Absence of Non-Canonical, Inhibitory MYD88 Splice Variants in B Cell Lymphomas Correlates With Sustained NF-κB Signaling

Gain-of-function mutations of the TLR adaptor and oncoprotein MyD88 drive B cell lymphomagenesis via sustained NF-κB activation. In myeloid cells, both short and sustained TLR activation and NF-κB activation lead to the induction of inhibitory MYD88 splice variants that restrain prolonged NF-κB activation. We therefore sought to investigate whether such a negative feedback loop exists in B cells. Analyzing MYD88 splice variants in normal B cells and different primary B cell malignancies, we observed that MYD88 splice variants in transformed B cells are dominated by the canonical, strongly NF-κB-activating isoform of MYD88 and contain at least three novel, so far uncharacterized signaling-competent splice isoforms. Sustained TLR stimulation in B cells unexpectedly reinforces splicing of NF-κB-promoting, canonical isoforms rather than the ‘MyD88s’, a negative regulatory isoform reported to be typically induced by TLRs in myeloid cells. This suggests that an essential negative feedback loop restricting TLR signaling in myeloid cells at the level of alternative splicing, is missing in B cells when they undergo proliferation, rendering B cells vulnerable to sustained NF-κB activation and eventual lymphomagenesis. Our results uncover MYD88 alternative splicing as an unappreciated promoter of B cell lymphomagenesis and provide a rationale why oncogenic MYD88 mutations are exclusively found in B cells.

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The failure of B cells to induce non-canonical MYD88 splice variants correlates with lymphomagenesis via sustained NF-κB signaling

10.1101/2020.06.18.154393 ◽

2020 ◽

Author(s):

Yamel Cardona Gloria ◽

Stephan H. Bernhart ◽

Sven Fillinger ◽

Olaf-Oliver Wolz ◽

Sabine Dickhöfer ◽

...

Keyword(s):

Alternative Splicing ◽

B Cells ◽

B Cell ◽

Negative Feedback ◽

Feedback Loop ◽

Splice Variants ◽

Myeloid Cells ◽

Negative Feedback Loop ◽

Tlr Signaling ◽

Splice Isoforms

AbstractGain-of-function mutations of the TLR adaptor and oncoprotein MyD88 drive B cell lymphomagenesis via sustained NF-κB activation. In myeloid cells, sustained TLR activation and NF-κB activation lead to the induction of inhibitory MYD88 splice variants that restrain prolonged NF-κB activation. We therefore sought to investigate whether such a negative feedback loop exists in B cells. Analyzing MYD88 splice variants in normal B cells and different primary B cell malignancies, we observed that MYD88 splice variants in transformed B cells are dominated by the canonical, strongly NF-κB-activating isoform of MYD88 and contain at least three novel, so far uncharacterized signaling-competent splice isoforms. TLR stimulation in B cells unexpectedly reinforces splicing of NF-κB-promoting, canonical isoforms rather than the ‘MyD88s’, a negative regulatory isoform that is typically induced by TLRs in myeloid cells. This suggests that an essential negative feedback loop restricting TLR signaling in myeloid cells at the level of alternative splicing, is missing in B cells, rendering B cells vulnerable to sustained NF-κB activation and eventual lymphomagenesis. Our results uncover MYD88 alternative splicing as an unappreciated promoter of B cell lymphomagenesis and provide a rationale why oncogenic MYD88 mutations are exclusively found in B cells.

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“A negative feedback loop mediated by the NR4A family of nuclear hormone receptors restrains expansion of B cells that receive signal one in the absence of signal two”

10.1101/2020.03.31.017434 ◽

2020 ◽

Author(s):

Corey Tan ◽

Ryosuke Hiwa ◽

James L. Mueller ◽

Vivasvan Vykunta ◽

Kenta Hibiya ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

Negative Feedback ◽

Feedback Loop ◽

Hormone Receptors ◽

Receive Signal ◽

Negative Feedback Loop ◽

Orphan Nuclear Receptors ◽

T Cell Help

ABSTRACTAg stimulation (signal 1) triggers B cell activation and proliferation, and primes B cells to recruit, engage, and respond to T cell help (signal 2). However, failure to receive signal 2 within a defined window of time results in an abortive round of proliferation, followed by anergy or apoptosis. Although the molecular basis of T cell help has been extensively dissected, the mechanisms that restrain Ag-stimulated B cells, and enforce dependence upon co-stimulation, are incompletely understood. Nr4a1-3 encode a small family of orphan nuclear receptors that are rapidly induced by B cell receptor (BCR) stimulation, yet little is known about their function in humoral immune responses. Here we use germline and conditional loss-of-function mouse models to show that Nr4a1 and Nr4a3 play partially redundant roles to restrain both the survival and proliferation of B cells that receive signal 1 in the absence of co-stimulatory signals, and do so in part by repressing expression of BATF and consequently c-MYC. Correspondingly, Ab responses to TI-2 immunogens are enhanced in the absence of Nr4a1, but are unaltered in response to immunogens that incorporate co-stimulatory signals. Unexpectedly, we also identify a role for the NR4A family in restraining B cell access to T cell help by repressing expression of the T cell chemokines CCL3/4, as well as CD86 and ICAM1, and show that this is relevant under conditions of competition for limiting T cell help. Our studies collectively reveal a novel negative feedback loop mediated by the NR4A family that increases B cell dependence upon T cell help and restrains strongly Ag-activated B cell clones from monopolizing limiting amounts of T cell help. We speculate that this imposes B cell tolerance and dampens immunodominance to facilitate preservation of clonal diversity during an immune response.

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Novel autoregulatory cases of alternative splicing coupled with nonsense-mediated mRNA decay

10.1101/464404 ◽

2018 ◽

Author(s):

Dmitri Pervouchine ◽

Yaroslav Popov ◽

Andy Berry ◽

Beatrice Borsari ◽

Adam Frankish ◽

...

Keyword(s):

Alternative Splicing ◽

Negative Feedback ◽

Feedback Loop ◽

Transcriptional Control ◽

Rna Binding ◽

Rna Binding Proteins ◽

Negative Feedback Loop ◽

Control Of Gene Expression ◽

Nonsense Mediated Decay ◽

Premature Termination Codons

AbstractNonsense-mediated decay (NMD) is a eukaryotic mRNA surveillance system that selectively degrades transcripts with premature termination codons (PTC). Many RNA-binding proteins (RBP) regulate their expression levels by a negative feedback loop, in which RBP binds its own pre-mRNA and causes alternative splicing to introduce a PTC. We present a bioinformatic framework to identify novel such autoregulatory feedback loops by combining eCLIP assays for a large panel of RBPs with the data on shRNA inactivation of NMD pathway, and shRNA-depletion of RBPs followed by RNA-seq. We show that RBPs frequently bind their own pre-mRNAs and respond prominently to NMD pathway disruption. Poison and essential exons, i.e., exons that trigger NMD when included in the mRNA or skipped, respectively, respond oppositely to the inactivation of NMD pathway and to the depletion of their host genes, which allows identification of novel autoregulatory mechanisms for a number of human RBPs. For example, SRSF7 binds its own pre-mRNA and facilitates the inclusion of two poison exons; SFPQ binding promotes switching to an alternative distal 3’-UTR that is targeted by NMD; RPS3 activates a poison 5’-splice site in its pre-mRNA that leads to a frame shift; U2AF1 binding activates one of its two mutually exclusive exons, leading to NMD; TBRG4 is regulated by cluster splicing of its two essential exons. Our results indicate that autoregulatory negative feedback loop of alternative splicing and NMD is a generic form of post-transcriptional control of gene expression.

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SKI Controls MDS-Associated Chronic TGFb Signaling, Aberrant Splicing, and Stem Cell Fitness

Blood ◽

10.1182/blood-2018-99-119441 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4350-4350

Author(s):

David Muench ◽

Kyle Ferchen ◽

Chinavenmeni S. Velu ◽

Kith Pradhan ◽

Kashish Chetal ◽

...

Keyword(s):

Stem Cell ◽

Alternative Splicing ◽

Negative Feedback ◽

Feedback Loop ◽

Early Stage ◽

Negative Feedback Loop ◽

Rna Seq ◽

Wild Type ◽

Tgfβ Signaling ◽

Tgfb Signaling

Abstract The transforming growth factor-beta (TGFβ) signaling pathway controls hematopoietic stem cell (HSC) behavior in the marrow niche; however, TGFb signaling becomes chronic in early-stage myelodysplasia (MDS) where it may select MDS-genic HSC clones. In MDS, the level of TGFb signaling may have prognostic value while TGFβ-receptor inhibitors improve hematopoiesis in MDS samples. Thus, to better understand MDS pathobiology, it is vital to understand the mechanisms underlying chronic TGFb signaling. Normally, TGFβ signaling is tightly controlled by antagonists (e.g. SMAD7, SKI) which block promiscuous activity. Upon ligand-receptor engagement, these antagonists are transiently eliminated to amplify the signal; however, they are re-induced by TGFβ signaling and subsequently terminate the signal (a negative feedback loop). Early-stage MDS marrow cells have significantly diminished expression of SMAD7 and elevated levels of microRNA-21 (miR-21), which targets SMAD7. Thus, miR-21 is one factor that interferes with the TGFβ negative feedback loop to generate a chronic TGFβ signal in MDS. We bioinformatically reanalyzed a recently published RNA-Seq dataset of MDS patient samples and find that a TGFb-signal-correlated gene signature is sufficient to identify a population of MDS patients with abnormal RNA splicing (e.g. CSF3R) independent of splicing factor mutations, and coincident with low HNRNPK activity. Elevated levels of SKI mRNA, encoding a transcriptional corepressor and TGFb-antagonist, are sufficient to identify these patients. We questioned why elevated SKI mRNA (encoding a TGFβ-antagonist) would be associated with chronic activation of TGFβ. We show that miR-21 targets SKI to block translation, and that event is associated with slight elevation in SKI mRNA abundance. Moreover, SKI protein is reduced in primary bone marrow samples from early-stage MDS patients with elevated miR-21 and chronic TGFβ signaling. To determine the impact of SKI loss, we examined murine Ski-/- HSC. Ski-/- HSC are initially specified and rescue transplant recipients in the absence of competitors, where they participate in multilineage hematopoiesis. However, when challenged with wild-type competitors, Ski-/- HSC display a profound defect in HSC fitness that can be rescued by increasing the number of Ski-/- HSC transplanted. Ski wild type and null embryonic stem cell - blastocyst complementation assays confirm an intrinsic Ski-/- HSC defect in the absence of transplantation. Using single-cell RNA-Seq, Ski-/- HSC exhibited striking upregulation of TGFb signaling, including Tgfb1 itself. Novel bioinformatics single-cell-splicing analyses revealed splicing alterations in Ski-/- HSC concordant with low HNRNPK activity. Strikingly, a large fraction of the differentially expressed genes and alternative splicing events in Ski-/- HSC are found in SKI-high MDS patients. We conclude that miR-21-mediated loss of SKI contributes to early stage MDS pathogenesis by activating TGFβ signaling and alternative splicing while hindering HSC fitness. Disclosures No relevant conflicts of interest to declare.

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Negative feedback by NUR77/Nr4a1 restrains B cell clonal dominance during early T-dependent immune responses

10.1101/2020.12.25.424409 ◽

2020 ◽

Author(s):

Jeremy F Brooks ◽

Corey Tan ◽

James L. Mueller ◽

Kenta Hibiya ◽

Ryosuke Hiwa ◽

...

Keyword(s):

B Cells ◽

Immune Responses ◽

B Cell ◽

Negative Feedback ◽

Feedback Loop ◽

Clonal Diversity ◽

Cell Precursor ◽

Immune Repertoire ◽

Cell Clones ◽

T Cell Help

AbstractB cell clones compete for entry into and dominance within germinal centers (GC), where the highest affinity BCRs are selected. However, diverse and low affinity B cells can enter and reside in GCs for extended periods. To reconcile these observations, we hypothesized that a negative feedback loop may operate within B cells to preferentially restrain high affinity clones from monopolizing the early GC niche. Here we report a role for the nuclear receptor NUR77/Nr4a1 in this process. We previously showed that NUR77 expression scales with antigen stimulation and restrains B cell expansion when T cell help is limiting. Here we show that, although NUR77 is dispensable for regulating GC size when GC are elicited in a largely clonal manner, it serves to curb immunodominance under conditions where diverse clonal populations must compete for a constrained niche. Moreover, this is independent of B cell precursor frequency and reflects, at least in part, a B cell-intrinsic role for NUR77. We propose that this is important to preserve early B cell clonal diversity in order to limit holes in the post-immune repertoire and to optimize GC selection.

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