Splicing Enhancers at Intron–Exon Borders Participate in Acceptor Splice Sites Recognition

Abstract

Acceptor splice site recognition (3′ splice site: 3′ss) is a fundamental step in precursor messenger RNA (pre-mRNA) splicing. Generally, the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF) heterodimer recognizes the 3′ss, of which U2AF35 has a dual function: (i) It binds to the intron–exon border of some 3′ss and (ii) mediates enhancer-binding splicing activators’ interactions with the spliceosome. Alternative mechanisms for 3′ss recognition have been suggested, yet they are still not thoroughly understood. Here, we analyzed 3′ss recognition where the intron–exon border is bound by a ubiquitous splicing regulator SRSF1. Using the minigene analysis of two model exons and their mutants, BRCA2 exon 12 and VARS2 exon 17, we showed that the exon inclusion correlated much better with the predicted SRSF1 affinity than 3′ss quality, which were assessed using the Catalog of Inferred Sequence Binding Preferences of RNA binding proteins (CISBP-RNA) database and maximum entropy algorithm (MaxEnt) predictor and the U2AF35 consensus matrix, respectively. RNA affinity purification proved SRSF1 binding to the model 3′ss. On the other hand, knockdown experiments revealed that U2AF35 also plays a role in these exons’ inclusion. Most probably, both factors stochastically bind the 3′ss, supporting exon recognition, more apparently in VARS2 exon 17. Identifying splicing activators as 3′ss recognition factors is crucial for both a basic understanding of splicing regulation and human genetic diagnostics when assessing variants’ effects on splicing.

LncRNA Pnky Positively Regulates Neural Stem Cell Migration via Modulating mRNA Splicing and Export of Target Genes

“…Spliceosome is identi ed to be composed of at least ve small nuclear ribonucleoprotein particles (snRNPs, U1, U2, U4/U6, and U5) that recognize intronic splice sites by base pairing, organize the assembly of protein splicing factors, and catalyze the cleavage and ligation reactions (Shi et al 2017b; van der Feltz and Hoskins 2019). U2AF1 is the small subunit of the U2 auxiliary factor (U2AF) that constitutes the U2 snRNP which is primarily responsible for 3' splice site selection during splicing (Kováčová et al 2020). Accumulating evidence showed that U2AF1 mutation caused altered pre-mRNA binding and splicing kinetics in a variety of cell types, such as hematological ).…”

The Many Roads from Alternative Splicing to Cancer: Molecular Mechanisms Involving Driver Genes

“…The cis elements are intronic splicing enhancers (ISEs), exonic splicing enhancers (ESEs), intronic splicing silencers (ISSs), and exonic splicing silencers (ESSs), according to their location and function ( Figure 2 ). The interplay between splice sites and cis regulatory elements results in a complex net of interactions [ 15 ]. The organisation of chromatin and its epigenetic modifications [ 16 , 17 ] and the phosphorylation of the C-terminal domain of RNA pol II [ 18 ] also have an influence on the regulation of spliceosome assembly.…”