the effects measured are small and sometimes hard to quantitate in the context of considerable variation between biological replicates (for example the results in Figure 4D).
The impact of the genetic modification on the CD28-IL2- Bcl2 axis is quantitatively small at the level of expression of individual proteins and there are likely to be additional components to this circuitry.
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