Splicing of long non-coding RNAs primarily depends on polypyrimidine tract and 5' splice-site sequences due to weak interactions with SR proteins.

Many nascent long non-coding RNAs (lncRNAs) undergo the same maturation steps as pre-mRNAs of protein-coding genes (PCGs), but they are often poorly spliced. To identify the underlying mechanisms for this phenomenon, we searched for putative splicing inhibitory sequences using the ncRNA-a2 as a model. Genome-wide analyses of intergenic lncRNAs (lincRNAs) revealed that lincRNA splicing efficiency positively correlates with 5'ss strength while no such correlation was identified for PCGs. In addition, efficiently spliced lincRNAs have higher thymidine content in the polypyrimidine tract (PPT) compared to efficiently spliced PCGs. Using model lincRNAs, we provide experimental evidence that strengthening the 5'ss and increasing the T content in PPT significantly enhances lincRNA splicing. We further showed that lincRNA exons contain less putative binding sites for SR proteins. To map binding of SR proteins to lincRNAs, we performed iCLIP with SRSF2, SRSF5 and SRSF6 and analyzed eCLIP data for SRSF1, SRSF7 and SRSF9. All examined SR proteins bind lincRNA exons to a much lower extent than expression-matched PCGs. We propose that lincRNAs lack the cooperative interaction network that enhances splicing, which renders their splicing outcome more dependent on the optimality of splice sites.

TALE-directed local modulation of H3K9 methylation shapes exon recognition.

In search for the function of local chromatin environment on pre-mRNA processing we established a new tool, which allows for the modification of chromatin using a targeted approach. Using Transcription Activator-Like Effector domains fused to histone modifying enzymes (TALE-HME), we show locally restricted alteration of histone methylation modulates the splicing of target exons. We provide evidence that a local increase in H3K9 di- and trimethylation promotes inclusion of the target alternative exon, while demethylation by JMJD2D leads to exon skipping. We further demonstrate that H3K9me3 is localized on internal exons genome-wide suggesting a general role in splicing. Consistently, targeting of the H3K9 demethylase to a weak constitutive exon reduced co-transcriptional splicing. Together our data show H3K9 methylation within the gene body is a factor influencing recognition of both constitutive and alternative exons.

Chromatin immunoprecipitation approaches to determine co-transcriptional nature of splicing.

Chromatin immunoprecipitation (ChIP) is a common method used to determine the position along DNA where an antigen is found. The method was initially devised for protein antigens that come in direct contact with genomic DNA, such as components of the transcriptional machinery and histones. However, ChIP can also be extended to antigens that bind RNA, as demonstrated by the specific localization of spliceosomal components to particular gene regions that correlate with when and where introns and exons are transcribed. The activities of any RNA binding protein can in principle be monitored using ChIP, and RNA dependency of binding can also be assessed through RNase treatment. Combined with qPCR or high-throughput sequencing, this method allows the detection of RNA bound proteins at individual genes or genome-wide. Here, we present a detailed protocol for "splicing factor ChIP" in tissue culture cells.

Dbf4 and Cdc7 proteins promote DNA replication through interactions with distinct Mcm2-7 protein subunits.

The essential cell cycle target of the Dbf4/Cdc7 kinase (DDK) is the Mcm2-7 helicase complex. Although Mcm4 has been identified as the critical DDK phosphorylation target for DNA replication, it is not well understood which of the six Mcm2-7 subunits actually mediate(s) docking of this kinase complex. We systematically examined the interaction between each Mcm2-7 subunit with Dbf4 and Cdc7 through two-hybrid and co-immunoprecipitation analyses. Strikingly different binding patterns were observed, as Dbf4 interacted most strongly with Mcm2, whereas Cdc7 displayed association with both Mcm4 and Mcm5. We identified an N-terminal Mcm2 region required for interaction with Dbf4. Cells expressing either an Mcm2 mutant lacking this docking domain (Mcm2ΔDDD) or an Mcm4 mutant lacking a previously identified DDK docking domain (Mcm4ΔDDD) displayed modest DNA replication and growth defects. In contrast, combining these two mutations resulted in synthetic lethality, suggesting that Mcm2 and Mcm4 play overlapping roles in the association of DDK with MCM rings at replication origins. Consistent with this model, growth inhibition could be induced in Mcm4ΔDDD cells through Mcm2 overexpression as a means of titrating the Dbf4-MCM ring interaction. This growth inhibition was exacerbated by exposing the cells to either hydroxyurea or methyl methanesulfonate, lending support for a DDK role in stabilizing or restarting replication forks under S phase checkpoint conditions. Finally, constitutive overexpression of each individual MCM subunit was examined, and genotoxic sensitivity was found to be specific to Mcm2 or Mcm4 overexpression, further pointing to the importance of the DDK-MCM ring interaction.

First exon length controls active chromatin signatures and transcription.

Here, we explore the role of splicing in transcription, employing both genome-wide analysis of human ChIP-seq data and experimental manipulation of exon-intron organization in transgenic cell lines. We show that the activating histone modifications H3K4me3 and H3K9ac map specifically to first exon-intron boundaries. This is surprising, because these marks help recruit general transcription factors (GTFs) to promoters. In genes with long first exons, promoter-proximal levels of H3K4me3 and H3K9ac are greatly reduced; consequently, GTFs and RNA polymerase II are low at transcription start sites (TSSs) and exhibit a second, promoter-distal peak from which transcription also initiates. In contrast, short first exons lead to increased H3K4me3 and H3K9ac at promoters, higher expression levels, accuracy in TSS usage, and a lower frequency of antisense transcription. Therefore, first exon length is predictive for gene activity. Finally, splicing inhibition and intron deletion reduce H3K4me3 levels and transcriptional output. Thus, gene architecture and splicing determines transcription quantity and quality as well as chromatin signatures.

Pause locally, splice globally.

Most eukaryotic protein-coding transcripts contain introns, which vary in number and position along the transcript body. Intron removal through pre-mRNA splicing is tightly linked to transcription by RNA polymerase II as it translocates along each gene. Here, we review recent evidence that transcription and splicing are functionally coupled. We focus on how RNA polymerase II elongation rates impact splicing through local regulation and transcriptional pausing within genes. Emerging concepts of how splicing-related changes in elongation might be achieved are highlighted. We place the interplay between transcription and splicing in the context of chromatin where nucleosome positioning influences elongation, and histone modifications participate directly in the recruitment of splicing regulators to nascent transcripts.

The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing.

RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3' processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end.