The coding sequence of each human pre-mRNA is interrupted, on average, by 11 introns that must be spliced out for proper gene expression. Each intron contains three obligate signals: a 5' splice site, a branch site, and a 3' splice site. Splice site usage has been mapped exhaustively across different species, cell types, and cellular states. In contrast, only a small fraction of branch sites have been identified even once. The few reported annotations of branch site are imprecise as reverse transcriptase skips several nucleotides while traversing a 2-5 linkage. Here, we report large-scale mapping of the branchpoints from deep sequencing data in three different species and in the SF3B1 K700E oncogenic mutant background. We have developed a novel method whereby raw lariat reads are refined by U2snRNP/pre-mRNA base-pairing models to return the largest current data set of branchpoint sequences with quality metrics. This analysis discovers novel modes of U2snRNA:pre-mRNA base-pairing conserved in yeast and provides insight into the biogenesis of intron circles. Finally, matching branch site usage with isoform selection across the extensive panel of ENCODE RNA-seq data sets offers insight into the mechanisms by which branchpoint usage drives alternative splicing.

Research into the problem of splice site selection has followed a reductionist approach focused on how individual splice sites are recognized. Early applications of information theory uncovered an inconsistency. Human splice signals do not contain enough information to explain the observed fidelity of splicing. Here, we conclude that introns do not necessarily contain ‘missing’ information but rather may require definition from neighboring processing events. For example, there are known cases where an intronic mutation disrupts the splicing of not only the local intron but also adjacent introns. We present a genome-wide measurement of the order of splicing within human transcripts. The observed order of splicing cannot be explained by a simple kinetic model. Simulations reveal a bias toward a particular, transcript-specific order of intron removal in human genes. We validate an extreme class of intron that can only splice in a multi-intron context. Special categories of splicing such as exon circularization, first and last intron processing, alternative 5 and 3′ss usage and exon skipping are marked by distinct patterns of ordered intron removal. Excessive intronic length and silencer density tend to delay splicing. Shorter introns that contain enhancers splice early.

BackgroundDuring abiotic stress many genes that are important for growth and adaptation to stress are expressed at elevated levels. However, the mechanisms that keep the stress responsive genes from expressing under non stress conditions remain elusive. Recent genetic characterization of the co-repressor LEUNIG_HOMOLOG (LUH) and transcriptional adaptor proteins SEUSS-LIKE1 (SLK1) and SLK2 have been proposed to function redundantly in diverse developmental processes; however their function in the abiotic stress response is unknown. Moreover, the molecular functions of LUH, SLK1 and SLK2 remain obscure. Here, we show the molecular function of LUH, SLK1 and SLK2 and the role of this complex in the abiotic stress response.ResultsThe luh, slk1 and slk2 mutant plants shows enhanced tolerance to salt and osmotic stress conditions. SLK1 and SLK2 interact physically with the LUFS domain in LUH forming SLK1-LUH and SLK2-LUH co-repressor complexes to inhibit the transcription. LUH has repressor activity, whereas SLK1 and SLK2 function as adaptors to recruit LUH, which in turn recruits histone deacetylase to the target sequences to repress transcription. The stress response genes RD20, MYB2 and NAC019 are expressed at elevated levels in the luh, slk1 and slk2 mutant plants. Furthermore, these stress response genes are associated with decreased nucleosome density and increased acetylation levels at H3K9 and H3K14 in the luh, slk1 and slk2 mutant plants.ConclusionsOur results indicate that SLK1, SLK2 and LUH form a co-repressor complex. LUH represses by means of an epigenetic process involving histone modification to facilitate the condensation of chromatin thus preventing transcription at the target genes.