Effective T cell responses against infections and tumors require a swift and ample production of cytokines, chemokines, and cytotoxic molecules. The production of these effector molecules relies on rapid changes of gene expression, determined by cell-intrinsic signals and environmental cues. Here, we review our current understanding of gene-specific regulatory networks that define the magnitude and timing of cytokine production in CD8 + T cells. We discuss the dynamic features of post-transcriptional control during CD8 + T cell homeostasis and activation, and focus on the crosstalk between cell signaling and RNA-binding proteins. Elucidating gene-specific regulatory circuits may help in the future to rectify dysfunctional T cell responses. Regulatory Mechanisms Driving Effective CD8 + T Cell ResponsesCytotoxic CD8 + T cells have a key role in fighting pathogenic insults and in immunosurveillance. This includes clearance of primary infections and killing of malignant cells, as well as long-term protection by memory T cells against secondary infections [1,2]. The effectiveness of CD8 + T cells to clear target cells is defined by their capacity to produce effector molecules, such as cytokines, chemokines, and cytotoxic granule contents. Whereas these effector molecules are essential for killing infected cells and for preventing pathogenic spread, they are also highly toxic. In fact, aberrant cytokine production strongly correlates with the development of autoimmune diseases and inflammatory pathologies, such as rheumatoid arthritis, multiple sclerosis, and various intestinal and skin disorders [3][4][5]. Stringent regulation of inflammatory gene expression is thus key for protective, yet balanced immune responses.Several regulatory nodes define the extent of cytokine production (i.e., protein production) upon T cell activation. Protein production generally initiates with the transcription of DNA into mRNA, a process that depends on the accessibility of genes to, and the availability of, transcription factors. The transcriptional regulatory networks that control mammalian T cell effector functions are well studied and described elsewhere [6,7]. However, the amount of newly transcribed mRNA is not solely defined by transcription rates [8]. Genome-wide studies in bacteria and mammalian cells demonstrated that mRNA and protein abundance do not follow a linear correlation [9][10][11]. For instance, in in vitro activated murine CD4 + T cells, the correlation coefficient is 0.49 [12]. This discordance between mRNA and protein expression has been attributed to several mechanisms of post-transcriptional regulation, including mRNA stability, translation efficiency, and protein degradation.Transcripts encoding effector molecules and regulatory proteins are generally unstable but become stabilized upon T cell activation [13,14]. This increased mRNA stability is required to augment the numbers of transcripts available for protein production and to prolong the immune HighlightsThe rapid remodeling of the T cell proteome upon acti...

Highlights d Loss-of-function mutations of DDX3X are frequent in MYCdriven B cell lymphomas d DDX3X promotes translation of mRNAs encoding the core protein synthesis machinery d Loss of DDX3X buffers MYC-driven global protein synthesis and proteotoxic stress d DDX3X loss is later rescued by ectopic expression of Ychromosome-encoded DDX3Y

Rrp6 is a conserved catalytic subunit of the eukaryotic nuclear exosome ribonuclease complex that functions in the productive 3’ end maturation of stable RNAs, the degradation of transiently expressed noncoding transcripts and in discard pathways that eradicate the cell of incorrectly processed or assembled RNAs. The function of Rrp6 in these pathways is at least partially dependent upon its interaction with a small nuclear protein called Rrp47/Lrp1, but the underlying mechanism(s) by which Rrp47 functions in concert with Rrp6 are not established. Previous work on yeast grown in rich medium has suggested that Rrp6 expression is not markedly reduced in strains lacking Rrp47. Here we show that Rrp6 expression in rrp47∆ mutants is substantially reduced during growth in minimal medium through effects on both transcript levels and protein stability. Exogenous expression of Rrp6 enables normal levels to be attained in rrp47∆ mutants. Strikingly, exogenous expression of Rrp6 suppresses many, but not all, of the RNA processing and maturation defects observed in an rrp47∆ mutant and complements the synthetic lethality of rrp47∆ mpp6∆ and rrp47∆ rex1∆ double mutants. Increased Rrp6 expression in the resultant rrp47∆ rex1∆ double mutant suppresses the defect in the 3’ maturation of box C/D snoRNAs. In contrast, increased Rrp6 expression in the rrp47∆ mpp6∆ double mutant diminishes the block in the turnover of CUTs and in the degradation of the substrates of RNA discard pathways. These results demonstrate that a principal function of Rrp47 is to facilitate appropriate expression levels of Rrp6 and support the conclusion that the Rrp6/Rrp47 complex and Rex1 provide redundant exonuclease activities for the 3’ end maturation of box C/D snoRNAs.

RhoG is a Rho family small GTPase implicated in cytoskeletal regulation, acting either upstream of or in parallel to Rac1. The precise function(s) of RhoG in vivo has not yet been defined. We have identified a novel role for RhoG in signaling the neutrophil respiratory burst stimulated by G protein-coupled receptor agonists. Bone marrow-derived neutrophils from RhoG knockout (RhoG−/−) mice exhibited a marked impairment of oxidant generation in response to C5a or fMLP, but normal responses to PMA or opsonized zymosan and normal bacterial killing. Activation of Rac1 and Rac2 by fMLP was diminished in RhoG−/− neutrophils only at very early (5 s) time points (by 25 and 32%, respectively), whereas chemotaxis in response to soluble agonists was unaffected by lack of RhoG. Additionally, fMLP-stimulated phosphorylation of protein kinase B and p38MAPK, activation of phospholipase D, and calcium fluxes were equivalent in wild-type and RhoG−/− neutrophils. Our results define RhoG as a critical component of G protein-coupled receptor-stimulated signaling cascades in murine neutrophils, acting either via a subset of total cellular Rac relevant to oxidase activation and/or by a novel and as yet undefined interaction with the neutrophil NADPH oxidase.

Crosslinking and Immunoprecipitation (CLIP) is a powerful technique to obtain transcriptome-wide maps of in vivo protein-RNA interactions, which are important to understand the post-transcriptional mechanisms mediated by RNA binding proteins (RBPs). Many variant CLIP protocols have been developed to improve the efficiency and convenience of cDNA library preparation. Here we describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions, and with public eCLIP and iCLIP PTBP1 data. We visualised enriched motifs surrounding the identified crosslink positions and RNA maps of these crosslinks around the alternative exons regulated by PTBP1. Notably, motif enrichment was higher in iiCLIP and iCLIP2 in comparison to public eCLIP and iCLIP, and we show how this impacts the specificity of RNA maps. In conclusion, iiCLIP is technically convenient and efficient, and enables production of highly specific datasets for identifying RBP binding sites.