The global Protein-RNA interaction map of ESRP1 defines a post-transcriptional program that is essential for epithelial cell function.

The epithelial splicing regulatory proteins, ESRP1 and ESRP2, are essential for mammalian development through the regulation of a global program of alternative splicing of genes involved in the maintenance of epithelial cell function. To further inform our understanding of the molecular functions of ESRP1, we performed enhanced crosslinking immunoprecipitation coupled with high-throughput sequencing (eCLIP) in epithelial cells of mouse epidermis. The genome-wide binding sites of ESRP1 were integrated with RNA-Seq analysis of alterations in splicing and total gene expression that result from epidermal ablation of Esrp1 and Esrp2. These studies demonstrated that ESRP1 functions in splicing regulation occur primarily through direct binding in a position-dependent manner to promote either exon inclusion or skipping. In addition, we also identified widespread binding of ESRP1 in 3' and 5' untranslated regions (UTRs) of genes involved in epithelial cell function, suggesting that its post-transcriptional functions extend beyond splicing regulation.

INTS13 variants causing a recessive developmental ciliopathy disrupt assembly of the Integrator complex.

Oral-facial-digital (OFD) syndromes are a heterogeneous group of congenital disorders characterized by malformations of the face and oral cavity, and digit anomalies. Mutations within 12 cilia-related genes have been identified that cause several types of OFD, suggesting that OFDs constitute a subgroup of developmental ciliopathies. Through homozygosity mapping and exome sequencing of two families with variable OFD type 2, we identified distinct germline variants in INTS13, a subunit of the Integrator complex. This multiprotein complex associates with RNA Polymerase II and cleaves nascent RNA to modulate gene expression. We determined that INTS13 utilizes its C-terminus to bind the Integrator cleavage module, which is disrupted by the identified germline variants p.S652L and p.K668Nfs*9. Depletion of INTS13 disrupts ciliogenesis in human cultured cells and causes dysregulation of a broad collection of ciliary genes. Accordingly, its knockdown in Xenopus embryos leads to motile cilia anomalies. Altogether, we show that mutations in INTS13 cause an autosomal recessive ciliopathy, which reveals key interactions between components of the Integrator complex.

The germ cell-specific RNA binding protein RBM46 is essential for spermatogonial differentiation in mice.

Control over gene expression is exerted, in multiple stages of spermatogenesis, at the post-transcriptional level by RNA binding proteins (RBPs). We identify here an essential role in mammalian spermatogenesis and male fertility for 'RNA binding protein 46' (RBM46). A highly evolutionarily conserved gene, Rbm46 is also essential for fertility in both flies and fish. We found Rbm46 expression was restricted to the mouse germline, detectable in males in the cytoplasm of premeiotic spermatogonia and meiotic spermatocytes. To define its requirement for spermatogenesis, we generated Rbm46 knockout (KO, Rbm46-/-) mice; although male Rbm46-/- mice were viable and appeared grossly normal, they were infertile. Testes from adult Rbm46-/- mice were small, with seminiferous tubules containing only Sertoli cells and few undifferentiated spermatogonia. Using genome-wide unbiased high throughput assays RNA-seq and 'enhanced crosslinking immunoprecipitation' coupled with RNA-seq (eCLIP-seq), we discovered RBM46 could bind, via a U-rich conserved consensus sequence, to a cohort of mRNAs encoding proteins required for completion of differentiation and subsequent meiotic initiation. In summary, our studies support an essential role for RBM46 in regulating target mRNAs during spermatogonia differentiation prior to the commitment to meiosis in mice.

Esrp1-Regulated Splicing of Arhgef11 Isoforms Is Required for Epithelial Tight Junction Integrity.

The epithelial-specific splicing regulators Esrp1 and Esrp2 are required for mammalian development, including establishment of epidermal barrier functions. However, the mechanisms by which Esrp ablation causes defects in epithelial barriers remain undefined. We determined that the ablation of Esrp1 and Esrp2 impairs epithelial tight junction (TJ) integrity through loss of the epithelial isoform of Rho GTP exchange factor Arhgef11. Arhgef11 is required for the maintenance of TJs via RhoA activation and myosin light chain (MLC) phosphorylation. Ablation or depletion of Esrp1/2 or Arhgef11 inhibits MLC phosphorylation and only the epithelial Arhgef11 isoform rescues MLC phosphorylation in Arhgef11 KO epithelial cells. Mesenchymal Arhgef11 transcripts contain a C-terminal exon that binds to PAK4 and inhibits RhoA activation byArhgef11. Deletion of the mesenchymal-specific Arhgef11 exon in Esrp1/2 KO epithelial cells using CRISPR/Cas9 restored TJ function, illustrating how splicing alterations can be mechanistically linked to disease phenotypes that result from impaired functions of splicing regulators.

Integrator subunit 4 is a 'Symplekin-like' scaffold that associates with INTS9/11 to form the Integrator cleavage module.

Integrator (INT) is a transcriptional regulatory complex associated with RNA polymerase II that is required for the 3'-end processing of both UsnRNAs and enhancer RNAs. Integrator subunits 9 (INTS9) and INTS11 constitute the catalytic core of INT and are paralogues of the cleavage and polyadenylation specificity factors CPSF100 and CPSF73. While CPSF73/100 are known to associate with a third protein called Symplekin, there is no paralog of Symplekin within INT raising the question of how INTS9/11 associate with the other INT subunits. Here, we have identified that INTS4 is a specific and conserved interaction partner of INTS9/11 that does not interact with either subunit individually. Although INTS4 has no significant homology with Symplekin, it possesses N-terminal HEAT repeats similar to Symplekin but also contains a ß-sheet rich C-terminal region, both of which are important to bind INTS9/11. We assess three functions of INT including UsnRNA 3'-end processing, maintenance of Cajal body structural integrity, and formation of histone locus bodies to conclude that INTS4/9/11 are the most critical of the INT subunits for UsnRNA biogenesis. Altogether, these results indicate that INTS4/9/11 compose a heterotrimeric complex that likely represents the Integrator 'cleavage module' responsible for its endonucleolytic activity.

ESRP1 Mutations Cause Hearing Loss due to Defects in Alternative Splicing that Disrupt Cochlear Development.

Alternative splicing contributes to gene expression dynamics in many tissues, yet its role in auditory development remains unclear. We performed whole-exome sequencing in individuals with sensorineural hearing loss (SNHL) and identified pathogenic mutations in Epithelial Splicing-Regulatory Protein 1 (ESRP1). Patient-derived induced pluripotent stem cells showed alternative splicing defects that were restored upon repair of an ESRP1 mutant allele. To determine how ESRP1 mutations cause hearing loss, we evaluated Esrp1-/- mouse embryos and uncovered alterations in cochlear morphogenesis, auditory hair cell differentiation, and cell fate specification. Transcriptome analysis revealed impaired expression and splicing of genes with essential roles in cochlea development and auditory function. Aberrant splicing of Fgfr2 blocked stria vascularis formation due to erroneous ligand usage, which was corrected by reducing Fgf9 gene dosage. These findings implicate mutations in ESRP1 as a cause of SNHL and demonstrate the complex interplay between alternative splicing, inner ear development, and auditory function.

Genome-Wide RNAi Screens for RNA Processing Events in Drosophila melanogaster S2 Cells.

Over the past 10 years, the design and application of genome-wide screening (GWS) has improved to the point that it can now be done at level of the individual laboratory. The advantages of GWSs compared to classical genetic screens include: immediate identification of a positive scoring gene, relatively short period of time necessary to conduct the screen (as little as 1 week), cell lines do not present developmental needs for gene expression that an organism normally would, and validation/confirmation of results is straightforward. Here, we describe a general protocol for GWS to be conducted in Drosophila melanogaster S2 cells. We provide specific details on what type of experiments must be done before initiating a screen, the materials that are required to conduct a screen, and make suggestions on methods to carry out secondary screening and counter-screening once the initial GWS is complete. Multiple considerations are also raised that focus on how to anticipate false positives/negatives and how to minimize their occurrence through intelligent design. Finally, we provide specific examples of data that our group has gathered from published genome-wide screens in order to exemplify how "hits" are scored and confirmed.

A distal auxiliary element facilitates cleavage and polyadenylation of Dux4 mRNA in the pathogenic haplotype of FSHD.

The degenerative muscle disorder facioscapulohumeral dystrophy (FSHD) is thought to be caused by the inappropriate expression of the Double Homeobox 4 (Dux4) protein in muscle cells leading to apoptosis. Expression of Dux4 in the major form of FSHD is a function of two contributing molecular changes: contractions in the D4Z4 microsatellite repeat region where Dux4 is located and an SNP present within a region downstream of the D4Z4. This SNP provides a functional, yet non-consensus polyadenylation signal (PAS) is used for the Dux4 mRNA 3' end processing. Surprisingly, the sequences flanking the Dux4 PAS do not resemble a typical cleavage and polyadenylation landscape with no recognizable downstream sequence element and a suboptimal cleavage site. Here, we conducted a systematic analysis of the cis-acting elements that govern Dux4 cleavage and polyadenylation. Using a transcriptional read-through reporter, we determined that sequences downstream of the SNP located within the ß-satellite region are critical for Dux4 cleavage and polyadenylation. We also demonstrate the feasibility of using antisense oligonucleotides to target these sequences as a means to reduce Dux4 expression. Our results underscore the complexity of the region immediately downstream of the D4Z4 and uncover a previously unknown function for the ß-satellite region in Dux4 cleavage and polyadenylation.

Triplex structures induce DNA double strand breaks via replication fork collapse in NER deficient cells.

Structural alterations in DNA can serve as natural impediments to replication fork stability and progression, resulting in DNA damage and genomic instability. Naturally occurring polypurine mirror repeat sequences in the human genome can create endogenous triplex structures evoking a robust DNA damage response. Failures to recognize or adequately process these genomic lesions can result in loss of genomic integrity. Nucleotide excision repair (NER) proteins have been found to play a prominent role in the recognition and repair of triplex structures. We demonstrate using triplex-forming oligonucleotides that chromosomal triplexes perturb DNA replication fork progression, eventually resulting in fork collapse and the induction of double strand breaks (DSBs). We find that cells deficient in the NER damage recognition proteins, XPA and XPC, accumulate more DSBs in response to chromosomal triplex formation than NER-proficient cells. Furthermore, we demonstrate that XPC-deficient cells are particularly prone to replication-associated DSBs in the presence of triplexes. In the absence of XPA or XPC, deleterious consequences of triplex-induced genomic instability may be averted by activating apoptosis via dual phosphorylation of the H2AX protein. Our results reveal that damage recognition by XPC and XPA is critical to maintaining replication fork integrity and preventing replication fork collapse in the presence of triplex structures.

CFIm25 regulates glutaminase alternative terminal exon definition to modulate miR-23 function.

Alternative polyadenylation (APA) and alternative splicing (AS) provide mRNAs with the means to avoid microRNA repression through selective shortening or differential usage of 3'UTRs. The two glutaminase (GLS) mRNA isoforms, termed KGA and GAC, contain distinct 3'UTRs with the KGA isoform subject to repression by miR-23. We show that depletion of the APA regulator CFIm25 causes a strong shift to the usage of a proximal poly(A) site within the KGA 3'UTR and also alters splicing to favor exclusion of the GAC 3'UTR. Surprisingly, we observe that while miR-23 is capable of down-regulating the shortened KGA 3'UTR, it has only minor impact on the full-length KGA 3'UTR, demonstrating that additional potent negative regulation of GLS expression exists beyond this single microRNA targeting site. Finally, we show that the apoptosis induced upon down-regulation of the GAC isoform can be alleviated through concurrent reduction in CFIm25 expression, revealing the sensitivity of glutaminase expression to the levels of RNA processing factors. These results exemplify the complex interplay between RNA processing and microRNA repression in controlling glutamine metabolism in cancer cells.

Gain-of-function reporters for analysis of mRNA 3'-end formation: Design and optimization.

The concept of mRNA 3'-end formation as a static, minimally regulated housekeeping process has undergone a paradigm shift. Many recent studies have shown that accurate and efficient 3'-end formation of mRNA is highly regulated and that dysregulation of this process is a hallmark of several diseases. While there are many global analysis methods for monitoring altered mRNA processing, methods for investigating specific RNA 3'-end processing events in cells have not significantly changed. Here we describe a facile gain-of-function cellular reporter for the analysis of mRNA 3'-end formation as an alternative to approaches that are technically challenging or use radioactivity. We also offer suggestions for optimization of our approach and enhancement of its reproducibility.

PMA induces SnoN proteolysis and CD61 expression through an autocrine mechanism.

Phorbol-12-myristate-13-acetate, also called PMA, is a small molecule that activates protein kinase C and functions to differentiate hematologic lineage cells. However, the mechanism of PMA-induced cellular differentiation is not fully understood. We found that PMA triggers global enhancement of protein ubiquitination in K562, a myelogenous leukemia cell line and one of the enhanced-ubiquitination targets is SnoN, an inhibitor of the Smad signaling pathway. Our data indicated that PMA stimulated the production of Activin A, a cytokine of the TGF-ß family. Activin A then activated the phosphorylation of both Smad2 and Smad3. In consequence, SnoN is ubiquitinated by the APC(Cdh1) ubiquitin ligase with the help of phosphorylated Smad2. Furthermore, we found that SnoN proteolysis is important for the expression of CD61, a marker of megakaryocyte. These results indicate that protein ubiquitination promotes megakaryopoiesis via degrading SnoN, an inhibitor of CD61 expression, strengths the roles of ubiquitination in cellular differentiation.

Non-mRNA 3' end formation: how the other half lives.

The release of nascent RNA from transcribing RNA polymerase complexes is required for all further functions carried out by RNA molecules. The elements and processing machinery involved in 3' end formation therefore represent key determinants in the biogenesis and accumulation of cellular RNA. While these factors have been well-characterized for messenger RNA, recent work has elucidated analogous pathways for the 3' end formation of other important cellular RNA. Here, we discuss four specific cases of non-mRNA 3' end formation-metazoan small nuclear RNA, Saccharomyces cerevisiae small nuclear RNA, Schizosaccharomyces pombe telomerase RNA, and the mammalian MALAT1 large noncoding RNA-as models of alternative mechanisms to generate RNA 3' ends. Comparison of these disparate processing pathways reveals an emerging theme of evolutionary ingenuity. In some instances, evidence for the creation of a dedicated processing complex exists; while in others, components are utilized from the existing RNA processing machinery and modified to custom fit the unique needs of the RNA substrate. Regardless of the details of how non-mRNA 3' ends are formed, the lengths to which biological systems will go to release nascent transcripts from their DNA templates are fundamental for cell survival.