scholarly journals Characterization of an Atypical eIF4E Ortholog in Leishmania, LeishIF4E-6

2021 ◽  
Vol 22 (23) ◽  
pp. 12720
Author(s):  
Nitin Tupperwar ◽  
Rohit Shrivastava ◽  
Nofar Baron ◽  
Orli Korchev ◽  
Irit Dahan ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Life Forms ◽  
Potential Effect ◽  
Binding Activity ◽  
Sand Fly ◽  
Translation Regulation ◽  
Genetic Profile ◽  
Mrna Decapping ◽  
Decapping Enzyme ◽  
Leishmania Parasites

Leishmania parasites are digenetic protists that shuffle between sand fly vectors and mammalian hosts, transforming from flagellated extracellular promastigotes that reside within the intestinal tract of female sand flies to the obligatory intracellular and non-motile amastigotes within mammalian macrophages. Stage differentiation is regulated mainly by post-transcriptional mechanisms, including translation regulation. Leishmania parasites encode six different cap-binding proteins, LeishIF4E1-6, that show poor conservation with their counterparts from higher eukaryotes and among themselves. In view of the changing host milieu encountered throughout their life cycle, we propose that each LeishIF4E has a unique role, although these functions may be difficult to determine. Here we characterize LeishIF4E-6, a unique eIF4E ortholog that does not readily associate with m7GTP cap in either of the tested life forms of the parasite. We discuss the potential effect of substituting two essential tryptophan residues in the cap-binding pocket, expected to be involved in the cap-binding activity, as judged from structural studies in the mammalian eIF4E. LeishIF4E-6 binds to LeishIF4G-5, one of the five eIF4G candidates in Leishmania. However, despite this binding, LeishIF4E-6 does not appear to function as a translation factor. Its episomal overexpression causes a general reduction in the global activity of protein synthesis, which was not observed in the hemizygous deletion mutant generated by CRISPR-Cas9. This genetic profile suggests that LeishIF4E-6 has a repressive role. The interactome of LeishIF4E-6 highlights proteins involved in RNA metabolism such as the P-body marker DHH1, PUF1 and an mRNA-decapping enzyme that is homologous to the TbALPH1.

scholarly journals LeishIF4E-5 Is a Promastigote-Specific Cap-Binding Protein in Leishmania

2021 ◽  
Vol 22 (8) ◽  
pp. 3979
Author(s):  
Rohit Shrivastava ◽  
Nitin Tupperwar ◽  
Bar Schwartz ◽  
Nofar Baron ◽  
Michal Shapira
Keyword(s):  
Environmental Conditions ◽  
Binding Protein ◽  
Binding Pocket ◽  
Binding Activity ◽  
Mass Spectrometry Analysis ◽  
Sand Fly ◽  
Translation Regulation ◽  
Binding Motif ◽  
Spectrometry Analysis ◽  
Cytochemical Analysis

Leishmania parasites cycle between sand fly vectors and mammalian hosts, transforming from extracellular promastigotes that reside in the vectors’ alimentary canal to obligatory intracellular non-motile amastigotes that are harbored by macrophages of the mammalian hosts. The transition between vector and host exposes them to a broad range of environmental conditions that induces a developmental program of gene expression, with translation regulation playing a key role. The Leishmania genome encodes six paralogs of the cap-binding protein eIF4E. All six isoforms show a relatively low degree of conservation with eIF4Es of other eukaryotes, as well as among themselves. This variability could suggest that they have been assigned discrete roles that could contribute to their survival under the changing environmental conditions. Here, we describe LeishIF4E-5, a LeishIF4E paralog. Despite the low sequence conservation observed between LeishIF4E-5 and other LeishIF4Es, the three aromatic residues in its cap-binding pocket are conserved, in accordance with its cap-binding activity. However, the cap-binding activity of LeishIF4E-5 is restricted to the promastigote life form and not observed in amastigotes. The overexpression of LeishIF4E-5 shows a decline in cell proliferation and an overall reduction in global translation. Immuno-cytochemical analysis shows that LeishIF4E-5 is localized in the cytoplasm, with a non-uniform distribution. Mass spectrometry analysis of proteins that co-purify with LeishIF4E-5 highlighted proteins involved in RNA metabolism, along with two LeishIF4G paralogs, LeishIF4G-1 and LeishIF4G-2. These vary in their conserved eIF4E binding motif, possibly suggesting that they can form different complexes.

The C. elegans mRNA decapping enzyme shapes morphology of cilia

2017 ◽  
Vol 493 (1) ◽  
pp. 382-387 ◽  
Author(s):  
Takeshi Adachi ◽  
Keigo Nagahama ◽  
Susumu Izumi
Keyword(s):  
Mrna Decapping ◽  
C Elegans ◽  
Decapping Enzyme

scholarly journals GPI- and transmembrane-anchored influenza hemagglutinin differ in structure and receptor binding activity

1993 ◽  
Vol 122 (6) ◽  
pp. 1253-1265 ◽  
Author(s):  
GW Kemble ◽  
YI Henis ◽  
JM White
Keyword(s):  
Cell Surface ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Binding Pocket ◽  
Binding Activity ◽  
Great Distance ◽  
Fusion Peptides ◽  
Wild Type ◽  
Influenza Hemagglutinin ◽  
Receptor Binding Activity

We investigated the influence of a glycosylphosphatidylinositol (GPI) anchor on the ectodomain of the influenza hemagglutinin (HA) by replacing the wild type (wt) transmembrane and cytoplasmic domains with a GPI lipid anchor. GPI-anchored HA (GPI-HA) was transported to the cell surface with equal efficiency and at the same rate as wt-HA. Like wt-HA, cell surface GPI-HA, and its ectodomain released with the enzyme PI-phospholipase C (PI-PLC), were 9S trimers. Compared to wt-HA, the GPI-HA ectodomain underwent additional terminal oligosaccharide modifications; some of these occurred near the receptor binding pocket and completely inhibited the ability of GPI-HA to bind erythrocytes. Growth of GPI-HA-expressing cells in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM) abrogated the differences in carbohydrate modification and restored the ability of GPI-HA to bind erythrocytes. The ectodomain of GPI-HA produced from cells grown in the presence or absence of dMM underwent characteristic low pH-induced conformational changes (it released its fusion peptides and became hydrophobic and proteinase sensitive) but at 0.2 and 0.4 pH units higher than wt-HA, respectively. These results demonstrate that although GPI-HA forms a stable trimer with characteristics of the wt, its structure is altered such that its receptor binding activity is abolished. Our results show that transmembrane and GPI-anchored forms of the same ectodomain can exhibit functionally important differences in structure at a great distance from the bilayer.

scholarly journals The hDcp2 protein is a mammalian mRNA decapping enzyme

2002 ◽  
Vol 99 (20) ◽  
pp. 12663-12668 ◽  
Author(s):  
Z. Wang ◽  
X. Jiao ◽  
A. Carr-Schmid ◽  
M. Kiledjian
Keyword(s):  
Mrna Decapping ◽  
Decapping Enzyme

scholarly journals Detection and Degradation of Nonsense-mediated mRNA Decay Substrates Involve Two Distinct Upf1-bound Complexes

2018 ◽  
Author(s):  
Marine Dehecq ◽  
Laurence Decourty ◽  
Abdelkader Namane ◽  
Caroline Proux ◽  
Joanne Kanaan ◽  
...  
Keyword(s):  
Large Scale ◽  
Mrna Decay ◽  
Affinity Purification ◽  
Rna Degradation ◽  
Degradation Pathway ◽  
Telomere Maintenance ◽  
Mrna Decapping ◽  
Nonsense Mediated Mrna Decay ◽  
Decapping Enzyme

AbstractNonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway involved in many cellular pathways and crucial for telomere maintenance and embryo development. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals, but a universal NMD model is lacking. We used affinity purification coupled with mass spectrometry and an improved data analysis protocol to obtain the first large-scale quantitative characterization of yeast NMD complexes in yeast (112 experiments). Unexpectedly, we identified two distinct complexes associated with Upf1: Detector (Upf1/2/3) and Effector. Effector contained the mRNA decapping enzyme, together with Nmd4 and Ebs1, two proteins that globally affected NMD and were critical for RNA degradation mediated by the Upf1 C-terminal helicase region. The fact that Nmd4 association to RNA was dependent on Detector components and the similarity between Nmd4/Ebs1 and mammalian Smg5-7 proteins suggest that in all eukaryotes NMD operates through successive Upf1-bound Detector and Effector complexes.

scholarly journals miR-4293 Upregulates lncRNA WFDC21P by Directly Suppressing mRNA-Decapping Enzyme 2 to Promote Lung Carcinoma Proliferation

2020 ◽  
Author(s):  
Qian Zhang ◽  
Yun-Fei Yan ◽  
Qing Lv ◽  
You-Jie Li ◽  
Ran-Ran Wang ◽  
...  
Keyword(s):  
Lung Carcinoma ◽  
Mrna Decapping ◽  
Lung Carcinoma Cells ◽  
Stat3 Phosphorylation ◽  
Rna Immunoprecipitation ◽  
Decapping Enzyme ◽  
Gain Loss ◽  
And Control ◽  
Relationship Of

Abstract Background: Emerging evidence shows that lncRNA WFDC21P could promote STAT3 phosphorylation and microRNA 4293 SNP is associated with the risk of carcinomas, but the oncogenic functions of WFDC21P and miR-4293 in lung carcinoma are unclear.Methods: mRNA sequencing of lung carcinoma and control para-carcinoma tissues was performed to screen the potential targets. WFDC21P and miR-4293 levels were evaluated in lung carcinoma cells and tissues by qRT-PCR. The function of WFDC21P and miR-4293 on proliferation, apoptosis and metastasis were assessed by MTT, FACS, western blot, transwell assays, colony formation assays and xenografts experiment. RNA immunoprecipitation assays were implemented to verify the relationship between WFDC21P and mRNA-decapping enzyme 2 (DCP2). Furthermore, gain/loss of miR-4293 functions were used to determine its targeting relationship of DCP2. Results: WFDC21P expression is markedly enhanced in lung carcinoma tissue and cells. Moreover, WFDC21P promotes tumor cell proliferation and metastasis but suppresses apoptosis. Mechanistic investigations identified DCP2 can directly bind to WFDC21P and down-regulates its expression. DCP2 as a direct target of miR-4293 and its expression is suppressed by miR-4293. Consequently, miR-4293 can further promote WFDC21P expression by regulating DCP2. With positive correlation to WFDC21P expression, miR-4293 also plays oncogenic role in lung carcinoma. Furthermore, knockdown of WFDC21P results in functional attenuation of miR-4293 on tumor promotion. In vivo xenograft growth is also promoted by both WFDC21P and miR-4293. Conclusion: Our results establish oncogenic roles for both WFDC21P and miR-4293, and demonstrate that interactions between miRNAs and lncRNAs through DCP2 are important in lung carcinoma pathogenesis.

scholarly journals Human Pumilio proteins directly bind the CCR4-NOT deadenylase complex to regulate the transcriptome

2020 ◽  
Author(s):  
Isioma I.I. Enwerem ◽  
Nathan D. Elrod ◽  
Chung-Te Chang ◽  
Ai Lin ◽  
Ping Ji ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Molecular Model ◽  
Direct Interaction ◽  
Vertebrate Development ◽  
Regulate Gene Expression ◽  
Mrna Decapping ◽  
Specific Mrnas ◽  
Decapping Enzyme ◽  
Pumilio Proteins

AbstractPumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome. We identified subunits of the CCR4-NOT (CNOT) deadenylase complex required for stable interaction with PUM1&2 and to elicit CNOT-dependent repression. Isoform-level RNA sequencing revealed broad co-regulation of target mRNAs through the PUM-CNOT repression mechanism.Functional dissection of the domains of PUM1&2 identified a conserved N-terminal region that confers the predominant repressive activity via direct interaction with CNOT. In addition, we show that the mRNA decapping enzyme, DCP2, has an important role in repression by PUM1&2 N-terminal regions. Our results support a molecular model of repression by human PUM1&2 via direct recruitment of CNOT deadenylation machinery in a decapping-dependent mRNA decay pathway.

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