Structural and Functional Peculiarities of Cytoplasmic Tropomyosin Isoforms, the Products of TPM1 and TPM4 Genes

Tropomyosin (Tpm) is one of the major protein partners of actin. Tpm molecules are α-helical coiled-coil protein dimers forming a continuous head-to-tail polymer along the actin filament. Human cells produce a large number of Tpm isoforms that are thought to play a significant role in determining actin cytoskeletal functions. Even though the role of these Tpm isoforms in different non-muscle cells is more or less studied in many laboratories, little is known about their structural and functional properties. In the present work, we have applied various methods to investigate the properties of five cytoplasmic Tpm isoforms (Tpm1.5, Tpm 1.6, Tpm1.7, Tpm1.12, and Tpm 4.2), which are the products of two different genes, TPM1 and TPM4, and also significantly differ by alternatively spliced exons: N-terminal exons 1a2b or 1b, internal exons 6a or 6b, and C-terminal exons 9a, 9c or 9d. Our results demonstrate that structural and functional properties of these Tpm isoforms are quite different depending on sequence variations in alternatively spliced regions of their molecules. The revealed differences can be important in further studies to explain why various Tpm isoforms interact uniquely with actin filaments, thus playing an important role in the organization and dynamics of the cytoskeleton.

A polyglutamine domain is required for de novo CIZ1 assembly formation at the inactive X chromosome

SummaryCIP1-interacting zinc finger protein 1 (CIZ1) forms large assemblies at the inactive X chromosome (Xi) in female fibroblasts in an Xist lncRNA-dependent manner. Here we address the requirements for assembly formation, and show that CIZ1 interacts directly with Xist via two independent domains in its N- and C-terminus. Interaction with Xist repeat E, assembly at Xi in cells, and the complexity of self-assemblies formed in vitro, are all modulated by alternatively-spliced exons that include two glutamine-rich prion-like domains (PLD1 and PLD2), both conditionally excluded from the N-terminal domain. Exclusion of PLD1 alone is sufficient to abrogate de novo establishment of new CIZ1 assemblies and Xi territories enriched for H3K27me3 in CIZ1-null fibroblasts. Together the data suggest that PLD1-driven CIZ1 assemblies form at Xi, are nucleated by interaction with Xist and amplified by multivalent interaction with RNA, so implicating a polyglutamine tract in the maintenance of epigenetic state.

Mutations primarily alter the inclusion of alternatively spliced exons

Genetic analyses and systematic mutagenesis have revealed that synonymous, non-synonymous and intronic mutations frequently alter the inclusion levels of alternatively spliced exons, consistent with the concept that altered splicing might be a common mechanism by which mutations cause disease. However, most exons expressed in any cell are highly-included in mature mRNAs. Here, by performing deep mutagenesis of highly-included exons and by analysing the association between genome sequence variation and exon inclusion across the transcriptome, we report that mutations only very rarely alter the inclusion of highly-included exons. This is true for both exonic and intronic mutations as well as for perturbations in trans. Therefore, mutations that affect splicing are not evenly distributed across primary transcripts but are focussed in and around alternatively spliced exons with intermediate inclusion levels. These results provide a resource for prioritising synonymous and other variants as disease-causing mutations.

Mutations primarily alter the inclusion of alternatively spliced exons

Genetic analyses and systematic mutagenesis have revealed that synonymous, non-synonymous and intronic mutations frequently alter the inclusion levels of alternatively spliced exons, consistent with the concept that altered splicing might be a common mechanism by which mutations cause disease. However, most exons expressed in any cell are highly-included in mature mRNAs. Here, by performing deep mutagenesis of highly-included exons and by analysing the association between genome sequence variation and exon inclusion across the transcriptome, we report that mutations only very rarely alter the inclusion of highly-included exons. This is true for both exonic and intronic mutations as well as for perturbations in trans. Therefore, mutations that affect splicing are not evenly distributed across primary transcripts but are focussed in and around alternatively spliced exons with intermediate inclusion levels. These results provide a resource for prioritising synonymous and other variants as disease-causing mutations.

The gene structure and hypervariability of the complete Penaeus monodon Dscam gene

Using two advanced sequencing approaches, Illumina and PacBio, we derive the entire Dscam gene from an M2 assembly of the complete Penaeus monodon genome. The P. monodon Dscam (PmDscam) gene is ~266 kbp, with a total of 44 exons, 5 of which are subject to alternative splicing. PmDscam has a conserved architectural structure consisting of an extracellular region with hypervariable Ig domains, a transmembrane domain, and a cytoplasmic tail. We show that, contrary to a previous report, there are in fact 26, 81 and 26 alternative exons in N-terminal Ig2, N-terminal Ig3 and the entirety of Ig7, respectively. We also identified two alternatively spliced exons in the cytoplasmic tail, with transmembrane domains in exon variants 32.1 and 32.2, and stop codons in exon variants 44.1 and 44.2. This means that alternative splicing is involved in the selection of the stop codon. There are also 7 non-constitutive cytoplasmic tail exons that can either be included or skipped. Alternative splicing and the non-constitutive exons together produce more than 21 million isoform combinations from one PmDscam locus in the P. monodon gene. A public-facing database that allows BLAST searches of all 175 exons in the PmDscam gene has been established at http://pmdscam.dbbs.ncku.edu.tw/.

Sequence and Evolutionary Features for the Alternatively Spliced Exons of Eukaryotic Genes

Alternative splicing of pre-mRNAs is a crucial mechanism for maintaining protein diversity in eukaryotes without requiring a considerable increase of genes in the number. Due to rapid advances in high-throughput sequencing technologies and computational algorithms, it is anticipated that alternative splicing events will be more intensively studied to address different kinds of biological questions. The occurrences of alternative splicing mean that all exons could be classified to be either constitutively or alternatively spliced depending on whether they are virtually included into all mature mRNAs. From an evolutionary point of view, therefore, the alternatively spliced exons would have been associated with distinctive biological characteristics in comparison with constitutively spliced exons. In this paper, we first outline the representative types of alternative splicing events and exon classification, and then review sequence and evolutionary features for the alternatively spliced exons. The main purpose is to facilitate understanding of the biological implications of alternative splicing in eukaryotes. This knowledge is also helpful to establish computational approaches for predicting the splicing pattern of exons.

Deep Splicing Code: Classifying Alternative Splicing Events Using Deep Learning

Alternative splicing (AS) is the process of combining different parts of the pre-mRNA to produce diverse transcripts and eventually different protein products from a single gene. In computational biology field, researchers try to understand AS behavior and regulation using computational models known as “Splicing Codes”. The final goal of these algorithms is to make an in-silico prediction of AS outcome from genomic sequence. Here, we develop a deep learning approach, called Deep Splicing Code (DSC), for categorizing the well-studied classes of AS namely alternatively skipped exons, alternative 5’ss, alternative 3’ss, and constitutively spliced exons based only on the sequence of the exon junctions. The proposed approach significantly improves the prediction and the obtained results reveal that constitutive exons have distinguishable local characteristics from alternatively spliced exons. Using the motif visualization technique, we show that the trained models learned to search for competitive alternative splice sites as well as motifs of important splicing factors with high precision. Thus, the proposed approach greatly expands the opportunities to improve alternative splicing modeling. In addition, a web-server for AS events prediction has been developed based on the proposed method and made available at https://home.jbnu.ac.kr/NSCL/dsc.htm.