CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

Abstract

32The exon junction complex (EJC) is an essential constituent and regulator of spliced messenger 33 ribonucleoprotein particles (mRNPs) in metazoans. As a core component of the EJC, CASC3 was 34 described to be pivotal for EJC-dependent nuclear and cytoplasmic processes. However, recent 35 evidence suggests that CASC3 functions differently from other EJC core proteins. Here, we have 36 established human CASC3 knockout cell lines to elucidate the cellular role of CASC3. In the knockout 37 cells, overall EJC composition and EJC-dependent splicing are unchanged, whereas mRNA isoforms 38 targeted by nonsense-mediated decay (NMD) are upregulated on a transcriptome-wide scale. 39Mechanistically, recruiting CASC3 to reporter mRNAs by direct tethering or via binding to the EJC 40 stimulates mRNA decay and endonucleolytic cleavage at the termination codon. Building on existing 41 EJC-NMD models, we propose that CASC3 equips the EJC with the ability to communicate with the 42

Ribosome Recycling by ABCE1 Links Lysosomal Function and Iron Homeostasis to 3ʹ UTR-Directed Regulation and Nonsense-Mediated Decay

“…Thus, steady-state EJC association with these transcripts largely or entirely represents binding of this complex downstream of the termination codon. CASC3 binding sites are highly specific markers of the location of EJCs (Hauer et al, 2016), and CASC3 knockout results in a global increase in NMD targets (Gerbracht et al, 2019). Indeed, CASC3 was the most highly ranked hit in our screen (Table S1) and was also identified in other published NMD screens (Alexandrov et al, 2017;Baird et al, 2018).…”

“…The EGFP and EGFP constructs were then placed under the control of a strong constitutive promoter and integrated into the AAVS1 locus, a region of open chromatin that allows high-level transgene expression (Hockemeyer et al, 2009;Lamartina et al, 2000), in HCT116 cells, a stably diploid human cell line amenable to pooled CRISPR screening (Golden et al, 2017;Manjunath et al, 2019). As expected, EGFP cells exhibited approximately 10-fold lower GFP fluorescence compared with EGFP PTC-35 cells ( Figure 1C).…”

“…At least 2 3 10 5 sorted cells and 4 3 10 7 unsorted cells for each clone were used for genomic DNA isolation. Genomic DNA isolation, sgRNA amplification, sequencing, and data analysis Genomic DNA (gDNA) was isolated from sorted cells by phenol-chloroform extraction as described (Golden et al, 2017). gDNA was isolated from unsorted cells using the MasterPureTM Complete DNA Purification Kit (Lucigen).…”

“…We recently developed a highly effective genome-wide CRISPR screening approach that utilizes fluorescent reporters coupled with a single round of high-stringency sorting that allows recovery of essential genes as hits (Golden et al, 2017;Manjunath et al, 2019). Here we describe application of this strategy to identify components of the NMD pathway in mammalian cells.…”

A Day in the Life of the Exon Junction Complex

“…Later in their life, around the time of their export to the cytoplasm, EJCs undergo a compositional switch and exist mainly as CASC3-containing monomeric EJCs ( Figure 1 B,C and Figure 4 ). In the second publication, CASC3 knockout (KO) cells were generated and the effects of CASC3 deficiency on EJC assembly and function were analyzed [ 20 ]. In contrast to the essential function of CASC3 during the assembly of recombinant EJCs in vitro [ 16 ], the absence of CASC3 did not quantitatively or qualitatively affect the assembly of EJCs during splicing in living cells [ 20 ].…”