An Ultraconserved Element (UCE) controls homeostatic splicing of ARGLU1 mRNA

Abstract

Arginine and Glutamate-Rich protein 1 (ARGLU1) is a protein whose function is poorly understood, but may act in both transcription and pre-mRNA splicing. We demonstrate that the ARGLU1 gene expresses at least three distinct RNA splice isoforms – a fully spliced isoform coding for the protein, an isoform containing a retained intron that is detained in the nucleus, and an isoform containing an alternative exon that targets the transcript for nonsense mediated decay. Furthermore, ARGLU1 contains a long, highly evolutionarily conserved sequence known as an Ultraconserved Element (UCE) that is within the retained intron and overlaps the alternative exon. Manipulation of the UCE, in a reporter minigene or via random mutations in the genomic context using CRISPR/Cas9, changed the splicing pattern. Further, overexpression of the ARGLU1 protein shifted the splicing of endogenous ARGLU1 mRNA, resulting in an increase in the retained intron isoform and nonsense mediated decay susceptible isoform and a decrease in the fully spliced isoform. Taken together with data showing that functional protein knockout shifts splicing toward the fully spliced isoform, our data are consistent with a model in which unproductive splicing complexes assembled at the alternative exon lead to inefficient splicing and intron retention.

Ultraconserved Elements Occupy Specific Arenas of Three-Dimensional Mammalian Genome Organization

“…We next used the Genomic Regions Enrichment of Annotations Tool (GREAT) (McLean et al, 2010) and discovered that exonic UCEs in pooled boundaries and pooled loop anchors are enriched for gene ontology (GO) terms associated with RNA processing (Figures S2A and S2B), and this is in line with previous reports that exonic UCEs are associated with RNA processing, including splicing (Baira et al, 2008; Bejerano et al, 2004; Lareau and Brenner, 2015; Lareau et al, 2007; Lupiáñez et al, 2016; Ni et al, 2007; Pirnie et al, 2017; Rödelsperger et al, 2009). Considering further the structure of exonic UCEs themselves, we found that 76% (16 out of 21; Table S5A) and 82% (43 out of 52; Table S5B) of exonic UCEs in pooled boundaries and loop anchors, respectively, partially overlap in-trons and hence cover splice sites, as compared to 57% in the full set of exonic UCEs (107 out of 185; Table S2E).…”

Ultraconserved elements occupy specific arenas of three-dimensional mammalian genome organization

“…We next used the Genomic Regions Enrichment of Annotations Tool (GREAT) (47) and discovered that exonic UCEs in Pooled boundaries and Pooled loop anchors are enriched for GO terms associated with RNA processing ( Supplementary Figures S3A and S3B), and this is in line with previous reports that exonic UCEs are associated with RNA processing, including splicing (1,12,14,51,(97)(98)(99)(100). Considering further the structure of exonic UCEs, themselves, we found that 76% (16 out of 21; Supplementary Table S6A) and 82% (43 out of 52; Supplementary Table S6B) of exonic UCEs in Pooled boundaries and loop anchors, respectively, partially overlap introns and hence cover splice sites, as compared to 57% in the full set of exonic UCEs (107 out of 185; Supplementary Table 3C).…”

Antisense targeting of decoy exons can reduce intron retention and increase protein expression in human erythroblasts

“…This design was expected to maximize the likelihood of blocking spliceosome assembly at the decoy. However, it eliminated from consideration strong decoys in ARGLU1 and DDX39B that possess alternative splice sites distributed over a wider range (Parra M. et al 2018;Pirnie et al 2017). Finally, we targeted 5' splice sites, because the relatively low GC content at typical 3' splice sites was predicted to reduce MO affinity and effectiveness.…”