Kristen W. Lynch

A compendium of RNA-binding motifs for decoding gene regulation

RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.

Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy

UNLABELLED The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. SIGNIFICANCE CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

Consequences of regulated pre-mRNA splicing in the immune system

Alternative splicing is widely recognized to be a ubiquitous and crucial mechanism for generating protein diversity and regulating protein expression. Numerous immunologically relevant genes have been found to undergo alternative splicing; however, there has been little effort to develop a coherent picture of how alternative splicing might be used as a general mechanism to regulate the function of the immune system. In this review, I summarize the mechanisms by which splicing is controlled in T cells, and discuss the role of alternative splicing and alternative isoform expression in the regulation of T-cell activation and function.

Common themes in the function of transcription and splicing enhancers.

Regulation of both transcription and RNA splicing requires enhancer elements, that is, cis-acting DNA or RNA sequences that promote the activities of linked promoters or splice sites, respectively. Both types of enhancer associate with regulatory proteins to form multicomponent enhancer complexes that recruit the necessary enzymatic machinery to promoter or splice site recognition sequences. This recruitment occurs as a result of direct interactions between regulatory proteins in the enhancer complexes and components of the basic enzymatic machineries. Recent advances suggest that the high degree of regulatory specificity observed for both transcription and splicing is due, in large part, to the multicomponent nature of enhancer complexes and to their cooperative assembly.

Regulation of Alternative Splicing: More than Just the ABCs

Alternative pre-mRNA splicing, the differential inclusion or exclusion of portions of a nascent transcript into the final protein-coding mRNA, is widely recognized to be a ubiquitous mechanism for controlling protein expression. Thus, understanding the molecular basis of alternative splicing is essential for deciphering post-transcriptional control of the genome. Pre-mRNA splicing in general is catalyzed by a large dynamic macromolecular machine known as the spliceosome. Notably, the recognition of the intron substrate by spliceosomal components and the assembly of these components to form a catalytic spliceosome occur through a network of highly combinatorial molecular interactions. Many, if not all, of these interactions are subject to regulation, forming the basis of alternative splicing. This minireview focuses on recent advances in our understanding of the diversity of mechanisms by which the spliceosome can be regulated so as to achieve precise control of alternative splicing under a range of cellular conditions.

HnRNP L represses exon splicing via a regulated exonic splicing silencer

Skipping of mammalian exons during pre‐mRNA splicing is commonly mediated by the activity of exonic splicing silencers (ESSs). We have recently identified a regulated ESS within variable exon 4 of the CD45 gene, named ESS1, that is necessary and sufficient for partial exon repression in resting T cells and has additional silencing activity upon T‐cell activation. In this study, we identify three heterogeneous nuclear ribonucleoproteins (hnRNPs) that bind specifically to ESS1. The binding of one of these proteins, hnRNP‐L, is significantly decreased by mutations that disrupt both the basal and induced activities of ESS1. Recombinant hnRNP‐L functions to repress exon inclusion in vitro in an ESS1‐dependent manner. Moreover, depletion of hnRNP‐L, either in vitro or in vivo, leads to increased exon inclusion. In contrast, the other ESS1‐binding proteins, PTB and hnRNP E2, do not discriminate between wild‐type and mutant ESS1 in binding studies, and do not specifically alter ESS1‐dependent splicing in vitro. Together, these studies demonstrate that hnRNP‐L is the primary protein through which CD45 exon 4 silencing is mediated by the regulatory sequence ESS1.

A CD45 Polymorphism Associated with Multiple Sclerosis Disrupts an Exonic Splicing Silencer

Previous studies have identified a single nucleotide polymorphism that significantly increases the splicing of variable exon 4 in transcripts of the human protein-tyrosine phosphatase CD45. Strikingly, the presence of this polymorphism correlates with susceptibility to the autoimmune disease multiple sclerosis. In this study we investigated the mechanism by which the polymorphism enhances splicing of CD45 exon 4. We found that at least four distinct splicing regulatory elements exist within exon 4 and that the strongest of these elements is an exonic splicing silencer (designated ESS1), which is disrupted by the polymorphism. We show that ESS1 normally functions to repress the weak 5′ splice site (ss) of CD45 exon 4. The ESS1 sequence also suppresses the splicing of a heterologous 5′ss and associates with a specific complex in nuclear extracts. We further demonstrate that ESS1 is juxtaposed to a purine-rich enhancer sequence that activates the use of the 5′ss of exon 4. Thus, proper functioning of the immune system is dependent on a complex interplay of regulatory activities that mediate the appropriate splicing of CD45 exon 4.

An optogenetic gene expression system with rapid activation and deactivation kinetics

Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems. However, current eukaryotic light-gated transcription systems are limited by toxicity, dynamic range, or slow activation/deactivation. Here we present an optogenetic gene expression system that addresses these shortcomings and demonstrate its broad utility. Our approach utilizes an engineered version of EL222, a bacterial Light-Oxygen-Voltage (LOV) protein that binds DNA when illuminated with blue light. The system has a large (>100-fold) dynamic range of protein expression, rapid activation (< 10 s) and deactivation kinetics (< 50 s), and a highly linear response to light. With this system, we achieve light-gated transcription in several mammalian cell lines and intact zebrafish embryos with minimal basal gene activation and toxicity. Our approach provides a powerful new tool for optogenetic control of gene expression in space and time.

A Model System for Activation-Induced Alternative Splicing of CD45 Pre-mRNA in T Cells Implicates Protein Kinase C and Ras

ABSTRACT Multiple isoforms of the protein tyrosine phosphatase CD45 are expressed on the surface of human T cells. Interestingly, the expression of these isoforms has been shown to vary significantly upon T-cell activation. In this report, we describe a novel cell line-based model system in which we can mimic the activation-induced alternative splicing of CD45 observed in primary T cells. Of the many proximal signaling events induced by T-cell stimulation, we show that activation of protein kinase C and activation of Ras are important for the switch toward the exclusion of CD45 variable exons, whereas events related to Ca2+ flux are not. In addition, the ability of cycloheximide to block the activation-induced alternative splicing of CD45 suggests a requirement for de novo protein synthesis. We further demonstrate that sequences which have previously been implicated in the tissue-specific regulation of CD45 variable exons are likewise necessary and sufficient for activation-induced splicing. These results provide an initial understanding of the requirements for CD45 alternative splicing upon T-cell activation, and they confirm the importance of this novel cell line in facilitating a more detailed analysis of the activation-induced regulation of CD45 than has been previously possible.

An exonic splicing silencer represses spliceosome assembly after ATP-dependent exon recognition

Precursor messenger RNA splicing is catalyzed by the spliceosome, a macromolecular complex that assembles in a stepwise process. The spliceosomes dynamic nature suggests the potential for regulation at numerous points along the assembly pathway; however, thus far, naturally occurring regulation of splicing has only been found to influence a small subset of spliceosomal intermediates. Here we report that the exonic splicing silencer (ESS1) that represses splicing of PTPRC (encoding CD45) exon 4 does not function by the typical mechanism of inhibiting binding of U1 or U2 small nuclear ribonucleoproteins (snRNPs) to the splice sites. Instead, a U1-, U2- and ATP-dependent complex forms across exon 4 that is required for inhibiting progression to the U4–U6–U5 tri-snRNP–containing B complex. Such inhibition represents a new mechanism for splicing regulation and suggests that regulation can probably occur at many of the transitions along the spliceosome assembly pathway.