RNA circuits and RNA-binding proteins in T cells.

RNA is integral to the regulatory circuits that control cell identity and behavior. Cis-regulatory elements in mRNAs interact with RNA-binding proteins (RBPs) that can alter RNA sequence, stability, and translation into protein. Similarly, long noncoding RNAs (lncRNAs) scaffold ribonucleoprotein complexes that mediate transcriptional and post-transcriptional regulation of gene expression. Indeed, cell programming is fundamental to multicellular life and, in this era of cellular therapies, it is of particular interest in T cells. Here, we review key concepts and recent advances in our understanding of the RNA circuits and RBPs that govern mammalian T cell differentiation and immune function.

The lncRNA Malat1 Inhibits miR-15/16 to Enhance Cytotoxic T Cell Activation and Memory Cell Formation.

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.

The lncRNA Malat1 inhibits miR-15/16 to enhance cytotoxic T cell activation and memory cell formation.

Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, 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 play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and memory. Comparative Argonaute-2 high-throughput sequencing of crosslinking immunoprecipitation (AHC) combined with gene expression profiling in normal and miR-15/16-deficient mouse T cells revealed a large network of hundreds of direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak. This binding site was 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 interleukin 2 (IL-2) and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence in mice following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of long non-coding 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.

A Mass Spectrometric Assay of METTL3/METTL14 Methyltransferase Activity.

A variety of covalent modifications of RNA have been identified and demonstrated to affect RNA processing, stability, and translation. Methylation of adenosine at the N6 position (m6A) in messenger RNA (mRNA) is currently the most well-studied RNA modification and is catalyzed by the RNA methyltransferase complex METTL3/METTL14. Once generated, m6A can modulate mRNA splicing, export, localization, degradation, and translation. Although potent and selective inhibitors exist for several members of the Type I S-adenosylmethionine (SAM)-dependent methyltransferase family, no inhibitors have been reported for METTL3/METTL14 to date. To facilitate drug discovery efforts, a sensitive and robust mass spectrometry-based assay for METTL3/METTL14 using self-assembled monolayer desorption/ionization (SAMDI) technology has been developed. The assay uses an 11-nucleotide single-stranded RNA compared to a previously reported 27-nucleotide substrate. IC50 values of mechanism-based inhibitors S-adenosylhomocysteine (SAH) and sinefungin (SFG) are comparable between the SAMDI and radiometric assays that use the same substrate. This work demonstrates that SAMDI technology is amenable to RNA substrates and can be used for high-throughput screening and compound characterization for RNA-modifying enzymes.