Evidence for the role of transcription factors in the co-transcriptional regulation of intron retention

Alternative splicing is a widespread regulatory phenomenon that enables a single gene to produce multiple transcripts. Among the different types of alternative splicing, intron retention is one of the least explored despite its high prevalence in both plants and animals. The recent discovery that the majority of splicing is co-transcriptional has led to the finding that chromatin state affects alternative splicing. Therefore it is plausible that transcription factors can regulate splicing outcomes. We provide evidence for this hypothesis by studying regions of open chromatin in retained and excised introns. Using deep learning models designed to distinguish between regions of open chromatin in retained introns and non-retained introns, we identified motifs enriched in IR events with significant hits to known human transcription factors. Our model predicts that the majority of transcription factors that affect intron retention come from the zinc finger family. We demonstrate the validity of these predictions using ChIP-seq data for multiple zinc finger transcription factors and find strong over-representation for their peaks in intron retention events. Availability: Source code available at https://github.com/fahadahaf/chromir

Quantifying mediation between omics layers and complex traits

Background: High-dimensional omics datasets provide valuable resources to determine the causal role of molecular traits in mediating the path from genotype to phenotype. Making use of quantitative trait loci (QTL) and genome-wide association studies (GWASs) summary statistics, we developed a multivariable Mendelian randomization (MVMR) framework to quantify the connectivity between three omics layers (DNA methylome (DNAm), transcriptome and proteome) and their cascading causal impact on complex traits and diseases. Results: Evaluating 50 complex traits, we found that on average 37.8% (95% CI: [36.0%-39.5%]) of DNAm-to-trait effects were mediated through transcripts in the cis-region, while only 15.8% (95% CI: [11.9%-19.6%]) are mediated through proteins in cis. DNAm sites typically regulate multiple transcripts, and while found to predominantly decrease gene expression, this was only the case for 53.4% across ~47,000 significant DNAm-transcript pairs. The average mediation proportion for transcript-to-trait effects through proteins (encoded for by the assessed transcript or located in trans) was estimated to be 5.27% (95%CI: [4.11%-6.43%]). Notable differences in the transcript and protein QTL architectures were detected with only 22% of protein levels being causally driven by their corresponding transcript levels. Several regulatory mechanisms were hypothesized including an example where cg10385390 (chr1:8,022,505) increases the risk of irritable bowel disease by reducing PARK7 transcript and protein expression. Conclusions: The proposed integrative framework identified putative causal chains through omics layers providing a powerful tool to map GWAS signals. Quantification of causal effects between successive layers indicated that molecular mechanisms can be more complex than what the central dogma of biology would suggest.

Molecular cloning, expression, and functional features of IGF1 splice variants in sheep

Insulin-like growth factor 1 (IGF1),also known as somatomedin C, is essential for the regulation of animal growth and development. In many species, the IGF1 gene can be alternatively spliced into multiple transcripts, encoding different pre-pro-IGF1 proteins. However, the exact alternative splicing patterns of IGF1 and the sequence information of different splice variants in sheep are still unclear. In this study, four splice variants (class 1-Ea, class 1-Eb, class 2-Ea, and class 2-Eb) were obtained, but no IGF1 Ec, similar to that found in other species, was discovered. Bioinformatics analysis showed that the four splice variants shared the same mature peptide (70 amino acids) and possessed distinct signal peptides and E peptides. Tissue expression analysis indicated that the four splice variants were broadly expressed in all tested tissues and were most abundantly expressed in the liver. In most tissues and stages, the expression of class 1-Ea was highest, and the expression of other splice variants was low. Overall, levels of the four IGF1 splice variants at the fetal and lamb stages were higher than those at the adult stage. Overexpression of the four splice variants significantly increased fibroblast proliferation and inhibited apoptosis (P < 0.05). In contrast, silencing IGF1 Ea or IGF1 Eb with siRNA significantly inhibited proliferation and promoted apoptosis (P < 0.05). Among the four splice variants, class 1-Ea had a more evident effect on cell proliferation and apoptosis. In summary, the four ovine IGF1 splice variants have different structures and expression patterns and might have different biological functions.

Identification of a Splice Variant (c.5074+3A>C) of BRCA1 by RNA Sequencing and TOPO Cloning

Grading the pathogenicity of BRCA1/2 variants has great clinical importance in patient treatment as well as in the prevention and screening of hereditary breast and ovarian cancer (HBOC). For accurate evaluation, confirming the splicing effect of a possible splice site variant is crucial. We report a significant splicing variant (c.5074+3A>C) in BRCA1 in a patient with recurrent ovarian cancer. Next-generation sequencing (NGS) of BRCA1/2 from patient’s peripheral blood identified the variant, which was strongly suspected of being a splicing mutation based on in silico predictions. Direct RNA analysis yielded multiple transcripts, and TOPO cloning of the complementary DNA (cDNA) and Sanger sequencing revealed an aberrant transcript with an insertion of the first 153 bp of intron 17, and another transcript with the 153 bp insertion along with an exon 18 deletion. A premature termination codon was presumed to be formed by the 153 bp partial intron retention common to the two transcripts. Therefore, BRCA1 c.5074+3A>C was classified as a likely pathogenic variant. Our findings show that active use of functional studies of variants suspected of altered splicing are of great help in classifying them.

Distinct developmental mechanisms influence sexual dimorphisms in the milkweed bug Oncopeltus fasciatus

Sexual dimorphism is a common feature of animals. Sex determination mechanisms vary widely among species and evolve rapidly. Until recently studies have found consistent mechanisms across the body of each individual determine female or male dimorphic body structures. In sexually dimorphic cells throughout the body of Drosophila, the relative dosage of autosomes and X chromosomes leads indirectly to alternatively spliced transcripts from the gene doublesex. The female Dsx isoform interacts with the mediator complex protein encoded by intersex to activate female development in flies. In males the transcription factor encoded by fruitless promotes male-specific behavior. In the milkweed bug Oncopeltus fasciatus, we find a requirement for different combinations of these genes during development of distinct dimorphic structures, within the same sex, suggesting a previously unappreciated level of diversity in sex determination. While intersex and fruitless are structurally conserved, doublesex has a history of duplication and divergence among Paraneoptera. Three doublesex paralogs in O. fasciatus produce multiple transcripts with sex- and tissue-specific expression. intersex and fruitless are expressed across the body, in females and males. RNA interference reveals only one doublesex paralog functions in somatic sex determination. Knockdown of doublesex and fruitless produces intersex phenotypic conditions in two sexually dimorphic structures: genitalia and abdominal sternites. In contrast, intersex is required for dimorphic development of female and male genitalia, but not for sternite dimorphism. These results reveal sex determination roles for intersex and fruitless distinct from their orthologs in other insects. Our results illuminate a novel form of developmental diversity in insect sex determination.

A programmable system to methylate and demethylate m6A on specific mRNAs

RNA N6-Methyladenosine (m6A) is the most abundant mRNA modification, and forms part of an epitranscriptomic system that modulates RNA function. RNA modifications can be reversibly catalyzed by several specific enzymes, and those modifications can be recognized by RNA binding proteins that in turn regulate biological processes. Although there are many reports demonstrating m6A participation in critical biological functions, this exploration has mainly been conducted through the global knockout or knockdown of the writers, erasers, or readers of m6A. Consequently, there is a lack of information about the role of m6A on single transcripts in biological processes, posing a challenge in understanding the biological functions of m6A. Here, we demonstrate a CRISPR/dCas13a-based RNA m6A-editor which can target mRNAs using single crRNA or multiple crRNAs array to methylate or demethylate m6A. We systematically assay its capabilities to enable the targeted rewriting of m6A dynamics, including modulation of circular RNA translation and transcript half-life. Finally, we demonstrate the utility of the system by specifically modulating XIST m6A levels, which can control X chromosome silencing and activation. Based on our editors, m6A on single and multiple transcripts can be modified to allow the exploration of the role of m6A on in biological processes.

A conserved role for the ALS-linked splicing factor SFPQ in repression of pathogenic cryptic last exons

The RNA-binding protein SFPQ plays an important role in neuronal development and has been associated with several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer’s disease. Here, we report that loss of sfpq leads to premature termination of multiple transcripts due to widespread activation of previously unannotated cryptic last exons (CLEs). These SFPQ-inhibited CLEs appear preferentially in long introns of genes with neuronal functions and can dampen gene expression outputs and/or give rise to short peptides interfering with the normal gene functions. We show that one such peptide encoded by the CLE-containing epha4b mRNA isoform is responsible for neurodevelopmental defects in the sfpq mutant. The uncovered CLE-repressive activity of SFPQ is conserved in mouse and human, and SFPQ-inhibited CLEs are found expressed across ALS iPSC-derived neurons. These results greatly expand our understanding of SFPQ function and uncover a gene regulation mechanism with wide relevance to human neuropathologies.

Differential Expression of BARD1 Isoforms in Melanoma

Melanoma comprises <5% of cutaneous malignancies, yet it causes a significant proportion of skin cancer-related deaths worldwide. While new therapies for melanoma have been developed, not all patients respond well. Thus, further research is required to better predict patient outcomes. Using long-range nanopore sequencing, RT-qPCR, and RNA sequencing analyses, we examined the transcription of BARD1 splice isoforms in melanoma cell lines and patient tissue samples. Seventy-six BARD1 mRNA variants were identified in total, with several previously characterised isoforms (γ, φ, δ, ε, and η) contributing to a large proportion of the expressed transcripts. In addition, we identified four novel splice events, namely, Δ(E3_E9), ▼(i8), IVS10+131▼46, and IVS10▼176, occurring in various combinations in multiple transcripts. We found that short-read RNA-Seq analyses were limited in their ability to predict isoforms containing multiple non-contiguous splicing events, as compared to long-range nanopore sequencing. These studies suggest that further investigations into the functional significance of the identified BARD1 splice variants in melanoma are warranted.

The first Conus genome assembly reveals a primary genetic central dogma of conopeptides in C. betulinus

Although there are various Conus species with publicly available transcriptome and proteome data, no genome assembly has been reported yet. Here, using Chinese tubular cone snail (C. betulinus) as a representative, we sequenced and assembled the first Conus genome with original identification of 133 genome-widely distributed conopeptide genes. After integration of our genomics, transcriptomics, and peptidomics data in the same species, we established a primary genetic central dogma of diverse conopeptides, assuming a rough number ratio of ~1:1:1:10s for the total genes: transcripts: proteins: post-translationally modified peptides. This ratio may be special for this worm-hunting Conus species, due to the high diversity of various Conus genomes and the big number ranges of conopeptide genes, transcripts, and peptides in previous reports of diverse Conus species. Only a fraction (45.9%) of the identified conotopeptide genes from our achieved genome assembly are transcribed with transcriptomic evidence, and few genes individually correspond to multiple transcripts possibly due to intraspecies or mutation-based variances. Variable peptide processing at the proteomic level, generating a big diversity of venom conopeptides with alternative cleavage sites, post-translational modifications, and N-/C-terminal truncations, may explain how the 133 genes and ~123 transcripts can generate thousands of conopeptides in the venom of individual C. betulinus. We also predicted many conopeptides with high stereostructural similarities to the putative analgesic ω-MVIIA, addiction therapy AuIB and insecticide ImI, suggesting that our current genome assembly for C. betulinus is a valuable genetic resource for high-throughput prediction and development of potential pharmaceuticals.

Regulatory roles of Escherichia coli 5' UTR and ORF-internal RNAs detected by 3' end mapping

Many bacterial genes are regulated by RNA elements in their 5´ untranslated regions (UTRs). However, the full complement of these elements is not known even in the model bacterium Escherichia coli. Using complementary RNA-sequencing approaches, we detected large numbers of 3´ ends in 5´ UTRs and open reading frames (ORFs), suggesting extensive regulation by premature transcription termination. We documented regulation for multiple transcripts, including spermidine induction involving Rho and translation of an upstream ORF for an mRNA encoding a spermidine efflux pump. In addition to discovering novel sites of regulation, we detected short, stable RNA fragments derived from 5´ UTRs and sequences internal to ORFs. Characterization of three of these transcripts, including an RNA internal to an essential cell division gene, revealed that they have independent functions as sRNA sponges. Thus, these data uncover an abundance of cis- and trans-acting RNA regulators in bacterial 5´ UTRs and internal to ORFs.