Analysis of intronless genes involved in oscillation and differentiation

The genomes of higher eukaryotes are replete with intron-containing genes. Transcription of these genes produces precursor mRNAs containing intervening sequences, which are subsequently removed and the exons spliced together to form the mature mRNA. However, a small proportion of eukaryotic protein-coding genes are intronless and therefore bypass post-transcriptional splicing events. Although a large proportion of intronless genes are known to code for certain types of proteins, their specific role in the genome of higher organism is perplexing. This research set out to elucidate the functions of intronless genes in humans by studying their involvement in the expression pattern of oscillatory gene that occurs in the pre-somitic mesoderm of developing embryo. Twenty-seven (27) human homologs of mouse oscillatory genes were analysed to determine the number of exons present in them using various bioinformatics databases. The result obtained identified two intronless genes –NRARP and ID1 – which are associated with the Notch signalling pathway of the segmentation clock. This represented 7.4% of the total oscillatory genes analysed. No intronless gene was found in the Wnt and FGF signalling pathways – two other pathways famous for oscillatory gene expression. The proteins encoded by the intronless genes are involved in several important biological processes including angiogenesis, cell cycle control and in the regulation of cellular senescence. Although oscillatory genes had fewer numbers of introns compared to the non-oscillatory genes, the intronless genes were not implicated in the regulation of the precise timing events of the segmentation clock. This result may also point to the fact that the rapid expression rate of the oscillatory genes in the PSM may favour the reduced intron length of the oscillatory genes.

Introns control stochastic allele expression bias

Monoallelic expression (MAE) or extreme allele bias can account for incomplete penetrance, missing heritability and non-Mendelian diseases. In cancer, MAE is associated with shorter patient survival times and higher tumor grade. Prior studies showed that stochastic MAE is caused by stochastic epigenetic silencing, in a gene and tissue-specific manner. Here, we used C. elegans to study stochastic MAE in vivo. We found allele bias/MAE to be widespread within C. elegans tissues, presenting as a continuum from fully biallelic to MAE. We discovered that the presence of introns within alleles robustly decreases MAE. We determined that introns control MAE at distinct loci, in distinct cell types, with distinct promoters, and within distinct coding sequences, using a 5’-intron position-dependent mechanism. Bioinformatic analysis showed human intronless genes are significantly enriched for MAE. Our experimental evidence demonstrates a role for introns in regulating MAE, possibly explaining why some mutations within introns result in disease.

Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing

The mRNA 5’ cap structure serves both to protect transcripts from degradation and promote their translation. Cap removal is thus an integral component of mRNA turnover that is carried out by cellular decapping enzymes, whose activity is tightly regulated and coupled to other stages of the mRNA decay pathway. The poxvirus vaccinia virus (VACV) encodes its own decapping enzymes, D9 and D10, that act on cellular and viral mRNA, but may be regulated differently than their cellular counterparts. Here, we evaluated the targeting potential of these viral enzymes using RNA sequencing from cells infected with wild-type and decapping mutant versions of VACV as well as in uninfected cells expressing D10. We found that D9 and D10 target an overlapping subset of viral transcripts but that D10 plays a dominant role in depleting the vast majority of human transcripts, although not in an indiscriminate manner. Unexpectedly, the splicing architecture of a gene influences how robustly its corresponding transcript is targeted by D10, as transcripts derived from intronless genes are less susceptible to enzymatic decapping by D10. As all VACV genes are intronless, preferential decapping of transcripts from intron-encoding genes provides an unanticipated mechanism for the virus to disproportionately deplete host transcripts and remodel the infected cell transcriptome.

Overexpression of the SETD2 WW domain inhibits the phosphor-IWS1/SETD2 interaction and the oncogenic AKT/IWS1 RNA splicing program.

Our earlier studies had shown that AKT phosphorylates IWS1, and that following phosphorylation, IWS1 recruits the histone methyltransferase SETD2 to an SPT6/IWS1/ALY complex, which assembles on the Ser2-phosphorylated CTD of RNA Pol II. Recruited SETD2 methylates histone H3 at K36, during transcriptional elongation of target genes, and this regulates multiple steps in RNA metabolism. By regulating the RNA splicing of U2AF2, it controls cell proliferation. Importantly, pathway activity correlates with grade, stage and metastatic potential of lung adenocarcinomas, especially those with EGFR mutations. By regulating nucleocytoplasmic mRNA transport of intronless genes, including those encoding type I IFNs, it regulates sensitivity to viral infection. Here, we show that SETD2 interacts with IWS1 via its WW domain, that the interaction is IWS1 phosphorylation-dependent and that WW domain overexpression blocks the interaction and inhibits the pathway and its biological outcomes. We conclude that blocking the phosphor-IWS1/SETD2 interaction is feasible and has significant therapeutic potential in human cancer.

Evolutionary Perspective and Expression Analysis of Intronless Genes Highlight the Conservation of Their Regulatory Role

The structure of eukaryotic genes is generally a combination of exons interrupted by intragenic non-coding DNA regions (introns) removed by RNA splicing to generate the mature mRNA. A fraction of genes, however, comprise a single coding exon with introns in their untranslated regions or are intronless genes (IGs), lacking introns entirely. The latter code for essential proteins involved in development, growth, and cell proliferation and their expression has been proposed to be highly specialized for neuro-specific functions and linked to cancer, neuropathies, and developmental disorders. The abundant presence of introns in eukaryotic genomes is pivotal for the precise control of gene expression. Notwithstanding, IGs exempting splicing events entail a higher transcriptional fidelity, making them even more valuable for regulatory roles. This work aimed to infer the functional role and evolutionary history of IGs centered on the mouse genome. IGs consist of a subgroup of genes with one exon including coding genes, non-coding genes, and pseudogenes, which conform approximately 6% of a total of 21,527 genes. To understand their prevalence, biological relevance, and evolution, we identified and studied 1,116 IG functional proteins validating their differential expression in transcriptomic data of embryonic mouse telencephalon. Our results showed that overall expression levels of IGs are lower than those of MEGs. However, strongly up-regulated IGs include transcription factors (TFs) such as the class 3 of POU (HMG Box), Neurog1, Olig1, and BHLHe22, BHLHe23, among other essential genes including the β-cluster of protocadherins. Most striking was the finding that IG-encoded BHLH TFs fit the criteria to be classified as microproteins. Finally, predicted protein orthologs in other six genomes confirmed high conservation of IGs associated with regulating neural processes and with chromatin organization and epigenetic regulation in Vertebrata. Moreover, this study highlights that IGs are essential modulators of regulatory processes, such as the Wnt signaling pathway and biological processes as pivotal as sensory organ developing at a transcriptional and post-translational level. Overall, our results suggest that IG proteins have specialized, prevalent, and unique biological roles and that functional divergence between IGs and MEGs is likely to be the result of specific evolutionary constraints.

Arginine Methyltransferases as Regulators of RNA-Binding Protein Activities in Pathogenic Kinetoplastids

A large number of eukaryotic proteins are processed by single or combinatorial post-translational covalent modifications that may alter their activity, interactions and fate. The set of modifications of each protein may be considered a “regulatory code”. Among the PTMs, arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), can affect how a protein interacts with other macromolecules such as nucleic acids or other proteins. In fact, many RNA-binding (RBPs) proteins are targets of PRMTs. The methylation status of RBPs may affect the expression of their bound RNAs and impact a diverse range of physiological and pathological cellular processes. Unlike most eukaryotes, Kinetoplastids have overwhelmingly intronless genes that are arranged within polycistronic units from which mature mRNAs are generated by trans-splicing. Gene expression in these organisms is thus highly dependent on post-transcriptional control, and therefore on the action of RBPs. These genetic features make trypanosomatids excellent models for the study of post-transcriptional regulation of gene expression. The roles of PRMTs in controlling the activity of RBPs in pathogenic kinetoplastids have now been studied for close to 2 decades with important advances achieved in recent years. These include the finding that about 10% of the Trypanosoma brucei proteome carries arginine methylation and that arginine methylation controls Leishmania:host interaction. Herein, we review how trypanosomatid PRMTs regulate the activity of RBPs, including by modulating interactions with RNA and/or protein complex formation, and discuss how this impacts cellular and biological processes. We further highlight unique structural features of trypanosomatid PRMTs and how it contributes to their singular functionality.

Evolutionary Perspective and Expression Analysis of Intronless Genes Highlight the Conservation on Their Regulatory Role

Eukaryotic gene structure is a combination of exons generally interrupted by intragenic non-coding DNA regions termed introns removed by RNA splicing to generate the mature mRNA. Thus, eukaryotic genes can be either single exon genes (SEGs) or multiple exon genes (MEGs). Among SEGs, intronless genes (IGs) are a subgroup that additionally lacks introns at their UTRs, and code for proteins essentially involved in development, growth, and cell proliferation. Gene expression of IGs has been proposed to be highly specialized for neuro-specific functions and linked to cancer, neuropathies, and developmental disorders. The abundant presence of introns in eukaryotic genomes is pivotal for the precise control of gene expression. Notwithstanding, IGs exempting splicing events entail a higher transcriptional fidelity, making them even more valuable for regulatory roles. This work aimed to infer the functional role and evolutionary history of IGs using the mouse genome. Intronless protein-coding genes consist of a subgroup of ~6 % of a total of 21,527 genes with one exon. To understand the prevalence, biological relevance, and evolution, we identified and studied their 1,116 functional proteins. We validated differential expression in transcriptomics data of early embryo stages using mouse telencephalon tissue. Our results showed that expression levels of IGs are lower compared to MEGs. However, strongly upregulated IGs include transcription factors (TFs) such as the class 3 of POU (HMG Box), Neurog1, Olig1, and BHLHe22, BHLHe23, among other essential genes including the beta cluster of protocadherins. Most striking was the finding that IG-encoded BHLH TFs qualify the criteria to be referred to as microprotein candidates. Finally, predicted protein orthologs in other six genomes confirmed a high conservancy of IGs associated with regulating neurobiological processes and with chromatin organization and epigenetic regulation in Vertebrata. Moreover, this study highlights that IGs are essential modulators of regulatory processes, as Wnt signaling pathway and biological processes as pivotal as sensory organs developing at a transcriptional and post-translational level. Overall, our results suggest that IG proteins have specialized, prevalent, and unique biological roles and that functional divergence between IGs and MEGs is likely to be the result of specific evolutionary constraints.

IWS1 phosphorylation by AKT3 controls nuclear export of type I IFN mRNAs and sensitivity to oncolytic viral infection, by regulating the alternative RNA splicing of U2AF2

Type I IFNs orchestrate the antiviral response. Interestingly, IFNA1 and IFNB1 genes are naturally intronless. Based on previous work, the splicing factor U2 Associated Factor 65 (U2AF65), encoded by U2AF2, and pre-mRNA Processing factor 19 (Prp19) function on the Cytoplasmic Accumulation Region Elements (CAR-E), affecting the nuclear export of intronless genes. We have previously shown that the loss of IWS1 phosphorylation by AKT3, promotes the alternative RNA splicing of U2AF2, resulting in novel transcripts lacking exon 2. This exon encodes part of the Serine-Rich (RS) domain of U2AF65, which is responsible for its binding with Prp19. Here, we show that IWS1 phosphorylation and the U2AF2 RNA splicing pattern affect the nuclear export of introless mRNAs. We also demonstrate that the same axis is required for the proper function of the CAR-Es. Mechanistically, whereas both U2AF65 isoforms bind CAR-E, the recruitment of Prp19 occurs only in cells expressing phosphorylated IWS, promoting intronless genes export. Moreover, analysis of Lung adenocarcinoma patients showed that high p-IWS1 activity correlates with the assembly of the U2AF65/Prp19 complex and export of intronless genes, in vivo. Accordingly, the expression of type I IFNs was decreased in cells deficient in IWS1 phosphorylation and the viral infection was increased. Furthermore, following infection with oncolytic virus, we observed reduced activation of p-STAT1 and expression of Interferon Stimulated Genes (ISG), in cells stimulated by shIWS1-derived supernatant, or cells treated with the pan-AKT inhibitor, MK2206. Consistently, killing curves and apoptosis assays after infection with oncolytic viruses, revealed increased susceptibility upon the loss of IWS1, with subsequent activation of Caspase-mediated death. The treatment of the lung adenocarcinoma cells with MK2206, phenocopied the loss of IWS1 phosphorylation. These data identify a novel mechanism by which the AKT/p-IWS1 axis, by hijacking the epigenetic regulation of RNA splicing and processing, contributes to the resistance to oncolytic viral infection, suggesting that combined inhibition of the splicing machinery and AKT/p-IWS1 signals would sensitize tumors to oncolytic viral treatment.