BackgroundThe 3′ splice site (SS) at the end of pre-mRNA introns has a consensus sequence (Y)nNYAG for constitutive splicing of mammalian genes. Deviation from this consensus could change or interrupt the usage of the splice site leading to alternative or aberrant splicing, which could affect normal cell function or even the development of diseases. We have shown that the position “N” can be replaced by a CA-rich RNA element called CaRRE1 to regulate the alternative splicing of a group of genes.ResultsTaking it a step further, we searched the human genome for purine-rich elements between the -3 and -10 positions of the 3′ splice sites of annotated introns. This identified several thousand such 3′SS; more than a thousand of them contain at least one copy of G tract. These sites deviate significantly from the consensus of constitutive splice sites and are highly associated with alterative splicing events, particularly alternative 3′ splice and intron retention. We show by mutagenesis analysis and RNA interference that the G tracts are splicing silencers and a group of the associated exons are controlled by the G tract binding proteins hnRNP H/F. Species comparison of a group of the 3′SS among vertebrates suggests that most (~87%) of the G tracts emerged in ancestors of mammals during evolution. Moreover, the host genes are most significantly associated with cancer.ConclusionWe call these elements together with CaRRE1 regulatory RNA elements between the Py and 3′AG (REPA). The emergence of REPA in this highly constrained region indicates that this location has been remarkably permissive for the emergence of de novo regulatory RNA elements, even purine-rich motifs, in a large group of mammalian genes during evolution. This evolutionary change controls alternative splicing, likely to diversify proteomes for particular cellular functions.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1143) contains supplementary material, which is available to authorized users.

N-Acetylglucosamine β-O-linked to nucleocytoplasmic proteins (O-GlcNAc) is implicated in the regulation of gene expression in organisms, from humans to Drosophila melanogaster. Within Drosophila, O-GlcNAc transferase (OGT) is one of the Polycomb group proteins (PcGs) that act through Polycomb group response elements (PREs) to silence homeotic (HOX) and other PcG target genes. Using Drosophila, we identify new O-GlcNAcylated PcG proteins and develop an antibody-free metabolic feeding approach to chemoselectively map genomic loci enriched in O-GlcNAc using next-generation sequencing. We find that O-GlcNAc is distributed to specific genomic loci both in cells and in vivo. Many of these loci overlap with PREs, but O-GlcNAc is also present at other loci lacking PREs. Loss of OGT leads to altered gene expression not only at loci containing PREs but also at loci lacking PREs, including several heterochromatic genes. These data suggest that O-GlcNAc acts through multiple mechanisms to regulate gene expression in Drosophila.

Advances in DNA sequencing are enabling new experimental modalities for studying chromatin. One emerging area is to use high-throughput DNA sequencing to monitor dynamic changes occurring to chromatin. O -Linked N -acetylglucosamine ( O -GlcNAc) is a reversible protein modification found on many chromatin-associated proteins. The mechanisms by which O -GlcNAc regulates gene transcription are of high interest. Here we use DNA precipitation methods to enable monitoring time-dependent turnover of O -GlcNAc modified proteins associated with chromatin. Using an antibody-free chemical reporter strategy to map O -GlcNAc to the genome, we performed time course metabolic feeding experiments with wild-type Drosophila larvae alongside larvae lacking O -GlcNAc hydrolase (OGA), which are accordingly unable to remove O -GlcNAc. Analysis of resulting next-generation DNA sequencing data revealed that O -GlcNAc on chromatin-associated proteins at most genomic loci is processed with a half-life in hours. Notably, loss of OGA only increases this half-life by ∼3-fold. Interestingly, a small set of genomic loci are particularly sensitive to loss of OGA. In addition to these observations and new strategies to permit monitoring turnover of O -GlcNAc on chromatin, we also detail methods for coded blinding of samples alongside new normalization strategies to enable time-resolved, genome-wide analyses using chemical genetic methods. We envision these general methods will be applicable to diverse protein and nucleic acid modifications.

Background: Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) and associated methods are widely used to define the genome wide distribution of chromatin associated proteins, post-translational epigenetic marks, and modifications found on DNA bases. An area of emerging interest is to study time dependent changes in the distribution of such proteins and marks by using serial ChIP-seq experiments performed in a time resolved manner. Despite such time resolved studies becoming increasingly common, software to facilitate analysis of such data in a robust automated manner is limited.