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EJC dependent regulation of splicing in human cells
In human cells, EJC components (eIF4A3, Y14, Acinus, SAP18 and RNPS1), rather than the assembled complex function as regulators of splicing of apoptosis regulator BCL‐X gene (also known as BCL21L1) pre‐mRNA (Michelle et al. 2012). Recently, transcriptome‐wide analysis from the lab revealed that the impact of EJC on alternative splicing is broader than previously expected (Wang et al. 2014). The reduction of EJC causes large numbers of splicing changes for variety of transcripts and for several of them the fully assembled EJC core is required for this effect. Interestingly, several constitutive exons were excluded in EJC depleted cells suggesting that EJCs contribute to the recognition of normal splicing events. The mechanism of splicing regulation in human cells can be different than that of Drosophila cells as most of the EJC dependent splicing changes are not dependent on ACINUS or SR proteins. Interestingly, RNA polymerase II (Pol II) transcription accelerates when the EJC proteins amount is reduced suggesting that some splicing changes can be attributed to the faster transcription rate. Variation of transcription elongation rates can change the time available for the recognition of competitive splice sites and thus can regulate alternative splicing (Kornblihtt et al. 2013). By slowing down the transcription elongation rate, EJC may allow more time for splicing factors to recognize alternative exons. How EJCs communicate with the transcription machinery is still a matter of investigation. However, we cannot exclude that EJCs in human also serve in some cases as direct splicing regulators as observed in drosophila. The mechanisms underlying the effect of EJC on specific splicing events may also rely on other direct or indirect factors, which are still unknown.
EJC enhances translation
The influence of splicing on protein synthesis is a phenomenon that has been observed in most organisms. Following the discovery of the EJC, which marks the spliced exon junctions, it was a matter of curiosity whether the increased expression of transcripts was related to splicing per se or to the presence of an EJC. Early studies showed that splicing can increase the expression of intron‐containing genes compared to their intron‐less counter parts, both at the mRNA and protein levels (Nott et al. 2003; Callis et al. 1987).
Then, studies of different reporters producing spliced mRNAs associated or not to EJCs allowed to attribute to EJCs a positive effect on translation independently of effects onto expression level (Wiegand et al. 2003; Nott et al. 2004). One study notably showed that EJCs increase the proportion of mRNAs associated to polysomes (Nott et al. 2004). Although EJCs are not part of translation machinery and are removed in the very first round of translation, it is interesting that EJCs offer a selective advantage to mRNAs that have never experienced translation.
This effect is most likely important to reduce the time window between gene activation at the transcriptional level and mRNA translation producing the final product (Nott et al. 2004). The molecular mechanisms linking the EJC and translation machinery are not clearly elucidated. However, few studies have attempted to clarify the role of the complex in the translation.
EJC enhances translation in mTOR pathway
The first mechanistic insights into EJC role in translation activation is linked to the mTOR pathway (Ma et al. 2008). mTOR is a stress‐sensing signaling pathway which enhances translation via kinase S6K1 to promote cell growth. eIF4A3‐bound SKAR on mRNAs serves to recruit activated S6K1 to the CBCmRNP, leading to phosphorylation of ribosomal proteins and translation initiation factors, thus activating translation initiation in mTOR signaling events (Figure 6). As EJC is loaded only on newly formed mRNAs but not on older templates, associated SKAR communicates with translation machinery as a signal of newly turned‐on gene. Thus mTOR/6K1 promptly targets the translation of freshly formed transcripts in order to reduce the time lag between transcription and translation in stress conditions. However, most EJC‐interactome studies could not identify SKAR as an EJC peripheral factor (Tange et al. 2005; Merz et al. 2007; Singh et al. 2012). This suggests that interaction between SKAR and EJC can be indirect or specific to stress conditions. Alternatively, the EJC‐SKAR synergy might be limited to specific transcripts only, making it difficult to detect in general.
EJC in quality control of mRNAs
The expression of a protein‐coding gene is controlled by balancing the synthesis, processing, translation and degradation of its corresponding mRNA. Additionally, mature mRNAs must carry all the information necessary to encode a functional protein. To accomplish this, eukaryotic cells have surveillance systems that scrutinize mRNA integrity (Kervestin & Jacobson 2012; Fatscher et al. 2015). There are several surveillance mechanisms, which ensure the degradation of defective mRNAs before they could be translated. Three major mRNA quality control mechanisms are (i) NSD (Non‐Stop Decay) that degrades mRNAs lacking stop codons; (ii) NGD (No‐Go Decay) which eliminates the mRNAs in which the ribosomes are blocked; and finally (iii) the NMD (Nonsense‐ Mediated‐Decay), which detects the presence of premature stop codons (PTCs) (Simms et al. 2016). Here, I will simply describe the process of NMD corresponding to the best‐documented function of EJCs.
The importance of NMD
In humans, many diseases originate from mutations causing premature termination codons (PTCs) (Khajavi et al. 2006; Kuzmiak & Maquat 2006). A PTC is distinguished by translation machinery from normal termination codon by the presence of EJC or long 3’ UTR downstream to it. If translated, these mRNAs would otherwise translate truncated proteins with potentially harmful dominant‐negative effects (Nicholson et al. 2010) (Figure 7). Approximately one third of all human genetic disorders of known etiology are caused by genes with germline or de novo mutations that generate PTCs (Karam et al. 2013).
Therefore, NMD plays an essential role in regulation of many human diseases. However, NMD is a double‐edged mechanism in the event of such diseases. When synthesized truncated proteins are sufficient for a normal phenotype, the intervention of NMD usually aggravates the clinical manifestations of the disease. In particular, the NMD worsens phenotypes of several diseases related to mutation of the dystrophin gene, Duchenne muscular dystrophy (Kerr et al. 2001). On the contrary, if the produced protein has a deleterious effect on the body, the NMD works as a lifeline.
Role of EJC in translation control of natural NMD targets
Apart from PTC containing mRNAs, recent studies show that NMD regulates the expression and abundance of transcripts encoding functional proteins. Genome‐wide studies done in S. cerevisiae cells, D. melanogaster and H. sapiens where NMD has been disrupted, reveal that the NMD regulates directly and indirectly the abundance of 3 to 20% of cellular transcripts (Mendell et al. 2004; Rehwinkel et al. 2005; Weischenfeldt et al. 2008; Chan et al. 2009; Weischenfeldt et al. 2012; Tani et al. 2012). Indeed, NMD can affect the expression of natural transcripts that present features triggering NMD like introns in the 3’‐UTR, uORF upstream of the main ORF or a long 3′ UTR. In this context, the mRNA is recognized as a target of the NMD and therefore degraded.
Arc (activity‐regulated cytoskeleton‐associated) mRNA takes advantage of this mechanism to be expressed in a spatiotemporal manner in neuronal synapses (Giorgi et al. 2007). Arc pre‐mRNA has two introns present in its 3’UTR that potentially lead to deposition of EJCs after splicing. This makes it a natural target for NMD. Arc mRNA is transported through the dendrites in a translational silent state. Its translation is activated in synapses where after few rounds of translation, it is degraded by NMD pathway. By this way, neurons maintain expression of Arc protein in a snapshot of time and at a specific place only. Also, it ensures that the protein is expressed in a limited turnover per molecule of mRNA, thus precisely regulating the total amount of protein based on number of mRNA targeted to the neuronal synapses.
Role of EJC in mRNA transport
Direct involvement of EJC in mRNA export was first established by testing whether spliced mRNAs, which do not harbor EJC, could be transported to cytoplasm or not in Xenopus laevis oocytes (Le Hir et al. 2001). The loading of EJC at the 5’ end of the mRNA was shown to enhance the export of short spliced mRNA reporters compared to similar reporters devoid of EJC. Immunoprecipitation experiments revealed that EJCs are associated to several export factors (Aly/Ref, UAP56, SRSF1 and SRSF7) as well as to NXF1‐ NXT1 (Le Hir et al. 2001; Zhou et al. 2000; Luo et al. 2001; Kim et al. 2001). The EJC could also facilitate export by stabilizing adaptor SR proteins as well as the overall mRNP structure (Singh et al. 2012). So far, only export of short transcripts (of a few hundred nucleotides long) was found to be highly dependent on splicing and the EJC (Le Hir et al. 2001). Since longer mRNAs have more binding sites for different adaptors, EJC is only one adaptor among others, which explains why the overall export of mRNAs only marginally depends on splicing and the EJC (Gatfield & Izaurralde 2002; Nott et al. 2003). Whether EJC is required for the nucleo‐cytoplasmic export of specific transcripts remains an open question.
Table of contents :
INTRODUCTION
1.0: mRNPs
2.0: EJC
2.1: The EJC core components
2.1.1: eIF4A3
2.1.2: MAGOH‐Y14
2.1.3: MLN51
2.2: The tetrameric EJC core
2.3: EJC assembly
2.3.1: The pre‐EJC core
2.3.2: The recruitment of eIF4A3 by CWC22
2.4: EJC peripheral factors
2.4.1: Splicing‐related peripheral factors
2.4.2: Export factors
2.4.3: NMD related factors
2.4.4: Other EJC related factors
3.0: EJC life cycle
3.1: The localization of EJC core components
3.2: EJC remodeling and variability
3.2.1: EJC disassembly
3.2.2: Re‐cycling of EJCs to the nucleus
4.0: EJC functions
4.1: EJC modulates splicing
4.1.1: EJC dependent regulation of splicing in human cells
4.2: EJC enhances translation
4.2.1: EJC enhances translation in mTOR pathway
4.2.2: Translation enhancement by MLN51
4.3: EJC in quality control of mRNAs
4.3.1: The importance of NMD
4.3.2: NMD components
4.3.3: NMD Mechanism
4.3.4: Role of EJC in NMD
4.3.5: Role of EJC in translation control of natural NMD targets
4.4: EJC participates to mRNA export
4.4.1: General mRNA export adaptors and receptors
4.4.2: Role of EJC in mRNA transport
5.0: Global view of EJC deposition on mRNAs
5.1: Differential EJC loading on human transcriptome
5.2: The canonical and non‐canonical EJC
6.0: mRNA localization and local translation
6.1: mRNA localization
6.2: Importance of mRNA localization
6.2.1: Energy efficiency for the cell
6.2.2: Spatio‐temporal translation
6.2.3: Storage
6.2.4: One transcript, multi‐functional protein
6.3: Examples of localized mRNAs
6.4: Mechanism of mRNA localization
6.4.1: Directed transport of mRNA in cytoplasm: Cis‐regulatory elements and trans‐acting factors
6.4.2: Directed transport of mRNA in cytoplasm: Recruitment of molecular motors
6.4.3: Role of EJC in sub‐cellular localization
7.0: EJC in physiological contexts
7.1: EJC in development
7.2: EJC in mammalian brain development
7.3: EJC in human diseases
8.0: Development of mammalian Brain
8.1: Primary progenitors
8.2: Intermediate progenitors
8.3: Role of NSC in brain development
8.4: Ependymal cells
8.4.1: Physiological Functions of ependymal cells in brain
8.4.1.1: Regulation of neuronal niche
8.4.1.2: CSF maintenance
8.4.1.3: Metabolic protection of brain
8.4.1.4: Protection of brain from infections
8.4.1.5: Repair of brain after stroke
8.4.2: Ependymal differentiation in mammalian brain
8.4.3: Centriole amplification in ependymal differentiation
9.0: Cell‐cycle and cell‐quiescence
10.0: Centrosome
10.1: Centrosome Composition
10.2: Centrosome duplication in cycling cells
10.3: Centrosome in post‐mitotic cells
10.3.1: Formation of primary cilia
10.3.1.1: Cilia structure and functions
10.3.1.2: Primary cilia in neurodevelopmental disorders
10.3.2: Centrosome amplification in post‐mitotic cells
10.4: Centrosome functions
10.4.1: Centrosome functions in cell fate
10.4.2: Centrosomes in human diseases
11.0: Questions Asked
RESULTS (Part 1): Article
RESULTS (Part 2): Additional results
1.0: Identification of EJC‐bound mRNAs in NSC
1.1: EJC RIP‐seq strategy
1.2: Cytoplasmic fractionation of NSC
1.3: Tests of immuno‐depletion
1.4: EJC‐RIP and RNA purification
1.5: Analysis of sequencing results
1.6: Statistical validation of RIPseq
1.7: Identity of EJC bound transcripts in NSC
2.0: Visualization of expression of mRNAs by smFISH
2.1: smFISH model
2.2: Design and Synthesis of Fluorescent Oligonucleotide Probe Sets
2.2.1. Design
2.3: Results obtained
2.3.1: smFISH in cycling MEF
2.3.2: smFISH in quiescent NSC
2.3.3: Distribution of total mRNAs in quiescent NSC
2.3.4: smFISH in quiescent MEF
DISCUSSION AND PERSPECTIVES
1.0: Resume of our results
2.0: Presence of mRNAs at centrosome in variety of cell types
3.0: Accumulation of untranslated mRNAs at centrosome
3.1: Hypothesis 1: Translation dependent disassembly of EJCs
3.2: Hypothesis 2: Degradation or diffusion of EJC bound mRNAs
4.0: Functions of untranslated mRNAs at centrosome
MATERIALS AND METHODS
1.0: CELL CULTURE
1.1: Neural Stem Cell culture
1.2: Mouse Embryonic Fibroblast culture
2.0: Transfection of plasmids
3.0: Immunofluorescence and Microscopy
3.1: Image analysis.
4.0: PROTEIN ANALYSIS
4.1: Protein extraction
4.2: Immunoprecipitation
4.3: SDS‐PAGE
4.4: Western Blot Analysis
5: RNA analysis
5.1: Cellular fractionation
5.2: RNA Immuno Precipitation
5.3: RNA isolation
5.3.1: Isolation of immunoprecipitated RNA
5.3.1.1: RNA precipitation
5.3.2: Total RNA isolation
5.4: Quantitative RT‐PCR
6.0: smFISH analysis
6.1: Probe synthesis
6.2: Hybridization of probes with FlapY‐Cy3
6.3: Cell fixation
6.4: In situ hybridization
7.0: List of buffers
REFERENCES
