Gene architecture directs splicing outcome in separate nuclear spatial regions.

How the splicing machinery defines exons or introns as the spliced unit has remained a puzzle for 30 years. Here, we demonstrate that peripheral and central regions of the nucleus harbor genes with two distinct exon-intron GC content architectures that differ in the splicing outcome. Genes with low GC content exons, flanked by long introns with lower GC content, are localized in the periphery, and the exons are defined as the spliced unit. Alternative splicing of these genes results in exon skipping. In contrast, the nuclear center contains genes with a high GC content in the exons and short flanking introns. Most splicing of these genes occurs via intron definition, and aberrant splicing leads to intron retention. We demonstrate that the nuclear periphery and center generate different environments for the regulation of alternative splicing and that two sets of splicing factors form discrete regulatory subnetworks for the two gene architectures. Our study connects 3D genome organization and splicing, thus demonstrating that exon and intron definition modes of splicing occur in different nuclear regions.

The importance of DNA methylation of exons on alternative splicing.

Alternative splicing (AS) contributes to proteome diversity. As splicing occurs cotranscriptionally, epigenetic determinants such as DNA methylation likely play a part in regulation of AS. Previously, we have shown that DNA methylation marks exons and that a loss of DNA methylation alters splicing patterns in a genome-wide manner. To investigate the influence of DNA methylation on splicing of individual genes, we developed a method to manipulate DNA methylation in vivo in a site-specific manner using the deactivated endonuclease Cas9 fused to enzymes that methylate or demethylate DNA. We used this system to directly change the DNA methylation pattern of selected exons and introns. We demonstrated that changes in the methylation pattern of alternatively spliced exons, but not constitutively spliced exons or introns, altered inclusion levels. This is the first direct demonstration that DNA methylation of exon-encoding regions is directly involved in regulation of AS.

The upstream 5' splice site remains associated to the transcription machinery during intron synthesis.

In the earliest step of spliceosome assembly, the two splice sites flanking an intron are brought into proximity by U1 snRNP and U2AF along with other proteins. The mechanism that facilitates this intron looping is poorly understood. Using a CRISPR interference-based approach to halt RNA polymerase II transcription in the middle of introns in human cells, we discovered that the nascent 5' splice site base pairs with a U1 snRNA that is tethered to RNA polymerase II during intron synthesis. This association functionally corresponds with splicing outcome, involves bona fide 5' splice sites and cryptic intronic sites, and occurs transcriptome-wide. Overall, our findings reveal that the upstream 5' splice sites remain attached to the transcriptional machinery during intron synthesis and are thus brought into proximity of the 3' splice sites; potentially mediating the rapid splicing of long introns.

DNA methylation directs microRNA biogenesis in mammalian cells.

MicroRNA (miRNA) biogenesis initiates co-transcriptionally, but how the Microprocessor machinery pinpoints the locations of short precursor miRNA sequences within long flanking regions of the transcript is not known. Here we show that miRNA biogenesis depends on DNA methylation. When the regions flanking the miRNA coding sequence are highly methylated, the miRNAs are more highly expressed, have greater sequence conservation, and are more likely to drive cancer-related phenotypes than miRNAs encoded by unmethylated loci. We show that the removal of DNA methylation from miRNA loci leads to their downregulation. Further, we found that MeCP2 binding to methylated miRNA loci halts RNA polymerase II elongation, leading to enhanced processing of the primary miRNA by Drosha. Taken together, our data reveal that DNA methylation directly affects miRNA biogenesis.

Calpain 12 Function Revealed through the Study of an Atypical Case of Autosomal Recessive Congenital Ichthyosis.

Congenital erythroderma is a rare and often life-threatening condition, which has been shown to result from mutations in several genes encoding important components of the epidermal differentiation program. Using whole exome sequencing, we identified in a child with congenital exfoliative erythroderma, hypotrichosis, severe nail dystrophy and failure to thrive, two heterozygous mutations in ABCA12 (c.2956C>T, p.R986W; c.5778+2T>C, p. G1900Mfs*16), a gene known to be associated with two forms of ichthyosis, autosomal recessive congenital ichthyosis, and harlequin ichthyosis. Because the patient displayed an atypical phenotype, including severe hair and nail manifestations, we scrutinized the exome sequencing data for additional potentially deleterious genetic variations in genes of relevance to the cornification process. Two mutations were identified in CAPN12, encoding a member of the calpain proteases: a paternal missense mutation (c.1511C>A; p.P504Q) and a maternal deletion due to activation of a cryptic splice site in exon 9 of the gene (c.1090_1129del; p.Val364Lysfs*11). The calpain 12 protein was found to be expressed in both the epidermis and hair follicle of normal skin, but its expression was dramatically reduced in the patient's skin. The downregulation of capn12 expression in zebrafish was associated with abnormal epidermal morphogenesis. Small interfering RNA knockdown of CAPN12 in three-dimensional human skin models was associated with acanthosis, disorganized epidermal architecture, and downregulation of several differentiation markers, including filaggrin. Accordingly, filaggrin expression was almost absent in the patient skin. Using ex vivo live imaging, small interfering RNA knockdown of calpain 12 in skin from K14-H2B GFP mice led to significant hair follicle catagen transformation compared with controls. In summary, our results indicate that calpain 12 plays an essential role during epidermal ontogenesis and normal hair follicle cycling and that its absence may aggravate the clinical manifestations of ABCA12 mutations.

HP1 is involved in regulating the global impact of DNA methylation on alternative splicing.

The global impact of DNA methylation on alternative splicing is largely unknown. Using a genome-wide approach in wild-type and methylation-deficient embryonic stem cells, we found that DNA methylation can either enhance or silence exon recognition and affects the splicing of more than 20% of alternative exons. These exons are characterized by distinct genetic and epigenetic signatures. Alternative splicing regulation of a subset of these exons can be explained by heterochromatin protein 1 (HP1), which silences or enhances exon recognition in a position-dependent manner. We constructed an experimental system using site-specific targeting of a methylated/unmethylated gene and demonstrate a direct causal relationship between DNA methylation and alternative splicing. HP1 regulates this gene's alternative splicing in a methylation-dependent manner by recruiting splicing factors to its methylated form. Our results demonstrate DNA methylation's significant global influence on mRNA splicing and identify a specific mechanism of splicing regulation mediated by HP1.