RNA circuits and RNA-binding proteins in T cells

Abstract

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.

Keywords: RNA-binding protein (RBP); T cell; immunity; long noncoding RNA (lncRNA); lymphocyte.

Figure 1.

Ribonucleoprotein capture methods performed in T cells (and other cell types). (A) RNA immunoprecipitation (RNA IP) captures RBP-RNA interactions using antibody-coated beads to pull down specific RBPs and their bound transcripts in the cell under native conditions. The bound transcript is processed for quantitative PCR to determine target transcripts by the specific RBP. (B) Crosslinking immunoprecipitation with high throughput sequencing (HITS-CLIP) methods use UV radiation to covalently bind RBPs to their transcripts. The ribonucleoprotein complexes (RNPs) are captured using antibody coated beads and undergo RNase digestion to generate small RNAs containing the bound region. These fragments are then sequenced to determine the transcriptomic binding profile. (C) RNA aptamers contain small, structured motifs that recognize small molecules and can be used to pull down and identify RBPs that bind to a sequence of interest. The illustration depicts an aptamer with modified streptavidin binding structures (S1m) and the sequence of interest. Streptavidin matrix is used to pull down the protein-bound aptamer and the proteins are processed for mass spectrometry. (D) RNA interactome capture methods (RNA IC) biotinylate the proteins and use streptavidin beads to extract RBP-bound RNA. Captured RPBs are identified using mass spectrometry and RNA undergoes library preparation and sequencing to determine RBP binding sites. (E) Organic phase separation can be used to systematically identify RBPs and RBP binding profiles of a cell. These methods use phenol phase separation which partitions proteins and RNA into the organic and aqueous phase respectively. RNPs that separate into the interphase are captured and processed for mass spectrometry and/or sequencing. Figure was created using Biorender.com.

Figure 2.

lncRNA Functions Identified in T cells (A) Transcription of lncRNAs has significant impacts on the expression of other genes in the same locus. In T cells the mechanisms of these cis-regulatory effects are still not well defined. (B) lncRNAs often regulate a host of other protein-coding genes via trans-regulatory mechanisms. This is often done via the scaffolding of various transcription or epigenetic factors and facilitating their binding to chromatin. This can influence the deposition of histone regulatory modifications such as H3K27me3. (C) lncRNAs can regulate transcription factors in a post-translational fashion by influencing the addition of ubiquitin or phosphoryl groups resulting in degradation, inhibition, or activation of the transcription factor. (D) lncRNAs can influence protein activity of the golgi vesicle trafficking network. In particular VPs13d activity is enhanced by the presence of a lncRNA and this is essential for the cell surface expression of important cytokine receptors such as CD127/IL7R. (E) lncRNAs act as ceRNAs for miRNAs, which reduces the amount of miRNA induced inhibition of protein coding targets. lncRNA acting as a ceRNA often leads to the degradation of the miRNA but in some cases may inhibit the miRNA solely via stoichiometric competition. Figure was created using Biorender.com.