RNA-binding proteins control gene expression and cell fate in the immune system.

RNA-binding proteins (RBPs) are essential for the development and function of the immune system. They interact dynamically with RNA to control its biogenesis and turnover by transcription-dependent and transcription-independent mechanisms. In this Review, we discuss the molecular mechanisms by which RBPs allow gene expression changes to occur at different speeds and to varying degrees, and which RBPs regulate the diversity of the transcriptome and proteome. These proteins are nodes for integration of transcriptional and signaling networks and are intimately linked to intermediary metabolism. They are essential components of regulatory feedback mechanisms that maintain immune tolerance and limit inflammation. The role of RBPs in malignancy and autoimmunity has led to their emergence as targets for the development of new therapeutic modalities.

The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers.

Antibody affinity maturation occurs in germinal centers (GCs), where B cells cycle between the light zone (LZ) and the dark zone. In the LZ, GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from helper T cells, which promotes their rapid proliferation. Here we found that the RNA-binding protein PTBP1 was needed for the progression of GC B cells through late S phase of the cell cycle and for affinity maturation. PTBP1 was required for proper expression of the c-MYC-dependent gene program induced in GC B cells receiving T cell help and directly regulated the alternative splicing and abundance of transcripts that are increased during positive selection to promote proliferation.

Translational repression of pre-formed cytokine-encoding mRNA prevents chronic activation of memory T cells.

Memory T cells are critical for the immune response to recurring infections. Their instantaneous reactivity to pathogens is empowered by the persistent expression of cytokine-encoding mRNAs. How the translation of proteins from pre-formed cytokine-encoding mRNAs is prevented in the absence of infection has remained unclear. Here we found that protein production in memory T cells was blocked via a 3' untranslated region (3' UTR)-mediated process. Germline deletion of AU-rich elements (AREs) in the Ifng-3' UTR led to chronic cytokine production in memory T cells. This aberrant protein production did not result from increased expression and/or half-life of the mRNA. Instead, AREs blocked the recruitment of cytokine-encoding mRNA to ribosomes; this block depended on the ARE-binding protein ZFP36L2. Thus, AREs mediate repression of translation in mouse and human memory T cells by preventing undesirable protein production from pre-formed cytokine-encoding mRNAs in the absence of infection.

Maintenance of the marginal-zone B cell compartment specifically requires the RNA-binding protein ZFP36L1.

RNA-binding proteins of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU-rich elements in the 3' untranslated region and promoting mRNA decay. Here we identified an indispensable role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal-zone B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene-expression program related to signaling, cell adhesion and locomotion; it achieved this in part by limiting expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RNA-binding proteins for maintaining cellular identity among closely related cell types.

Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven lymphomagenesis.

DDX3X is a ubiquitously expressed RNA helicase involved in multiple stages of RNA biogenesis. DDX3X is frequently mutated in Burkitt lymphoma, but the functional basis for this is unknown. Here, we show that loss-of-function DDX3X mutations are also enriched in MYC-translocated diffuse large B cell lymphoma and reveal functional cooperation between mutant DDX3X and MYC. DDX3X promotes the translation of mRNA encoding components of the core translational machinery, thereby driving global protein synthesis. Loss-of-function DDX3X mutations moderate MYC-driven global protein synthesis, thereby buffering MYC-induced proteotoxic stress during early lymphomagenesis. Established lymphoma cells restore full protein synthetic capacity by aberrant expression of DDX3Y, a Y chromosome homolog, the expression of which is normally restricted to the testis. These findings show that DDX3X loss of function can buffer MYC-driven proteotoxic stress and highlight the capacity of male B cell lymphomas to then compensate for this loss by ectopic DDX3Y expression.

The RNA-binding protein HuR is essential for the B cell antibody response.

Post-transcriptional regulation of mRNA by the RNA-binding protein HuR (encoded by Elavl1) is required in B cells for the germinal center reaction and for the production of class-switched antibodies in response to thymus-independent antigens. Transcriptome-wide examination of RNA isoforms and their abundance and translation in HuR-deficient B cells, together with direct measurements of HuR-RNA interactions, revealed that HuR-dependent splicing of mRNA affected hundreds of transcripts, including that encoding dihydrolipoamide S-succinyltransferase (Dlst), a subunit of the 2-oxoglutarate dehydrogenase (α-KGDH) complex. In the absence of HuR, defective mitochondrial metabolism resulted in large amounts of reactive oxygen species and B cell death. Our study shows how post-transcriptional processes control the balance of energy metabolism required for the proliferation and differentiation of B cells.

Noncoding RNA and its associated proteins as regulatory elements of the immune system.

The rapid changes in gene expression that accompany developmental transitions, stress responses and proliferation are controlled by signal-mediated coordination of transcriptional and post-transcriptional mechanisms. In recent years, understanding of the mechanics of these processes and the contexts in which they are employed during hematopoiesis and immune challenge has increased. An important aspect of this progress is recognition of the importance of RNA-binding proteins and noncoding RNAs. These have roles in the development and function of the immune system and in pathogen life cycles, and they represent an important aspect of intracellular immunity.

Zfp36l1 establishes the high-affinity CD8 T-cell response by directly linking TCR affinity to cytokine sensing.

How individual T cells compete for and respond to IL-2 at the molecular level, and, as a consequence, how this shapes population dynamics and the selection of high-affinity clones is still poorly understood. Here we describe how the RNA binding protein ZFP36L1, acts as a sensor of TCR affinity to promote clonal expansion of high-affinity CD8 T cells. As part of an incoherent feed-forward loop, ZFP36L1 has a nonredundant role in suppressing multiple negative regulators of cytokine signaling and mediating a selection mechanism based on competition for IL-2. We suggest that ZFP36L1 acts as a sensor of antigen affinity and establishes the dominance of high-affinity T cells by installing a hierarchical response to IL-2.

Regulation of IFN-γ production by ZFP36L2 in T cells is time-dependent.

CD8+ T cells kill target cells by releasing cytotoxic molecules and proinflammatory cytokines, such as TNF and IFN-γ. The magnitude and duration of cytokine production are defined by posttranscriptional regulation, and critical regulator herein are RNA-binding proteins (RBPs). Although the functional importance of RBPs in regulating cytokine production is established, the kinetics and mode of action through which RBPs control cytokine production are not well understood. Previously, we showed that the RBP ZFP36L2 blocks the translation of preformed cytokine encoding mRNA in quiescent memory T cells. Here, we uncover that ZFP36L2 regulates cytokine production in a time-dependent manner. T cell-specific deletion of ZFP36L2 (CD4-cre) had no effect on T-cell development or cytokine production during early time points (2-6 h) of T-cell activation. In contrast, ZFP36L2 specifically dampened the production of IFN-γ during prolonged T-cell activation (20-48 h). ZFP36L2 deficiency also resulted in increased production of IFN-γ production in tumor-infiltrating T cells that are chronically exposed to antigens. Mechanistically, ZFP36L2 regulates IFN-γ production at late time points of activation by destabilizing Ifng mRNA in an AU-rich element-dependent manner. Together, our results reveal that ZFP36L2 employs different regulatory nodules in effector and memory T cells to regulate cytokine production.

The microRNA miR-155 controls CD8(+) T cell responses by regulating interferon signaling.

We found upregulation of expression of the microRNA miR-155 in primary effector and effector memory CD8(+) T cells, but low miR-155 expression in naive and central memory cells. Antiviral CD8(+) T cell responses and viral clearance were impaired in miR-155-deficient mice, and this defect was intrinsic to CD8(+) T cells, as miR-155-deficient CD8(+) T cells mounted greatly diminished primary and memory responses. Conversely, miR-155 overexpression augmented antiviral CD8(+) T cell responses in vivo. Gene-expression profiling showed that miR-155-deficient CD8(+) T cells had enhanced type I interferon signaling and were more susceptible to interferon's antiproliferative effect. Inhibition of the type I interferon-associated transcription factors STAT1 or IRF7 resulted in enhanced responses of miR-155-deficient CD8(+) T cells in vivo. We have thus identified a previously unknown role for miR-155 in regulating responsiveness to interferon and CD8(+) T cell responses to pathogens in vivo.

Regulation and function of poised mRNAs in lymphocytes.

Pre-existing but untranslated or 'poised' mRNA exists as a means to rapidly induce the production of specific proteins in response to stimuli and as a safeguard to limit the actions of these proteins. The translation of poised mRNA enables immune cells to express quickly genes that enhance immune responses. The molecular mechanisms that repress the translation of poised mRNA and, upon stimulation, enable translation have yet to be elucidated. They likely reflect intrinsic properties of the mRNAs and their interactions with trans-acting factors that direct poised mRNAs away from or into the ribosome. Here, I discuss mechanisms by which this might be regulated.

Data-independent acquisition proteomics of cerebrospinal fluid implicates endoplasmic reticulum and inflammatory mechanisms in amyotrophic lateral sclerosis.

While unbiased proteomics of human cerebrospinal fluid (CSF) has been used successfully to identify biomarkers of amyotrophic lateral sclerosis (ALS), high-abundance proteins mask the presence of lower abundance proteins that may have diagnostic and prognostic value. However, developments in mass spectrometry (MS) proteomic data acquisition methods offer improved protein depth. In this study, MS with library-free data-independent acquisition (DIA) was used to compare the CSF proteome of people with ALS (n = 40), healthy (n = 15) and disease (n = 8) controls. Quantified protein groups were subsequently correlated with clinical variables. Univariate analysis identified 7 proteins, all significantly upregulated in ALS versus healthy controls, and 9 with altered abundance in ALS versus disease controls (FDR < 0.1). Elevated chitotriosidase-1 (CHIT1) was common to both comparisons and was proportional to ALS disability progression rate (Pearson r = 0.41, FDR-adjusted p = 0.035) but not overall survival. Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1; upregulated in ALS versus healthy controls) was proportional to disability progression rate (Pearson r = 0.53, FDR-adjusted p = 0.003) and survival (Kaplan Meier log-rank p = 0.013) but not independently in multivariate proportional hazards models. Weighted correlation network analysis was used to identify functionally relevant modules of proteins. One module, enriched for inflammatory functions, was associated with age at symptom onset (Pearson r = 0.58, FDR-adjusted p = 0.005) and survival (Hazard Ratio = 1.78, FDR = 0.065), and a second module, enriched for endoplasmic reticulum proteins, was negatively correlated with disability progression rate (r = -0.42, FDR-adjusted p = 0.109). DIA acquisition methodology therefore strengthened the biomarker candidacy of CHIT1 and UCHL1 in ALS, while additionally highlighted inflammatory and endoplasmic reticulum proteins as novel sources of prognostic biomarkers.

Premorbid brain structure influences risk of amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a disease of the motor network associated with brain structure and functional connectivity alterations that are implicated in disease progression. Whether such changes have a causal role in ALS, fitting with a postulated influence of premorbid cerebral architecture on the phenotypes associated with neurodegenerative disorders is not known. METHODS: This study considered causal effects and shared genetic risk of 2240 structural and functional MRI brain scan imaging-derived phenotypes (IDPs) on ALS using two sample Mendelian randomisation, with putative associations further examined with extensive sensitivity analysis. Shared genetic predisposition between IDPs and ALS was explored using genetic correlation analysis. RESULTS: Increased white matter volume in the cerebral hemispheres was causally associated with ALS. Weaker causal associations were observed for brain stem grey matter volume, parieto-occipital white matter surface and volume of the left thalamic ventral anterior nucleus. Genetic correlation was observed between ALS and intracellular volume fraction and isotropic free water volume fraction within the posterior limb of the internal capsule. CONCLUSIONS: This study provides evidence that premorbid brain structure, in particular white matter volume, contributes to the risk of ALS.

Venous thromboembolism risk in amyotrophic lateral sclerosis: a hospital record-linkage study.

BACKGROUND: Venous thromboembolism (VTE) can occur in amyotrophic lateral sclerosis (ALS) and pulmonary embolism causes death in a minority of cases. The benefits of preventing VTE must be weighed against the risks. An accurate estimate of the incidence of VTE in ALS is crucial to assessing this balance. METHODS: This retrospective record-linkage cohort study derived data from the Hospital Episode Statistics database, covering admissions to England's hospitals from 1 April 2003 to 31 December 2019 and included 21 163 patients with ALS and 17 425 337 controls. Follow-up began at index admission and ended at VTE admission, death or 2 years (whichever came sooner). Adjusted HRs (aHRs) for VTE were calculated, controlling for confounders. RESULTS: The incidence of VTE in the ALS cohort was 18.8/1000 person-years. The relative risk of VTE in ALS was significantly greater than in controls (aHR 2.7, 95% CI 2.4 to 3.0). The relative risk of VTE in patients with ALS under 65 years was five times higher than controls (aHR 5.34, 95% CI 4.6 to 6.2), and higher than that of patients over 65 years compared with controls (aHR 1.86, 95% CI 1.62 to 2.12). CONCLUSIONS: Patients with ALS are at a higher risk of developing VTE, but this is similar in magnitude to that reported in other chronic neurological conditions associated with immobility, such as multiple sclerosis, which do not routinely receive VTE prophylaxis. Those with ALS below the median age of symptom onset have a notably higher relative risk. A reappraisal of the case for routine antithrombotic therapy in those diagnosed with ALS now requires a randomised controlled trial.

Neurofilament light chain in drug development for amyotrophic lateral sclerosis: a critical appraisal.

Interest in amyotrophic lateral sclerosis (ALS) biomarkers has grown exponentially over the course of the last 25 years, with great hope that they might serve as tools to facilitate the development of meaningful therapies for this otherwise inexorably progressive and invariably fatal disease. Effective use of biomarkers, however, requires an understanding of what it means for them to be 'fit-for-purpose' as well as an appreciation of the nuances of the clinical context(s) in which they will be applied. Neurofilament light chain (NfL) has emerged as a leading candidate with enormous potential to aid ALS therapy development; it is, however, also profoundly misunderstood. Within the conceptual framework of the BEST (Biomarkers, EndpointS, and other Tools) Resource, developed by the National Institutes of Health and the Food and Drug Administration in the USA, we consider the evidence supporting the use of NfL for a variety of purposes in different clinical contexts. We conclude that: (i) it may serve as a susceptibility/risk biomarker in populations at elevated risk for ALS; (ii) it has value as a prognostic biomarker when measured early in the course of established disease, empowering stratification or dynamic randomization to amplify the signal-to-noise ratio of promising therapeutics; and (iii) there is sufficient evidence to support the use of a reduction in NfL in response to an experimental therapeutic as a pharmacodynamic biomarker that may aid in phase 2 trial go/no-go decisions. Moreover, the basis for expecting that a reduction in NfL is a reasonably likely surrogate end-point (i.e. reasonably likely to predict clinical benefit-which may be more than simply survival) is nuanced, and depends on when in the course of disease the experimental therapeutic is administered.

Fluctuating salience in those living with genetic risk of motor neuron disease: A qualitative interview study.

BACKGROUND: Motor neuron disease (MND) (also known as amyotrophic lateral sclerosis) is a life-limiting neurodegenerative condition. In up to 20% of people with MND, a pathogenic variant associated with autosomal dominant inheritance can be identified. Children of people carrying a pathogenic variant have a 50% chance of inheriting this and a higher, although harder to predict, chance of developing the disease compared to the general adult population. This paper explores the experience of living with the genetic risk of MND. METHODS: We undertook a UK-based interview study with 35 individuals, including: 7 people living with genetically-mediated forms of MND; 24 asymptomatic relatives, the majority of whom had an increased risk of developing the disease; and 4 unrelated partners. RESULTS: We explore how individuals make sense of genetic risk, unpacking the interplay between genetic knowledge, personal perception, experiences of the disease in the family, age and life stage and the implications that living with risk has for different aspects of their lives. We balance an emphasis on the emotional and psychological impact described by participants, with a recognition that the salience of risk fluctuates over time. Furthermore, we highlight the diverse strategies and approaches people employ to live well in the face of uncertainty and the complex ways they engage with the possibility of developing symptoms in the future. Finally, we outline the need for open-ended, tailored support and information provision. CONCLUSIONS: Drawing on wider literature on genetic risk, we foreground how knowledge of MND risk can disrupt individuals' taken-for-granted assumptions on life and perceptions of the future, but also its contextuality, whereby its relevance becomes more prominent at critical junctures. This research has been used in the development of a public-facing resource on the healthtalk.org website. PATIENT OR PUBLIC CONTRIBUTION: People with experience of living with genetic risk were involved throughout the design and conduct of the study and advised on aspects including the topic guide, sampling and recruitment and the developing analysis. Two patient and public involvement contributors joined a formal advisory panel.

Experiences of predictive genetic testing in inherited motor neuron disease: Findings from a qualitative interview study.

Predictive genetic testing is increasingly available for individuals with a heightened risk of motor neuron disease (MND). However, little is known about how they decide whether or not to get tested, and how they experience this process. This paper reports findings from a constructivist grounded theory-informed interview study with 24 family members of people with identified or suspected inherited MND (iMND). Fourteen did not know their genetic status, and nine had decided to have predictive testing, of whom six tested positive for the pathogenic gene variant identified in their family and three tested negative. One additional person was identified as negative through a parent's negative result. This paper explores the diverse ways people approached testing, and the many factors and motivations involved, based on personal attitudes and goals, experiences of living with genetic risk, and wider family considerations and circumstances. Results were met with a range of emotions; whatever the outcome, the news disrupted each person's view of the future, and they adapted in their own way and time. Support after results was variable and a perceived lack of support impacted coping and the ability to move forwards. This paper situates findings against literature on other genetic conditions, highlighting experiences as grounded in the unique characteristics of iMND. Thus, it emphasizes the need for disease-specific guidelines and support structures around predictive genetic testing in this context. Understanding people's experiences and responding to these needs is particularly timely given the uptake of testing amongst this group is anticipated to rise with increasing access to genetic testing for people with MND, and gene-specific clinical trials.

Tensor image registration library: Deformable registration of stand-alone histology images to whole-brain post-mortem MRI data.

BACKGROUND: Accurate registration between microscopy and MRI data is necessary for validating imaging biomarkers against neuropathology, and to disentangle complex signal dependencies in microstructural MRI. Existing registration methods often rely on serial histological sampling or significant manual input, providing limited scope to work with a large number of stand-alone histology sections. Here we present a customisable pipeline to assist the registration of stand-alone histology sections to whole-brain MRI data. METHODS: Our pipeline registers stained histology sections to whole-brain post-mortem MRI in 4 stages, with the help of two photographic intermediaries: a block face image (to undistort histology sections) and coronal brain slab photographs (to insert them into MRI space). Each registration stage is implemented as a configurable stand-alone Python script using our novel platform, Tensor Image Registration Library (TIRL), which provides flexibility for wider adaptation. We report our experience of registering 87 PLP-stained histology sections from 14 subjects and perform various experiments to assess the accuracy and robustness of each stage of the pipeline. RESULTS: All 87 histology sections were successfully registered to MRI. Histology-to-block registration (Stage 1) achieved 0.2-0.4 mm accuracy, better than commonly used existing methods. Block-to-slice matching (Stage 2) showed great robustness in automatically identifying and inserting small tissue blocks into whole brain slices with 0.2 mm accuracy. Simulations demonstrated sub-voxel level accuracy (0.13 mm) of the slice-to-volume registration (Stage 3) algorithm, which was observed in over 200 actual brain slice registrations, compensating 3D slice deformations up to 6.5 mm. Stage 4 combined the previous stages and generated refined pixelwise aligned multi-modal histology-MRI stacks. CONCLUSIONS: Our open-source pipeline provides robust automation tools for registering stand-alone histology sections to MRI data with sub-voxel level precision, and the underlying framework makes it readily adaptable to a diverse range of microscopy-MRI studies.

The contribution of Neanderthal introgression and natural selection to neurodegenerative diseases.

Humans are thought to be more susceptible to neurodegeneration than equivalently-aged primates. It is not known whether this vulnerability is specific to anatomically-modern humans or shared with other hominids. The contribution of introgressed Neanderthal DNA to neurodegenerative disorders remains uncertain. It is also unclear how common variants associated with neurodegenerative disease risk are maintained by natural selection in the population despite their deleterious effects. In this study, we aimed to quantify the genome-wide contribution of Neanderthal introgression and positive selection to the heritability of complex neurodegenerative disorders to address these questions. We used stratified-linkage disequilibrium score regression to investigate the relationship between five SNP-based signatures of natural selection, reflecting different timepoints of evolution, and genome-wide associated variants of the three most prevalent neurodegenerative disorders: Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease. We found no evidence for enrichment of positively-selected SNPs in the heritability of Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease, suggesting that common deleterious disease variants are unlikely to be maintained by positive selection. There was no enrichment of Neanderthal introgression in the SNP-heritability of these disorders, suggesting that Neanderthal admixture is unlikely to have contributed to disease risk. These findings provide insight into the origins of neurodegenerative disorders within the evolution of Homo sapiens and addresses a long-standing debate, showing that Neanderthal admixture is unlikely to have contributed to common genetic risk of neurodegeneration in anatomically-modern humans.