recombinant human eif4e protein fused to his-tag at n-terminus  (Creative BioMart)

Journal: The Journal of Biological Chemistry

Article Title: Mechanistic differences in eukaryotic initiation factor requirements for eIF4GI-driven cap-independent translation of structured mRNAs

doi: 10.1016/j.jbc.2024.107866

Figure Lengend Snippet: Dissociation constant values of uncapped 5′UTR mRNAs binding with eIFs at 25 ± 0.5 °C

Article Snippet: Each 25 μl translation reaction mixture contained 70% v/v of RRL (Promega) supplemented with 0.5 mM MgCl 2 , 0.02 mM amino acid mixture, 10 U/μl RiboLock RNase Inhibitor (Thermo Fisher Scientific), and varying concentrations of purified eIF4A and eIF4E proteins in presence of RocA (2 mM stock in dimethylsulfoxide (MedChemExpress, catalog number HY-19356) and 4EGI-1 chemical (10 mM stock in dimethylsulfoxide) (Selleck Chemicals, catalog number S7369), ( and ).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanistic differences in eukaryotic initiation factor requirements for eIF4GI-driven cap-independent translation of structured mRNAs

doi: 10.1016/j.jbc.2024.107866

Figure Lengend Snippet: Comparison of fluorescein-labeled uncapped 5′UTRs of FGF-9 and HIF-1α binding with eIF4G1, eIF4G1-eIF4A, and eIF4G1-eIF4A-eIF4E complexes. Equilibrium binding titrations of fluorescein-labeled uncapped 5′UTRs of ( A ) FGF-9 and ( B ) HIF-1α mRNAs with eIF4GI 557-1599 alone, or a mixture of eIF4GI 557-1599 and eIF4A (eIF4GI 557-1599 •eIF4A) or eIF4GI 557-1599 and eIF4E (eIF4GI 557-1599 •eIF4E). Briefly, 10 nM fluorescein-labeled uncapped 5′UTR RNA oligonucleotide was titrated with increasing concentrations of protein/protein mixtures in the titration buffer at 25 ± 0.5 °C and the anisotropy at each titration point was measured using excitation and emission wavelengths of 495 nm and 520 nm, respectively. Data points correspond to the average of three independent anisotropy measurements. The curves represent the nonlinear fits used to obtain the corresponding K D values. eIF, eukaryotic initiation factor; FGF, fibroblast-growth factor; HIF-1α, hypoxia inducible factor 1 subunit alpha.

Article Snippet: Each 25 μl translation reaction mixture contained 70% v/v of RRL (Promega) supplemented with 0.5 mM MgCl 2 , 0.02 mM amino acid mixture, 10 U/μl RiboLock RNase Inhibitor (Thermo Fisher Scientific), and varying concentrations of purified eIF4A and eIF4E proteins in presence of RocA (2 mM stock in dimethylsulfoxide (MedChemExpress, catalog number HY-19356) and 4EGI-1 chemical (10 mM stock in dimethylsulfoxide) (Selleck Chemicals, catalog number S7369), ( and ).

Techniques: Comparison, Labeling, Binding Assay, Titration

Journal: The Journal of Biological Chemistry

Article Title: Mechanistic differences in eukaryotic initiation factor requirements for eIF4GI-driven cap-independent translation of structured mRNAs

doi: 10.1016/j.jbc.2024.107866

Figure Lengend Snippet: Effect of eIF4A and eIF4E on the cap-independent activities of FGF-9 and HIF-1α ApppG-capped-UTR-Luc-mRNA reporters. Translation yields of ( A ) ApppG-capped-FGF-9-UTR-Luc-mRNA and ( B ) ApppG-capped- HIF-1α-UTR-Luc-mRNA following treatment of the RRL with 60 nM of Rocaglamide (RRL (+)RocA ) and increasing concentration of eIF4A. C , translation yields of ApppG-capped-HIF-1α-UTR-Luc-mRNA following treatment of the RRL with 60 μM 4EGI-1 molecule (RRL (+)4EGI-1 ) and increasing concentration of eIF4E. Bar heights and error bars correspond to the average and standard deviations, respectively, of three independent luciferase activity measurements with control (DMSO) set at 100%. Data were analyzed by two-tailed unpaired Student’s t test: n.s, p = 0.12; ∗, p < 0.033; ∗∗, p = 0.002; ∗∗∗, p < 0.001. FGF-9, fibroblast-growth factor 9; HIF-1α, hypoxia inducible factor 1 subunit alpha; eIF, eukaryotic initiation factor; RRL, rabbit reticulocyte lysate; DMSO, dimethylsulfoxide.

Article Snippet: Each 25 μl translation reaction mixture contained 70% v/v of RRL (Promega) supplemented with 0.5 mM MgCl 2 , 0.02 mM amino acid mixture, 10 U/μl RiboLock RNase Inhibitor (Thermo Fisher Scientific), and varying concentrations of purified eIF4A and eIF4E proteins in presence of RocA (2 mM stock in dimethylsulfoxide (MedChemExpress, catalog number HY-19356) and 4EGI-1 chemical (10 mM stock in dimethylsulfoxide) (Selleck Chemicals, catalog number S7369), ( and ).

Techniques: Concentration Assay, Luciferase, Activity Assay, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Mechanistic differences in eukaryotic initiation factor requirements for eIF4GI-driven cap-independent translation of structured mRNAs

doi: 10.1016/j.jbc.2024.107866

Figure Lengend Snippet: Proposed model describing two possible mechanisms of cap-independent translation initiation of 5′UTRs of HIF-1α and FGF-9 encoding mRNAs employed by cells to mitigate cellular stress conditions. Under stress, cap-dependent translation is compromised due to m 7 G cap unavailability caused by the sequestration of eIF4E by hypophosphorylated 4E-binding proteins (4E-BP1) ( , ). Top panel shows when bound by eIF4A and eIF4E, eIF4GI protein undergoes a conformational change that enhances binding to CITE - elements in the HIF-1α 5′UTR. Using the CITE-like pathway, HIF-1α 5′UTR recruits the 43S PIC at or near its 5′ end. This complex scans downstream until it encounters a start codon and begins translation ( Top panel). In contrast, FGF-9 mRNA uses its IRES-like element to stably bind eIF4GI, which allows it to recruit the 43 S PIC within the vicinity of its start codon and initiate translation with little or no scanning ( Middle panel). Bottom panel showed nucleotide-bound eIF4A regulates the recruitment of unstructured RNA to eIF4GI. The 43S PIC scans through 5′ to 3′ on mRNA until it recognizes a start codon, facilitating translation initiation (Modified from Izidoro et al. 2022). CITE, cap-independent translation enhancer; IRES, internal ribosome entry site; eIF, eukaryotic initiation factor; FGF-9, fibroblast-growth factor 9; HIF-1α, hypoxia inducible factor 1 subunit alpha; m 7 G, methyl guanosine; PIC, preinitiation complex.

Article Snippet: Each 25 μl translation reaction mixture contained 70% v/v of RRL (Promega) supplemented with 0.5 mM MgCl 2 , 0.02 mM amino acid mixture, 10 U/μl RiboLock RNase Inhibitor (Thermo Fisher Scientific), and varying concentrations of purified eIF4A and eIF4E proteins in presence of RocA (2 mM stock in dimethylsulfoxide (MedChemExpress, catalog number HY-19356) and 4EGI-1 chemical (10 mM stock in dimethylsulfoxide) (Selleck Chemicals, catalog number S7369), ( and ).

Techniques: Binding Assay, Stable Transfection, Modification

Journal: BMC Molecular and Cell Biology

Article Title: Exonuclease resistant 18S and 25S ribosomal RNA components in yeast are possibly newly transcribed by RNA polymerase II

doi: 10.1186/s12860-020-00303-z

Figure Lengend Snippet: Presence of caps on 18S and 25S RNAs from stationary and BMH21 treated cells. a SYBR-gold stained gel showing that decapping did not affect RNA integrity. b Immunoblot of the gel ( a ) showing decrease in intensity after treatment with decapping enzyme. Some cross reactivity can be seen in lanes 1 and 3 (see text). c SYBR gold stained gel and corresponding Northern blot showing decapping of 18S, 25S and mRNAs. d SYBR gold stained gel showing total RNA extracted from mid-log (ML) and stationary (ST) C. albicans (lanes 2 and 3) and immunoblot (lanes 4 and 5) using anti-m7G-cap mAb to detect bands precipitated by cap binding protein (CBP) eIF4F. e Conditions in lanes 2 and 3 are the same as in ( d ), lanes 4 and 5 are untreated mid-log and stationary RNA. In lanes 6 and 7 RNA was decapped prior to precipitating with CBP, showing that decapping removes the target of eIF4E protein

Article Snippet: Two micrograms of total RNA from C. albicans were incubated with 1 μg of recombinant human eIF4E protein fused to His-tag at N-terminus (Creative BioMart) in binding buffer (25 mM Tris, pH 8.0, 150 mM NaCl, 1 mM DTT, 5 mM imidazole) and incubated at 4 °C overnight.

Techniques: Staining, Western Blot, Northern Blot, Binding Assay

Journal: BMC Molecular and Cell Biology

Article Title: Exonuclease resistant 18S and 25S ribosomal RNA components in yeast are possibly newly transcribed by RNA polymerase II

doi: 10.1186/s12860-020-00303-z

Figure Lengend Snippet: CBP and RIP qPCR analysis. ( a and b ) RT-qPCR quantification of 18S, 25S and ITS-1 molecules precipitated from total RNA by ( a ) eIF4E (CBP) and (B) anti-m7G-cap mAb, both from mid-log (ML) and stationary (St) organisms. ITS-1 was used as a negative control for the assay. Error bars represent standard deviation from three different experiments. P values generated by Student’s test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Two micrograms of total RNA from C. albicans were incubated with 1 μg of recombinant human eIF4E protein fused to His-tag at N-terminus (Creative BioMart) in binding buffer (25 mM Tris, pH 8.0, 150 mM NaCl, 1 mM DTT, 5 mM imidazole) and incubated at 4 °C overnight.

Techniques: Quantitative RT-PCR, Negative Control, Standard Deviation, Generated

Journal: PLoS ONE

Article Title: The Parkinson's Disease Associated LRRK2 Exhibits Weaker In Vitro Phosphorylation of 4E-BP Compared to Autophosphorylation

doi: 10.1371/journal.pone.0008730

Figure Lengend Snippet: (A) Time-course assays were performed for LRRK2 wild-type (upper panel) by measuring the incorporation of [γ 33 P] ATP at different time points (0, 5, 15, 30 and 60 mins) and at 3 µM concentrations of 4E-BP (upper left panel), 3 µM concentrations of eIF4E (upper middle panel) and 3 µM concentrations of 4E-BP and 3 µM concentrations of eIF4E (upper right panel), respectively, followed by SDS-PAGE and autoradiography. (B) Phosphorylation of 4E-BP by LRRK2-wild type were plotted as a function of time.

Article Snippet: Recombinant eIF4E protein was purchased from Origene (Rockville, MD, USA).

Techniques: SDS Page, Autoradiography, Phospho-proteomics