exon junction
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EMBO Reports ◽  
2021 ◽  
Author(s):  
Cheuk Hei Ho ◽  
Chiara Paolantoni ◽  
Praveen Bawankar ◽  
Zuojian Tang ◽  
Stuart Brown ◽  
...  

2021 ◽  
Vol 8 (4) ◽  
Author(s):  
Annapurna Sahoo ◽  
Kunja Bihari Satapathy

Exon junction complexes (EJCs) associate with mRNAs, mediate the pre-mRNA splicing and eventually gets displaced by ribosomes during the initial phase of translation. EJCs are involved in several critical physiological pathways. The functional nature of EJCs and the underlying molecular mechanism(s) still needs to be elucidated particularly in case of plants. Here, we report that the putative core protein factors of the EJC differentially express under short-day and long-day conditions. Since, plants are constantly exposed to biotic and abiotic factor(s), it would be significant to see how the EJCs respond to different stress inducing conditions. The protein levels of EJC core proteins under short-day conditions were 1.25 times higher relative to the protein levels under long-day conditions. Similar results were observed for the mRNA transcripts of the EJC core protein factors. These results signify that under short-day conditions, the EJC proteins are more activated and might be involved in few events which are yet to be revealed.


2021 ◽  
Author(s):  
Lena Pia Schlautmann ◽  
Volker Boehm ◽  
Jan-Wilm Lackmann ◽  
Janine Altmueller ◽  
Christoph Dieterich ◽  
...  

The exon junction complex (EJC) is an RNA-binding multi-protein complex with critical functions in post-transcriptional gene regulation. It is deposited on the mRNA during splicing and regulates diverse processes including pre-mRNA splicing, mRNA export, mRNA translation, and nonsense-mediated mRNA decay (NMD) via various interacting peripheral proteins. The EJC-binding protein RNPS1 might serve two functions: it suppresses mis-splicing of cryptic splice sites and activates NMD in the cytoplasm. When analyzing the transcriptome-wide effects of EJC and RNPS1 knockdowns in different human cell lines, we find no evidence for RNPS1 being a globally essential NMD factor. However, various aberrant splicing events strongly suggest that the main function of RNPS1 is splicing regulation. Rescue analyses revealed that about half of these RNPS1-dependent splicing events was fully or partially rescued by the expression of the isolated RRM domain of RNPS1, whereas other splicing events are regulated by its C-terminal domain. We identified many splicing-regulatory factors, including SR proteins and U1 snRNP components, that specifically interact with the C-terminus or with the RRM of RNPS1. Thus, RNPS1 emerges as a multifunctional splicing regulator that promotes correct and efficient splicing of different vulnerable splicing events via the formation of diverse splicing-promoting complexes.


2021 ◽  
Vol 7 (32) ◽  
pp. eabf7561
Author(s):  
Dimitris C. Kanellis ◽  
Jaime A. Espinoza ◽  
Asimina Zisi ◽  
Elpidoforos Sakkas ◽  
Jirina Bartkova ◽  
...  

Eukaryotic initiation factor 4A-III (eIF4A3), a core helicase component of the exon junction complex, is essential for splicing, mRNA trafficking, and nonsense-mediated decay processes emerging as targets in cancer therapy. Here, we unravel eIF4A3’s tumor-promoting function by demonstrating its role in ribosome biogenesis (RiBi) and p53 (de)regulation. Mechanistically, eIF4A3 resides in nucleoli within the small subunit processome and regulates rRNA processing via R-loop clearance. EIF4A3 depletion induces cell cycle arrest through impaired RiBi checkpoint–mediated p53 induction and reprogrammed translation of cell cycle regulators. Multilevel omics analysis following eIF4A3 depletion pinpoints pathways of cell death regulation and translation of alternative mouse double minute homolog 2 (MDM2) transcript isoforms that control p53. EIF4A3 expression and subnuclear localization among clinical cancer specimens correlate with the RiBi status rendering eIF4A3 an exploitable vulnerability in high-RiBi tumors. We propose a concept of eIF4A3’s unexpected role in RiBi, with implications for cancer pathogenesis and treatment.


2021 ◽  
Author(s):  
Damaris Wallmeroth ◽  
Volker Boehm ◽  
Jan-Wilm Lackmann ◽  
Janine Altmueller ◽  
Christoph Dieterich ◽  
...  

The paralogous human proteins UPF3A and UPF3B are involved in recognizing mRNAs targeted by nonsense-mediated mRNA decay (NMD). While UPF3B has been demonstrated to support NMD, contradicting reports describe UPF3A either as an NMD activator or inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using combinatory experimental approaches. Overexpression or knockout of UPF3A as well as knockout of UPF3B did not detectably change global NMD activity. In contrast, the co-depletion of UPF3A and UPF3B resulted in a marked NMD inhibition and a transcriptome-wide upregulation of NMD substrates, demonstrating a functional redundancy between both NMD factors. Although current models assume that UPF3 bridges NMD-activating exon-junction complexes (EJC) to the NMD factor UPF2, UPF3B exhibited normal NMD activity in rescue experiments when UPF2 or EJC binding was impaired. Further rescue experiments revealed partially redundant functions of UPF3B domains in supporting NMD, involving both UPF2 and EJC interaction sites and the central region of UPF3. Collectively, UPF3A and UPF3B serve as fault-tolerant NMD activators in human cells.


2021 ◽  
Author(s):  
Zhongxia Yi ◽  
René M Arvola ◽  
Sean Myers ◽  
Corinne N Dilsavor ◽  
Rabab Abu Alhasan ◽  
...  

Nonsense-mediated mRNA decay (NMD) is governed by the three conserved factors - UPF1, UPF2 and UPF3. While all three are required for NMD in yeast, UPF3B is dispensable for NMD in mammals, with its paralog UPF3A suggested to only weakly activate or even repress NMD due to its weaker binding to the exon junction complex (EJC). Here we characterize the UPF3B-dependent and -independent NMD in human cell lines knocked-out of one or both UPF3 paralogs. We show that in human colorectal cancer HCT116 cells, EJC-mediated NMD can operate in UPF3B-dependent and -independent manner. While UPF3A is almost completely dispensable for NMD in wild-type cells, it strongly activates EJC-mediated NMD in cells lacking UPF3B. Surprisingly, this major NMD branch can operate in UPF3-independent manner questioning the idea that UPF3 is needed to bridge UPF proteins to the EJC during NMD. Complementation studies in UPF3 knockout cells further show that EJC-binding domain of UPF3 paralogs is not essential for NMD. Instead, the conserved mid domain of UPF3B, previously shown to engage with ribosome release factors, is required for its full NMD activity. Altogether, UPF3 plays a more active role in NMD than simply being a bridge between the EJC and the UPF complex.


2021 ◽  
Vol 22 (12) ◽  
pp. 6519
Author(s):  
Yuta Otani ◽  
Ken-ichi Fujita ◽  
Toshiki Kameyama ◽  
Akila Mayeda

Using TSG101 pre-mRNA, we previously discovered cancer-specific re-splicing of mature mRNA that generates aberrant transcripts/proteins. The fact that mRNA is aberrantly re-spliced in various cancer cells implies there must be an important mechanism to prevent deleterious re-splicing on the spliced mRNA in normal cells. We thus postulated that mRNA re-splicing is controlled by specific repressors, and we searched for repressor candidates by siRNA-based screening for mRNA re-splicing activity. We found that knock-down of EIF4A3, which is a core component of the exon junction complex (EJC), significantly promoted mRNA re-splicing. Remarkably, we could recapitulate cancer-specific mRNA re-splicing in normal cells by knock-down of any of the core EJC proteins, EIF4A3, MAGOH, or RBM8A (Y14), implicating the EJC core as the repressor of mRNA re-splicing often observed in cancer cells. We propose that the EJC core is a critical mRNA quality control factor to prevent over-splicing of mature mRNA.


PLoS Genetics ◽  
2021 ◽  
Vol 17 (5) ◽  
pp. e1009563
Author(s):  
Brian Joseph ◽  
Eric C. Lai

Accurate splice site selection is critical for fruitful gene expression. Recently, the mammalian EJC was shown to repress competing, cryptic, splice sites (SS). However, the evolutionary generality of this remains unclear. Here, we demonstrate the Drosophila EJC suppresses hundreds of functional cryptic SS, even though most bear weak splicing motifs and are seemingly incompetent. Mechanistically, the EJC directly conceals cryptic splicing elements by virtue of its position-specific recruitment, preventing aberrant SS definition. Unexpectedly, we discover the EJC inhibits scores of regenerated 5’ and 3’ recursive SS on segments that have already undergone splicing, and that loss of EJC regulation triggers faulty resplicing of mRNA. An important corollary is that certain intronless cDNA constructs yield unanticipated, truncated transcripts generated by resplicing. We conclude the EJC has conserved roles to defend transcriptome fidelity by (1) repressing illegitimate splice sites on pre-mRNAs, and (2) preventing inadvertent activation of such sites on spliced segments.


2021 ◽  
Author(s):  
Vasily V Grinev ◽  
Natalia Siomava ◽  
Laurent Vallar ◽  
Petr V Nazarov

Alternative splicing is an essential characteristic of living cells that usually infers a various exon-exon junction governed by different splice sites. The traditional classification based on the mode of use designates splice site to one of the two groups, constitutive or alternative. Here, we considered another criterion and reorganized splice sites into "unisplice" and "multisplice" groups according to the number of undertaken splicing events. This approach provided us with a new insight in the organization and functionality of leukemia cells. We determined features associated with uni- and multisplice sites and found that combinatorics of these sites follows strict rules of the power-law in the t(8;21)-positive leukemia cells. We also found that system splicing characteristics of the transcriptome of leukemia cells remained persistent after drastic changes in the transcript composition caused by knockdown of the RUNX1-RUNX1T1 oncogene. In this work, we show for the first time that leukemia cells possess a sub-set of unisplice sites with a hidden multisplice potential. These findings reveal a new side in organization and functioning of the leukemic cells and open up new perspectives in the study of the t(8;21)-positive leukemia.


2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Carrie Kovalak ◽  
Scott Donovan ◽  
Alicia A. Bicknell ◽  
Mihir Metkar ◽  
Melissa J. Moore

Abstract Background Alternative splicing, which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing splicing pathways cannot be determined by traditional RNA-Seq, however, because different mRNA isoforms can have widely differing decay rates. Indeed, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses. Results RNA immunoprecipitation in tandem (RIPiT) of exon junction complex components allows for purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and, therefore, translation-dependent mRNA decay. Here we compare exon junction complex RIPiT-Seq to whole cell RNA-Seq data from HEK293 cells. Consistent with expectation, the flux through known AS-NMD pathways is substantially higher than that captured by RNA-Seq. Our RIPiT-Seq also definitively demonstrates that the splicing machinery itself has no ability to detect reading frame. We identify thousands of previously unannotated splicing events; while many can be attributed to splicing noise, others are evolutionarily conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis. Conclusions Deep sequencing of RNAs in post-splicing, pre-translational mRNPs provides a means to identify and quantify splicing events without the confounding influence of differential mRNA decay. For many known AS-NMD targets, the nonsense-mediated decay-linked alternative splicing pathway predominates. Exon junction complex RIPiT-Seq also revealed numerous conserved but previously unannotated AS-NMD events.


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