Co-transcriptional regulation of alternative pre-mRNA splicing

Abstract

While studies of alternative pre-mRNA splicing regulation have typically focused on RNA-binding proteins and their target sequences within nascent message, it is becoming increasingly evident that mRNA splicing, RNA polymerase II (pol II) elongation and chromatin structure are intricately intertwined. The majority of introns in higher eukaryotes are excised prior to transcript release in a manner that is dependent on transcription through pol II. As a result of co-transcriptional splicing, variations in pol II elongation influence alternative splicing patterns, wherein a slower elongation rate is associated with increased inclusion of alternative exons within mature mRNA. Physiological barriers to pol II elongation, such as repressive chromatin structure, can thereby similarly impact splicing decisions. Surprisingly, pre-mRNA splicing can reciprocally influence pol II elongation and chromatin structure. Here, we highlight recent advances in co-transcriptional splicing that reveal an extensive network of coupling between splicing, transcription and chromatin remodeling complexes. This article is part of a Special Issue entitled: Chromatin in time and space.

Figure 1. Nucleosomes are co-transcriptionally remodeled

Nucleosomal arrays pose a barrier to pol II elongation. Phosphorylation of serine 2 of pol II CTD recruits the histone chaperone, FACT. FACT destabilizes nucleosomes in front of pol II through removal of one H2A–H2B dimer. FACT also prevents cryptic transcription by redepositing histones in pol II’s wake, facilitating nucleosome reassembly.

Figure 2. Potential mechanisms for chromatin-associated spliceosome assembly

Exons are marked by increased nucleosome occupancy, distinct histone modifications and elevated DNA methylation relative to introns. These modifications at the DNA level may influence splice site selection by a) modulating elongation or b) through direct recruitment of auxiliary factors. a) A slow rate of pol II elongation favors spliceosome assembly at weak exons, whereas a rapid rate may not provide a sufficient spatiotemporal window prior to synthesis of competing downstream splice sites. Intragenic chromatin structure may act to locally modulate elongation rate. As shown here, the upstream exon is excluded from mRNA as a result of a locally rapid pol II elongation rate, thereby shifting spliceosome assembly to the downstream exon. b) Chromatin modifications may recruit chromatin binding proteins (CBP) to exonic DNA, which thereby act as adaptor molecules for RNA binding proteins (RBP) that promote or inhibit spliceosome assembly. As shown here, the chromatin context of the upstream exon binds a CBP that recruits an RNA binding splicing repressor, thereby shifting spliceosome assembly to the downstream exon.

Figure 3. Intragenic CTCF and 5-methylcytosine reciprocally influence exon inclusion in spliced mRNA

Intragenic assembly of DNA binding proteins can influence co-transcriptional pre-mRNA splicing. a) The zinc-finger DNA binding protein CTCF acts as a direct barrier to pol II elongation, resulting in pol II pausing and spliceosome assembly at weak upstream splice sites. b) DNA methylation inhibits CTCF binding and associated pol II pausing, culminating in reduced spliceosome assembly at weak upstream splice sites and exclusion of exons from spliced mRNA.