Interaction of the spliced Oskar localization element of Oskar mRNA with the protein PYM

mRNAs and the process of mRNA localization are the fundamental and pivotal parts of cellular functions. mRNA localization encompasses an important role in cellular differentiation and site-specific cellular functions, from the basic cellular biochemical mechanism to advanced abdomen formation. The study of mRNA, its localization mechanism along its binding partners have always been the main focus of study for several years. As they define life, in terms of cellular and sub-cellular mechanisms. Our study also involves one of the binding partners of the localization complex, which is Pym protein. Pym protein and exon junction complex are the common localization binding partners to many mRNA localization and Oskar mRNA is one of them. Pym being one of the recycling factors of the Exon Junction Complex shows binding interactions with many components, such as RNAs, Exon junction Complex, and Ribosomes. Our results show interesting structural and binding features of the protein Pym. NMR studies reveal that Pym160, the shorter construct of Pym is structurally unfolded, with the general characteristic of an intrinsically disordered protein. It has the long helical structural element in the middle part of the protein, while both N-terminal and C-terminal ends remain highly flexible with the structurally unfolded regions. The C-terminal part of the protein is not showing any direct involvement in the interaction with the SOLE RNA. However, it is structurally a very important part of the protein, as it stabilizes the ionic and hydrophobic interactions of the protein, so that protein could able to be a stable soluble protein. We have studied the binding motifs of the protein Pym160 with SOLE RNA and its isomers. Pym160 has binding motifs in the N-terminal region and in the middle helical region. Studies have confirmed that the N-terminal part of the protein binds to the Y14-Mago heterodimer, which is an essential part of the exon junction complex. In the absence of an Exon Junction Complex, the N-terminal part of the protein binds to the RNA. So, the study of the protein Pym160 is very much interesting and essential as it is a common protein for the wide range of mRNA localization mechanisms. Our studies explain the widespread binding nature of the Pym160, which might be due to its functional significance of being a structurally unfolded protein

Investigating the mechanism behind the feedback regulation of the Exon Junction Complex component MAGOH

RNA binding proteins are critical for regulation of RNA function, and therefore maintaining appropriate levels of these proteins is important for cellular regulation. I focus on MAGOH, an essential subunit of the Exon Junction Complex (EJC) that is necessary for stable binding of the EJC to mRNA. Like all key regulatory proteins, MAGOH levels in the cell are tightly controlled. Reductions in MAGOH can lead to defects in embryonic development. We have found that, in human embryonic kidney (HEK293) cells, when an externally introduced copy of MAGOH (exogenous FLAG-MAGOH) is forcibly expressed from a tetracycline-inducible promoter, it causes the levels of endogenous MAGOH protein to go down. Thus, there is a feedback mechanism that maintains appropriate levels of MAGOH. The molecular details of this mechanism remain largely unknown and are the subject of my research. Here, I test if the regulation of MAGOH occurs at the transcriptional or post-transcriptional level. My results show that when increasing amounts of FLAG-MAGOH mRNA and protein are expressed in the cell, the endogenous MAGOH mRNA and pre-mRNA levels are unchanged. This data suggests that neither the degradation nor the synthesis of endogenous MAGOH mRNA is affected by more FLAG-MAGOH protein expression. Interestingly, I find that when cells are treated with a proteasome inhibitor (MG-132), the endogenous MAGOH protein does not decrease as observed in the untreated cells. Therefore, MAGOH protein levels are regulated by proteasome-mediated degradation of the protein. Additionally, the incorporation of MAGOH into the EJC seems to play a role in its regulation.No embargoAcademic Major: Molecular Genetic

Identification of antiviral roles for the exon-junction complex and nonsense-mediated decay in flaviviral infection.

West Nile virus (WNV) is an emerging mosquito-borne flavivirus, related to dengue virus and Zika virus. To gain insight into host pathways involved in WNV infection, we performed a systematic affinity-tag purification mass spectrometry (APMS) study to identify 259 WNV-interacting human proteins. RNA interference screening revealed 26 genes that both interact with WNV proteins and influence WNV infection. We found that WNV, dengue and Zika virus capsids interact with a conserved subset of proteins that impact infection. These include the exon-junction complex (EJC) recycling factor PYM1, which is antiviral against all three viruses. The EJC has roles in nonsense-mediated decay (NMD), and we found that both the EJC and NMD are antiviral and the EJC protein RBM8A directly binds WNV RNA. To counteract this, flavivirus infection inhibits NMD and the capsid-PYM1 interaction interferes with EJC protein function and localization. Depletion of PYM1 attenuates RBM8A binding to viral RNA, suggesting that WNV sequesters PYM1 to protect viral RNA from decay. Together, these data suggest a complex interplay between the virus and host in regulating NMD and the EJC

Splicing-dependent NMD does not require the EJC in Schizosaccharomyces pombe

Nonsense-mediated mRNA decay (NMD) is a translation-linked process that destroys mRNAs with premature translation termination codons (PTCs). In mammalian cells, NMD is also linked to pre-mRNA splicing, usually PTCs trigger strong NMD only when positioned upstream of at least one intron. The exon junction complex (EJC) is believed to mediate the link between splicing and NMD in these systems. Here, we report that in Schizosaccharomyces pombe splicing also enhances NMD, but against the EJC model prediction, an intron stimulated NMD regardless of whether it is positioned upstream or downstream of the PTC and EJC components are not required. Still the effect of splicing seems to be direct—we have found that the important NMD determinant is the proximity of an intron to the PTC, not just the occurrence of splicing. On the basis of these results, we propose a new model to explain how splicing could affect NMD

Identification and characterization of novel factors that act in the nonsense-mediated mRNA decay pathway in nematodes, flies and mammals

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs harboring premature termination codons (PTCs). We have conducted a genome-wide RNAi screen in Caenorhabditis elegans that resulted in the identification of five novel NMD genes that are conserved throughout evolution. Two of their human homologs, GNL2 (ngp-1) and SEC13 (npp-20), are also required for NMD in human cells. We also show that the C. elegans gene noah-2, which is present in Drosophila melanogaster but absent in humans, is an NMD factor in fruit flies. Altogether, these data identify novel NMD factors that are conserved throughout evolution, highlighting the complexity of the NMD pathway and suggesting that yet uncovered novel factors may act to regulate this process

Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1.

One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity

Protecting the proteome: Eukaryotic cotranslational quality control pathways.

The correct decoding of messenger RNAs (mRNAs) into proteins is an essential cellular task. The translational process is monitored by several quality control (QC) mechanisms that recognize defective translation complexes in which ribosomes are stalled on substrate mRNAs. Stalled translation complexes occur when defects in the mRNA template, the translation machinery, or the nascent polypeptide arrest the ribosome during translation elongation or termination. These QC events promote the disassembly of the stalled translation complex and the recycling and/or degradation of the individual mRNA, ribosomal, and/or nascent polypeptide components, thereby clearing the cell of improper translation products and defective components of the translation machinery

Full UPF3B function is critical for neuronal differentiation of neural stem cells

Acknowledgments We thank Fred H Gage (Salk Institute, La Jolla, CA, USA) for HCN-A94 cells and Niels Gehring (University of Cologne, Germany) for constructs. We gratefully acknowledge Tenovus Scotland (Project Grant G11-06), Moonlight Prowl (FS) and the Saudi Arabian Ministry of Higher Education via King Abdullah Program for Scholarships for support (TA). JA is supported by a PhD studentship from Medical Research Scotland (PhD-654-2012) and Dundee Cell Products.Peer reviewedPublisher PD

Mago Nashi, Tsunagi/Y14, and Ranshi form a complex that influences oocyte differentiation in Drosophila melanogaster

AbstractDuring Drosophila melanogaster oogenesis, a germline stem cell divides forming a cyst of 16 interconnected cells. One cell enters the oogenic pathway, and the remaining 15 differentiate as nurse cells. Although directed transport and localization of oocyte differentiation factors within the single cell are indispensible for selection, maintenance, and differentiation of the oocyte, the mechanisms regulating these events are poorly understood. Mago Nashi and Tsunagi/Y14, core components of the exon junction complex (a multiprotein complex assembled on spliced RNAs), are essential for restricting oocyte fate to a single cell and for localization of oskar mRNA. Here we provide evidence that Mago Nashi and Tsunagi/Y14 form an oogenic complex with Ranshi, a protein with a zinc finger-associated domain and zinc finger domains. Genetic analyses of ranshi reveal that (1) 16-cell cysts are formed, (2) two cells retain synaptonemal complexes, (3) all cells have endoreplicated DNA (as observed in nurse cells), and (4) oocyte-specific cytoplasmic markers accumulate and persist within a single cell but are not localized within the posterior pole of the presumptive oocyte. Our results indicate that Ranshi interacts with the exon junction complex to localize components essential for oocyte differentiation within the posterior pole of the presumptive oocyte

A Competition between Stimulators and Antagonists of Upf Complex Recruitment Governs Human Nonsense-Mediated mRNA Decay

The nonsense-mediated decay (NMD) pathway subjects mRNAs with premature termination codons (PTCs) to rapid decay. The conserved Upf1–3 complex interacts with the eukaryotic translation release factors, eRF3 and eRF1, and triggers NMD when translation termination takes place at a PTC. Contrasting models postulate central roles in PTC-recognition for the exon junction complex in mammals versus the cytoplasmic poly(A)-binding protein (PABP) in other eukaryotes. Here we present evidence for a unified model for NMD, in which PTC recognition in human cells is mediated by a competition between 3′ UTR–associated factors that stimulate or antagonize recruitment of the Upf complex to the terminating ribosome. We identify cytoplasmic PABP as a human NMD antagonizing factor, which inhibits the interaction between eRF3 and Upf1 in vitro and prevents NMD in cells when positioned in proximity to the termination codon. Surprisingly, only when an extended 3′ UTR places cytoplasmic PABP distally to the termination codon does a downstream exon junction complex enhance NMD, likely through increasing the affinity of Upf proteins for the 3′ UTR. Interestingly, while an artificial 3′ UTR of >420 nucleotides triggers NMD, a large subset of human mRNAs contain longer 3′ UTRs but evade NMD. We speculate that these have evolved to concentrate NMD-inhibiting factors, such as PABP, in spatial proximity of the termination codon