Florian Heyd

Context-Dependent Regulatory Mechanism of the Splicing Factor hnRNP L

Splicing regulatory proteins often have distinct activities when bound to exons versus introns. However, less clear is whether variables aside from location can influence activity. HnRNP L binds to a motif present in both CD45 variable exons 4 and 5 to affect their coordinate repression. Here, we show that, in contrast to its direct repression of exon 4, hnRNP L represses exon 5 by countering the activity of a neighboring splicing enhancer. In the absence of the enhancer, hnRNP L unexpectedly activates exon inclusion. As the splice sites flanking exon 4 and 5 are distinct, we directly examined the effect of varying splice site strength on the mechanism of hnRNP L function. Remarkably, binding of hnRNP L to an exon represses strong splice sites but enhances weak splice sites. A model in which hnRNP L stabilizes snRNP binding can explain both effects in a manner determined by the inherent snRNP-substrate affinity.

DEGRADE, MOVE, REGROUP: signaling control of splicing proteins

With recent advances in microarrays and sequencing it is now relatively straightforward to compare pre-mRNA splicing patterns in different cellular conditions on a genome-wide scale. Such studies have revealed extensive changes in cellular splicing programs in response to stimuli such as neuronal depolarization, DNA damage, immune signaling and cellular metabolic changes. However, for many years our understanding of the signaling pathways responsible for such splicing changes was greatly lacking. Excitingly, over the past few years this gap has begun to close. Recent studies now suggest notable trends in the mechanisms that link cellular stimuli to downstream alternative splicing events. These include regulated synthesis or degradation of splicing factors, differential protein-protein interactions, altered nuclear translocation and changes in transcription elongation.

Alternative splicing networks regulated by signaling in human T cells

The formation and execution of a productive immune response requires the maturation of competent T cells and a robust change in cellular activity upon antigen challenge. Such changes in cellular function depend on regulated alterations to protein expression. Previous research has focused on defining transcriptional changes that regulate protein expression during T-cell maturation and antigen stimulation. Here, we globally analyze another critical process in gene regulation during T-cell stimulation, alternative splicing. Specifically, we use RNA-seq profiling to identify 178 exons in 168 genes that exhibit robust changes in inclusion in response to stimulation of a human T-cell line. Supporting an important role for the global coordination of alternative splicing following T-cell stimulation, these signal-responsive exons are significantly enriched in genes with functional annotations specifically related to immune response. The vast majority of these genes also exhibit differential alternative splicing between naive and activated primary T cells. Comparison of the responsiveness of splicing to various stimuli in the cultured and primary T cells further reveals at least three distinct networks of signal-induced alternative splicing events. Importantly, we find that each regulatory network is specifically associated with distinct sequence features, suggesting that they are controlled by independent regulatory mechanisms. These results thus provide a basis for elucidating mechanisms of signal pathway-specific regulation of alternative splicing during T-cell stimulation.

Prox1 interacts with Atoh1 and Gfi1, and regulates cellular differentiation in the inner ear sensory epithelia

Inner ear hair cells and supporting cells arise from common precursors and, in mammals, do not show phenotypic conversion. Here, we studied the role of the homeodomain transcription factor Prox1 in the inner ear sensory epithelia. Adenoviral-mediated Prox1 transduction into hair cells in explant cultures led to strong repression of Atoh1 and Gfi1, two transcription factors critical for hair cell differentiation and survival. Luciferase assays showed that Prox1 can repress transcriptional activity of Gfi1 independently of Atoh1. Prox1 transduction into cochlear outer hair cells resulted in degeneration of these cells, consistent with the known phenotype of Gfi1-deficient mice. These results together with the widespread expression of endogenous Prox1 within the population of inner ear supporting cells point to the role for Prox1 in antagonizing the hair cell phenotype in these non-sensory cells. Further, in vivo analyses of hair cells from Gfi1-deficient mice suggest that the cyclin-dependent kinase inhibitor p57(Kip2) mediates the differentiation- and survival-promoting functions of Gfi1. These data reveal novel gene interactions and show that these interactions regulate cellular differentiation within the inner ear sensory epithelia. The data point to the tight regulation of phenotypic characteristics of hair cells and supporting cells.

Auxiliary splice factor U2AF26 and transcription factor Gfi1 cooperate directly in regulating CD45 alternative splicing.

By alternative splicing, different isoforms of the transmembrane tyrosine phosphatase CD45 are generated that either enhance or limit T cell receptor signaling. We report here that CD45 alternative splicing is regulated by cooperative action of the splice factor U2AF26 and the transcription factor Gfi1. U2AF26 promoted formation of the less-active CD45RO by facilitating exon exclusion. Gfi1 antagonized that process by directly interacting with U2AF26, identifying a previously unknown link between a transcription factor and alternative splicing. The presence of Gfi1 led to formation of the more-active CD45RB, whereas loss of Gfi1 favored CD45RO production. We propose that the relative abundance of U2AF26 and Gfi1 determines the ratio of CD45 isoforms, thereby regulating T cell activation.

Differential impact of the transcriptional repressor Gfi1 on mature CD4+ and CD8+ T lymphocyte function

The transcriptional repressor Gfi1 is a nuclear zinc‐finger protein that is expressed in T cell precursors in the thymus, but is down‐regulated in mature, resting T cells. Gfi1 expression rises transiently to levels seen in thymocytes upon antigenic activation. We show here that lack of Gfi1 causes delayed cell cycle entry and apoptosis after antigenic stimulation in both mature CD4+ and CD8+ T cells ex vivo. DNA micro‐array analysis demonstrated that this correlated with an up‐regulation of the death receptor CD95, the proapoptotic factors Bad and Apaf1 and the cell cycle inhibitor p21, and a down‐regulation of Bcl‐2 expression in Gfi1–/– T cells. Surprisingly, while Gfi1‐deficient CD4+ T cells showed the same defective behavior in vivo, Gfi1‐deficient CD8+ T cells showed no aberration in vivo and were fully able to mount an anti‐viral immune response. This indicates that Gfi1 exerts different functions in CD4+ and CD8+ T cells very likely by maintaining different genetic programs in both cell types, and appears to be essential for the CD4 helper T cell immune response but dispensable for the function of cytotoxic CD8+ T cells.

Alternative splicing of MALT1 controls signalling and activation of CD4 + T cells

MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4+ T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation.

Signal- and development-dependent alternative splicing of LEF1 in T cells is controlled by CELF2.

ABSTRACT The HMG-box transcription factor LEF1 controls many developmentally regulated genes, including genes that activate expression of the T-cell antigen receptor alpha chain (TCR-alpha) in developing thymocytes. At least two distinct isoforms of LEF1 are expressed, resulting from variable inclusion of LEF1 exon 6; however, the expression pattern of these isoforms and mechanism of splicing regulation have not been explored. Here we demonstrate that inclusion of LEF1 exon 6 is increased during thymic development and in response to signaling in a cultured T-cell line in a manner which temporally correlates with increased expression of TCR-alpha. We further find that inclusion of exon 6 is dependent on the signal-induced increase in expression and binding of the splicing factor CELF2 to two intronic sequences flanking the regulated exon. Importantly, loss of exon 6 inclusion, through knockdown of CELF2 or direct block of the exon 6 splice site, results in reduced expression of TCR-alpha mRNA. Together, these data establish the mechanistic basis of LEF1 splicing regulation and demonstrate that LEF1 alternative splicing is a contributing determinant in the optimal expression of the TCR-alpha chain.

Alternative Splicing Controlled by Heterogeneous Nuclear Ribonucleoprotein L Regulates Development, Proliferation, and Migration of Thymic Pre-T Cells

The regulation of posttranscriptional modifications of pre-mRNA by alternative splicing is important for cellular function, development, and immunity. The receptor tyrosine phosphatase CD45, which is expressed on all hematopoietic cells, is known for its role in the development and activation of T cells. CD45 is known to be alternatively spliced, a process that is partially regulated by heterogeneous nuclear ribonucleoprotein (hnRNP) L. To investigate the role of hnRNP L further, we have generated conditional hnRNP L knockout mice and found that LckCre-mediated deletion of hnRNP L results in a decreased thymic cellularity caused by a partial block at the transition stage between double-negative 4 and double-positive cells. In addition, hnRNP L−/− thymocytes express aberrant levels of the CD45RA splice isoforms and show high levels of phosphorylated Lck at the activator tyrosine Y394, but lack phosphorylation of the inhibitory tyrosine Y505. This indicated an increased basal Lck activity and correlated with higher proliferation rates of double-negative 4 cells in hnRNP L−/− mice. Deletion of hnRNP L also blocked the migration and egress of single-positive thymocytes to peripheral lymphoid organs in response to sphingosine-1-phosphate and the chemokines CCL21 and CXCL12 very likely as a result of aberrant splicing of genes encoding GTPase regulators and proteins affecting cytoskeletal organization. Our results indicate that hnRNP L regulates T cell differentiation and migration by regulating pre-TCR and chemokine receptor signaling.

The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.

The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation.