Lexus Dickson
Dr. Ely
BIOL303
20 October 2014
Genotypic and Phenotypic Effects Caused by Alterations in Genes Associated with the Exon
Junction Complex and Nonsense Mediated Decay Pathways
Over the past twenty years, genetics researchers have made multiple discoveries concerning the processing of RNA and its role in the cell. RNA processing consists of the biochemical pathways and systems involved in the manipulation of RNA following transcription and preceding translation, thus making the process significant in determining the expression of genes (Haremaki et al., 2010). A group of proteins called the Exon Junction Complex (EJC) is one of the major signaling loci for several methods of RNA processing in eukaryotes (Moore and
Proudfoot, 2009). The EJC facilitates a number of pathways including translational regulation, mRNA localization within the cell, and nonsense-mediated decay (NMD) (Tange et al., 2004).
Geneticists have recently examined the effects of the EJC and its pathways in more detail, especially involving the effects of EJC and NMD protein deficiencies and profusions in vertebrate animals, including humans. Their findings provide insight into novel functions of the
EJC and its pathways, and reveal how mutations in the gene sequences coding for EJC proteins can result in phenotypic abnormalities during growth.
The EJC is made up of several “core” proteins and a series of accessory proteins (Figure
1). These “core” proteins include EIF4A3, Y14, Magoh, and MLN51 (Le Hir et al., 2000).
EIF4A3, otherwise known as eukaryotic translation initiation factor 4a3, comes into direct

contact with RNA while the EJC is assisting in RNA processing pathways such as translational regulation and NMD (Shibuya et al., 2004; Ballut et al., 2005). The EJC attaches to RNA 20-25 nucleotides upstream of exon-exon splice junctions (Le Hir et al., 2000). After attaching, the EJC remains attached to RNA as it is shuttled out of the nucleus of the cell and aids it in localization, or the organization of the RNA, within the cytoplasm (Ballut et al., 2005). The EJC can also participate in translational regulation or function within NMD. NMD is a system which degrades mRNAs with nonsense mutations. EJC functions within this system by attaching to the mRNA’s stop codon and signaling its degradation, preventing a buildup of truncated proteins within the body (Chang et al., 2007). UPF2 and UPF3 both participate in NMD, helping to detect nonsense
Figure 1 . A diagram representing the exon junction complex, its core and accessory proteins, and the different functions of each. The top half of the diagram also shows the attachment of the EJC to mRNA 20-24 nucleotides before an exon-exon splice junction. mutations within a segment of mRNA and initiate NMD (Tange et al., 2004).

A study performed by Favaro et al. (2014) studied the origins of Richieri-Costa-Pereira
Syndrome (RCPS), a homozygous recessive craniofacial disorder that can also be associated with limb defects and learning and language disabilities. The researchers used homozygosity mapping analysis, a method used to identify recessive copies of disease alleles and their markers through comparison with individuals of a family, to find a unique extended region of homozygosity at 17q25.3 in affected individuals. LOD Scores, a statistical estimate of the odds that two genes are linked, were then taken of the region and were found to be significant, suggesting that the two genes were linked. Segregation analysis with the microsatellite markers found at 17q25.3 further confirmed the linkage, and they were able to refine the locus to a 122 kb region containing the genes CCdC40, GAA, EIF4A3, and CARD14. By comparing and analyzing the symptoms of various known mutations in these genes, researchers determined that
EIF4A3 was the most likely candidate of a mutation resulting in RCPS.
PCR amplification of exon 1 of EIF4A3 revealed that the exon had a larger homozygous allele only among affected individuals (Favaro et al. 2014). Both affected and control individuals tested had similar motifs containing 18 to 20 nucleotides in 3 different types: CACA-20-nt, CA-
18-nt, and CGCA-20-nt, each named after the type of variation in the sequence and the number of nucleotides. However, the affected individuals had a different arrangement of these motifs that led them to have a total of around 15 to 16 motif repeats upstream of the first methionine, which was remarkably larger than the 5 to 12 repeats noted in control individuals (Figure 2). Seventeen affected individuals were homozygous for the 16 repeat allele and 3 others were compound heterozygotes, meaning they had two different recessive alleles for the gene. All tested parents of

the affected individuals were heterozygous for the 16-repeat allele and all unaffected siblings lacked the allele entirely or were heterozygous.
Figure 2.
Diagram representing the different extensions of the EIF4A3 gene within control and affected individuals. The graph on the bottom represents the average number of repeats found on each allele, with blue representing the controls and red representing the affected individuals.
RT-qPCR of EIF4A3 transcripts using mRNA from white blood cells and mesenchymal cells showed that EIF4A3 transcript abundance was about 30%-40% lower in the affected groups than the control groups within both cells. Favaro et al. (2014) thus concluded that the expanded allele in affected individuals reduces the abundance of EIF4A3 mRNA in the RCPS cells tested, but does not affect splicing. They also hypothesized that the reduced RNA levels were the cause of RCPS syndrome. In order to test this hypothesis, researchers then performed EIF4A3 knockdowns using morpholinos to block either EIF4A3 mRNA translation (TRA1-MO and
TRA2-MO) or pre-mRNA splicing (Spl-MO) in zebrafish. The morphants showed phenotypic

similarities to RCPS affected individuals (Figure 3). When mRNA coding for zebrafish EIF4A3
Figure 3. Staining showing the craniofacial abnormalities within zebrafish embryos injected with the morpholino (Tra1-
MO), injected with a placebo version of the morpholino (Tra1-MO-Mis), and those coinjected with the morpholino and
EIF4A3 mRNA. Note different in craniofacial structures on the right, and the left shows eyes reduced in size and dark opaque zones in all brain structures (a sign of apoptosis). was coinjected with each of the three MOs, the abnormalities were rescued, demonstrating that
EIF4A3 knockdown was the cause of the phenotypic changes.

Due to the changes in craniofacial cartilage and bone deformations in the morpholinos,
Favaro et al. (2014) performed an RT-qPCR analysis on RNA extracted from the cranial neural crest (CNC), where the structures are developed. The results showed that the loss of EIF4A3 affected the transcription of neural crest gene markers. These data indicate that EIF4A3 depletion could lead to a failure in the EJC assembly, leading to CNC cell death and underdevelopment of pharyngeal arches, the tissues in vertebrate embryos that lead to organ development. Thus,
Favaro et al. (2014) were able to conclude that a knockdown or reduction of EIF4A3 could reduce EJC assembly by association and result in craniofacial deformities due to the connection of EIF4A3 with cranial neural crest markers. As for the limb deformities and language and learning impediments also accompanying RCPS, they concluded that more research needed to be performed, as they did not look into the possible causes of these phenotypes.
Haremaki et al. (2010) had performed an earlier study involving EIF4A3 and other EJC proteins with the African clawed toad, Xenopus laevis . They used morpholinos to knockdown

EIF4A3 in Xenopus embryos, which led to defects in pigment forming cells, paralysis, lack of response to touch stimuli (Figure 4), and heart defects. These phenotypes were all rescued, partially or fully, when coinjected with both the morpholino and EIF4A3 mRNA without the morpholino binding site. Knockdown of other core EJC proteins (Y14 and MAGOH) resulted in an almost identical phenotype and were also rescued with co-expression of their RNA, suggesting to researchers that EIF4A3’s function in the EJC is what makes it critical for proper embryonic development, not simply the presence of EIF4A3.
Figure 4. Phenotypes of morphant (4a3MO), nonknockdown morphants (5MM), and rescue morphants. A shows the change in pigmentation, B shows the touchresponsiveness of the embryos based on the amount of MO and RNA given, and C shows heart formation in each.
Haremaki et al. (2010) found that paralysis could be due to a number of factors, including defects in skeletal muscle or neuron development. Islet-1, a cardiac marker capable of differentiating into several different cell types including smooth muscle and cardiac tissue, was found to occupy a wider zone in the back and stomach areas than seen in controls and also had a weaker signal as well. A marker for Rohon-Beard neurons, specialized neurons that respond to mechanical stimuli, and the cranial neural crest was also reduced in intensity. From these data, the researchers concluded that a lack of touch response may be a result of EIF4A3 effecting

Rohon-Beard neurons, although they also believe that it is secondary to the complete paralysis, suggesting that the improper bundling and decreased Islet-1 signal probably causes the paralysis phenotype.
To further investigate the effect of EIF4A3 knockdown on embryonic development, researchers looked at the cranial neural crest, which originated from the same cells as Rohon-
Beard cells (Artinger et al., 1999; Cornell and Eisen, 2000; Cornell and Eisen 2002). They examined the expression and localization of neural crest markers, but found them all to be normal. Unlike the Favaro et al. (2014) study, the normal development of the CNC markers suggests that EIF4A3 was not required for differentiation or migration of the CNC nor for all of the tissues it developed. However, Haremaki et al. found that the expression of tyrosinase (an enzyme that aids melanophores in melanin production to create pigmentation) and tyrosinaserelated-protein-2 are both reduced in morphants that lack pigmentation. The restoration of this expression with co-expression of EIF4A3 RNA, suggests that EIF4A3 does play a part in at least some development in melanophores, which are a type of neural crest derivative (Kumosaka et al., 2003).
The inconsistencies in the findings between Favaro et al. (2014) and Haremaki et al.
(2010) on the phenotypic developments of different neural crest markers may be due to the fact that the role of NMD and the EJC in different animals varies greatly, especially in consideration to neural development (Nguyen et al., 2013). Fruit flies with deleted NMD genes were unable to completely form neuromuscular junction synapses, had reduced neurotransmission responses, and had reduced synaptic vesicle cycling, resulting in difficulty sending neurological signals in
Long et al.’s study (2010). When EIF4A3 was depleted in rats, synaptic strength of the neurons increased (Giorgi et al., 2007). Thus, the role that EJC and NMD proteins play in various species

seems to differ enough to give rise to a variety of phenotypes, although there is an underlying trend of EJC and NMD protein participation in a variety of functions associated with embryonic development.
After collecting data on both Rohon-Beard and melanophore cells, Haremaki et al. (2010) theorized that reductions of both Rohon Beard cells and tyrosinase may be due to increased rates of apoptic death noted among these cell types. TUNEL assays performed on the cells found an increase in pro-apoptic elements within the head and eyes of morphant embryos, so they concluded that loss of pigmentation within the anterior region of the morphants may be due to apoptic cell death, although the data does not support the theory that apoptosis results in defects in the Rohon-Beard sensory neurons or in loss of pigmentation within the trunk of morphants.
They proposed that these phenotypes may result from inappropriate differentiation or migration of subpopulation of trunk neural crest. Increased apoptosis was a common phenotype of morphants in Favaro et al.’s study as well (2014).
In conclusion, the data from Haremaki et al. (2010) show that EIF4A3 is required for embryonic movement, pigment formation, and cardiac development in Xenopus laevis , due to its role as an EJC core. These findings suggest that EIF4A3 may play a role in the development of certain neural-crest derived cells in embryonic development, including Rohon-Beard sensory neurons and melanophores. EIF4A3 may also regulate cardiac development through actions on the neural crest, shown by heart defects, but some derivatives of the neural crest (such as cartilage from the CNC, which was studied in Favaro et al. 2014) may be entirely independent of
EIF4A3 and EJC regulation.
Nguyen et al. (2013) took a different direction in their studies by examining various forms of intellectual disabilities (ID) that result from the reduction of proteins associated with

NMD pathways. They chose to focus mainly on UPF3B and RBM8A, both accessory EJC proteins that function within NMD that have been associated with ID in previous studies, but they also looked at copy number variants of other EJC- and NMD-associated genes. Copy number variations (CNVs) are the number of copies of a specific gene that gained or lost, and which can also be connected with human disease. They found that CNV losses and gains were enriched within the individuals tested. These CNV losses and gains occurred in many different locations and genes, including UPF2, UPF3, RBM8A, and EIF4A3, which may explain the multiple phenotypes classified as ID. The abundance of these CNVs, and their presence in IDaffected individuals, suggested to Nguyen et al. (2013) that duplications and deletions of NMD and EJC factors could contribute to pathology. To further analyze the CNVs, They examined the effects of heterozygous deletions of UPF2, which had resulted in developmental delay, deformities, speech and language problems, and deafness within tested individuals. UPF2 was chosen because it is expressed in all tissues, including the brain. It acts as a bridge between
UPF3B and the EJC, and it is essential for NMD function and cell development (Witman et al.,
2006; Weischenfeldt et al. 2008; Chamieh et al., 2008; Anderson and Le Hir, 2008).

Nguyen et al. (2013) then used RNA-Seq on polyA+ RNA from the lymphoblast cell
Table 1.
A chart showing the genes tested and the copy number variations among them (gain/loss). P values are from
Fisher’s exact test, which determines if the association between two values is random or nonrandom. A P < 0.05 shows there is a significant association between the two variables (in this case, the UPF2 groups and the UPF3B groups).

lines (LCLs) of those with UPF2 deletions, and they found 1009 differently expressed genes
(DEG) that were deregulated or upregulated by at least two-fold compared to the control. Also,
38% of these DEGs were also deregulated by two-fold in patients with UPF3B mutations assessed against the same controls, and 95% of the DEGs identified in either UPF2 or UPF3B patients had the same trend of deregulation. Using Pearson’s correlation efficiency tests, they found the gene expression changes between the two were correlated. When specific genes were examined to validate this correlation, they found that the genes tested were similarly upregulated in both patient groups, leading them to conclude that NMD was at least partially due to UPF2 deletions and that the phenotypic transcriptome outcomes in patients with UPF2 deletions and
UPF3B mutations are comparable—similar to how Haremaki et al. (2013) found the phenotypic outcomes of EJC core gene knockdowns to be nearly identical. They also identified the DEGs deregulated in both patient groups with known neuronal functions and found that the DEGs represent the most consistently deregulated genes when NMD is compromised.
Since Nguyen et al. (2013) found connections between ID and NMD factors within humans, similar analyses of CNVs will be needed with the EIF4A3 and RCPS genes to determine if they affect learning and language disabilities in affected individuals. Nguyen et al.
(2013) were able to identify CNVs throughout five NMD factors associated with neurodevelopmental disorders, which could possibly contribute to UPF3A neural tube defects, SMG6 and RNPS1 duplication syndromes, and pathogenic regions involving UPF2 and EIF4A3. They also found that UPF2 deletions had similar effects on the transcriptome as found in other ID patients with different genotypes, such as those with a loss of UPF3B function, and slightly different phenotypes. These similarities are most likely due to their common molecular pathway,
NMD, being deregulated. However, when NMD studies on humans are compared with UPF2

knockout in mouse tissues, the complexes targeted by NMD are not the same in each group, showing that NMD functions differently across species. This complexity of the NMD possibly provides some explanation for the differences in phenotypes between Favaro et al. (2014) and
Haremaki et al. (2010) when the same gene was deregulated in different species. CNV gains of
UPF2, SMG6, RBM8A, EIF4A3 and RNSP1—proteins either associated with the EJC or
NMD—were also significantly enriched in ID patients, suggesting that NMD function may be key to normal neuronal development and function, although this hypothesis requires further investigation. Finally, the entirety of Nguyen et al.’s data demonstrates that NMD efficiency can be influenced by copy numbers and point mutations in NMD and EJC genes, causing different specific phenotypic outcomes, but all relating back to the overarching ID spectrum.
The studies done by Favaro et al. (2014), Haremaki et al. (2010), and Nguyen et al.
(2013) all demonstrate the different phenotypic and genotypic effects of deregulation of EJC and
NMD associated genes. Both Favaro et al. (2014) and Nguyen et al. (2013) connected known pathological phenotypes with their respective genetic causations, providing deeper insight into the role of the EJC and NMD pathway within humans and the effects of its deregulation.
Haremaki et al. (2010) looked at the role of the EJC and NMD pathway, albeit in frogs, through a similar deregulation of EIF4A3. Therefore, each of these studies provides insight into the novel roles of the EJC RNA processing within different vertebrate species, showing the variety of systems that the EJC influences and providing a backbone for further research on the phenotypic abnormalities investigated in each case.

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