Hox gene-specific cellular targeting using split intein Trojan exons.

The Trojan exon method, which makes use of intronically inserted T2A-Gal4 cassettes, has been widely used in Drosophila to create thousands of gene-specific Gal4 driver lines. These dual-purpose lines provide genetic access to specific cell types based on their expression of a native gene while simultaneously mutating one allele of the gene to enable loss-of-function analysis in homozygous animals. While this dual use is often an advantage, the truncation mutations produced by Trojan exons are sometimes deleterious in heterozygotes, perhaps by creating translation products with dominant negative effects. Such mutagenic effects can cause developmental lethality as has been observed with genes encoding essential transcription factors. Given the importance of transcription factors in specifying cell type, alternative techniques for generating specific Gal4 lines that target them are required. Here, we introduce a modified Trojan exon method that retains the targeting fidelity and plug-and-play modularity of the original method but mitigates its mutagenic effects by exploiting the self-splicing capabilities of split inteins. "Split Intein Trojan exons" (siTrojans) ensure that the two truncation products generated from the interrupted allele of the native gene are trans-spliced to create a full-length native protein. We demonstrate the efficacy of siTrojans by generating a comprehensive toolkit of Gal4 and Split Gal4 lines for the segmentally expressed Hox transcription factors and illustrate their use in neural circuit mapping by targeting neurons according to their position along the anterior-posterior axis. Both the method and the Hox gene-specific toolkit introduced here should be broadly useful.

The novel HLA-B*44:48:02 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-B*44:48:02 differs from HLA-B*44:48:01 by one synonymous nucleotide substitution in exon 3.

The novel HLA-C*07:02:147 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-C*07:02:147 differs from HLA-C*07:02:01:01 by one synonymous nucleotide substitution in exon 2.

The novel HLA-A*36:14 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-A*36:14 differs from HLA-A*36:01:01:01 by one non-synonymous nucleotide substitution in exon 4.

The novel HLA-DRB1*03:210 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-DRB1*03:210 differs from HLA-DRB1*03:01:01:01 by one non-synonymous nucleotide substitution in exon 3.

The novel HLA-DRB1*11:323 allele characterized by two different sequencing-based typing techniques.

The novel allele HLA-DRB1*11:323 differs from HLA-DRB1*11:01:02:01 by one non-synonymous nucleotide substitution in exon 2.

The novel HLA-A*30:01:23 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-A*30:01:23 differs from HLA-A*30:01:01:01 by one synonymous nucleotide substitution in exon 2.

The novel HLA-B*35:01:77 allele identified in a Russian volunteer bone marrow donor.

Nucleotide substitutions in the 5'UTR and exon 2 of HLA-B*35:01:01:05 result in a novel allele, HLA-B*35:01:77.

Characterisation of RAET1E/ULBP4 exon 4 and 3' untranslated region genetic architecture reveals further diversity and allelic polymorphism.

NKG2D is a natural killer cell activating receptor recognising ligands on infected or tumorigenic cells, leading to their cytolysis. There are eight known genes encoding NKG2D ligands: MICA, MICB and ULBP1-6. MICA and MICB are highly polymorphic and well characterised, whilst ULBP ligands are less polymorphic and the functional implication of their diversity is not well understood. Using International HLA and Immunogenetics Workshop (IHIW) cell line DNA, we previously characterised alleles of the RAET1E gene (encoding ULBP4 proteins), including the 5' UTR promoter region and exons 1-3. We found 11 promoter haplotypes associating with alleles based on exons 1-3, revealing 19 alleles overall. The current study extends this analysis using 87 individual DNA samples from IHIW cell lines or cord blood to include RAET1E exon 4 and the 3' UTR, as polymorphism in these regions have not been previously investigated. We found two novel exon 4 polymorphisms encoding amino acid substitutions altering the transmembrane domain. An amino acid substitution at residue 233 was unique to the RAET1E*008 allele whereas the substitution at residue 237 was shared between groups of alleles. Additionally, four haplotypes were found based on 3' UTR sequences, which were unique to certain alleles or shared with allele groups based on exons 1-4 polymorphisms. Furthermore, putative microRNAs were identified that may interact with these polymorphic sites, repressing transcription and potentially affecting expression levels.

The novel HLA-DPB1*1467:01 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-DPB1*1467:01 differs from HLA-DPB1*09:01:01:01 by one non-synonymous nucleotide substitution in exon 2.

Recognition of the HLA-C*07:359 allele in Taiwanese individuals.

One nucleotide substitution in codon 264 of HLA-C*07:02:01:01 results in a novel allele, HLA-C*07:359.

On a kneading theory for gene-splicing.

Two well-known facets in protein synthesis in eukaryotic cells are transcription of DNA to pre-RNA in the nucleus and the translation of messenger-RNA (mRNA) to proteins in the cytoplasm. A critical intermediate step is the removal of segments (introns) containing ∼97% of the nucleic-acid sites in pre-RNA and sequential alignment of the retained segments (exons) to form mRNA through a process referred to as splicing. Alternative forms of splicing enrich the proteome while abnormal splicing can enhance the likelihood of a cell developing cancer or other diseases. Mechanisms for splicing and origins of splicing errors are only partially deciphered. Our goal is to determine if rules on splicing can be inferred from data analytics on nucleic-acid sequences. Toward that end, we represent a nucleic-acid site as a point in a plane defined in terms of the anterior and posterior sub-sequences of the site. The "point-set" representation expands analytical approaches, including the use of statistical tools, to characterize genome sequences. It is found that point-sets for exons and introns are visually different, and that the differences can be quantified using a family of generalized moments. We design a machine-learning algorithm that can recognize individual exons or introns with 91% accuracy. Point-set distributions and generalized moments are found to differ between organisms.

Characterization of the novel HLA-B*40:538 allele by next-generation sequencing.

The HLA-B*40:538 allele differs from HLA-B*40:01:02:01 at position 905 C→T in exon 5.

Antisense oligonucleotide therapeutic approach for Timothy syndrome.

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A2-6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed7, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.

Alternative splicing for tuneable expression of protein subunits at desired ratios.

The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.

Identification of the novel allele, HLA-A*30:01:22, in a Saudi individual.

A single nucleotide substitution in exon 5 of HLA-A*30:01:01:01 results in the novel HLA-A*30:01:22 allele.

Splice modulators target PMS1 to reduce somatic expansion of the Huntington's disease-associated CAG repeat.

Huntington's disease (HD) is a dominant neurological disorder caused by an expanded HTT exon 1 CAG repeat that lengthens huntingtin's polyglutamine tract. Lowering mutant huntingtin has been proposed for treating HD, but genetic modifiers implicate somatic CAG repeat expansion as the driver of onset. We find that branaplam and risdiplam, small molecule splice modulators that lower huntingtin by promoting HTT pseudoexon inclusion, also decrease expansion of an unstable HTT exon 1 CAG repeat in an engineered cell model. Targeted CRISPR-Cas9 editing shows this effect is not due to huntingtin lowering, pointing instead to pseudoexon inclusion in PMS1. Homozygous but not heterozygous inactivation of PMS1 also reduces CAG repeat expansion, supporting PMS1 as a genetic modifier of HD and a potential target for therapeutic intervention. Although splice modulation provides one strategy, genome-wide transcriptomics also emphasize consideration of cell-type specific effects and polymorphic variation at both target and off-target sites.

KIS counteracts PTBP2 and regulates alternative exon usage in neurons.

Alternative RNA splicing is an essential and dynamic process in neuronal differentiation and synapse maturation, and dysregulation of this process has been associated with neurodegenerative diseases. Recent studies have revealed the importance of RNA-binding proteins in the regulation of neuronal splicing programs. However, the molecular mechanisms involved in the control of these splicing regulators are still unclear. Here, we show that KIS, a kinase upregulated in the developmental brain, imposes a genome-wide alteration in exon usage during neuronal differentiation in mice. KIS contains a protein-recognition domain common to spliceosomal components and phosphorylates PTBP2, counteracting the role of this splicing factor in exon exclusion. At the molecular level, phosphorylation of unstructured domains within PTBP2 causes its dissociation from two co-regulators, Matrin3 and hnRNPM, and hinders the RNA-binding capability of the complex. Furthermore, KIS and PTBP2 display strong and opposing functional interactions in synaptic spine emergence and maturation. Taken together, our data uncover a post-translational control of splicing regulators that link transcriptional and alternative exon usage programs in neuronal development.

Identification of the novel null allele HLA-DPB1*1449:01N by next-generation sequencing.

The novel HLA-DPB1*1449:01N allele differs from HLA-DPB1*16:01:01:01 by a nucleotide at codon 92 in exon 2.