Rapid Creation of Stable Mammalian Cell Lines for Regulated Expression of Proteins Using the Gateway® Recombination Cloning Technology and Flp-In T-REx® Lines

Spitzer, Jessica; Landthaler, Markus; Tuschl, Thomas

doi:10.1016/b978-0-12-418687-3.00008-2

Cited by 25 publications

(25 citation statements)

References 6 publications

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“…11965118) supplemented with 5% FBS, 5% Cosmic calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 100 μg/mL zeocin, and 15 μg/mL blasticidin. Cell lines expressing inducible BirA-4EHP or -eIF4E were generated as described (45) and selected and maintained in medium supplemented with 100 μg/mL hygromycin. Expression of tagged proteins was induced for 24 h by addition of tetracycline to 1 μg/mL final concentration.…”

Section: Methodsmentioning

confidence: 99%

Cap-binding protein 4EHP effects translation silencing by microRNAs

Chapat

Jafarnejad

Matta‐Camacho

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

111

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MicroRNAs (miRNAs) play critical roles in a broad variety of biological processes by inhibiting translation initiation and by destabilizing target mRNAs. The CCR4-NOT complex effects miRNA-mediated silencing, at least in part through interactions with 4E-T (eIF4E transporter) protein, but the precise mechanism is unknown. Here we show that the cap-binding eIF4E-homologous protein 4EHP is an integral component of the miRNA-mediated silencing machinery. We demonstrate that the cap-binding activity of 4EHP contributes to the translational silencing by miRNAs through the CCR4-NOT complex. Our results show that 4EHP competes with eIF4E for binding to 4E-T, and this interaction increases the affinity of 4EHP for the cap. We propose a model wherein the 4E-T/4EHP interaction engenders a closed-loop mRNA conformation that blocks translational initiation of miRNA targets.M icroRNAs (miRNAs) are short, ∼22-nucleotide noncoding RNAs that affect gene expression in most eukaryotes. miRNAs mediate posttranscriptional silencing by guiding the miRNA-induced silencing complex (miRISC), an assembly of Argonautes and GW182/TNRC6 proteins, to target mRNAs. Target recognition initiates a succession of events: mRNA translational repression, deadenylation, and mRNA decay (1). miRNAs impair the function of eIF4F, a three-subunit complex composed of eIF4E, the m 7 GTP (cap)-interacting factor; eIF4G, a scaffolding protein; and eIF4A, a DEAD-box RNA helicase (2-5). The silencing activity of miRISC is mediated by the CCR4-NOT deadenylase complex through the scaffolding subunit, CNOT1 (6-8). CNOT1 recruits the DDX6 and 4E-T (eIF4E transporter, also known as EIF4ENIF1) proteins, which are important for miRNA-mediated silencing (9-16). The 4E-T protein is a conserved eIF4E-binding protein, which directly binds to the dorsal surface of eIF4E through its canonical eIF4E-binding YX 4 LL (Y 30 TKEELL) motif and impairs the eIF4E/eIF4G interaction and translation initiation (17). The 4E-T protein also facilitates the decay of CCR4-NOT-targeted mRNAs by linking the 3′-terminal mRNA decay machinery to the cap via its interaction with eIF4E (13).In mammals, eIF4E is the best-studied member of a family of proteins composed of eIF4E (eIF4E1), 4EHP (4E-homologous protein; eIF4E2), and eIF4E3. The 4EHP and eIF4E proteins share 28% sequence identity (18,19). The 4EHP protein is ubiquitously expressed, and it is 5-10 times less abundant than eIF4E in a number of mammalian cell lines (18)(19)(20). Like eIF4E, 4EHP binds to 4E-T, but in sharp contrast to eIF4E, it does not associate with eIF4G (18, 21). The 4EHP protein has a 30-to 100-fold weaker affinity for the cap than eIF4E due to a twoamino acid substitution in its cap-binding pocket (22).The 4EHP protein has primarily been documented as a translation repressor. In the Drosophila embryo, 4EHP associates with the RNA binding protein Bicoid to repress caudal mRNA translation (23). Similarly, 4EHP also represses the hunchback mRNA by binding to the nanos repressive element complex, which consists of nanos...

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Section: Methodsmentioning

confidence: 99%

Cap-binding protein 4EHP effects translation silencing by microRNAs

Chapat

Jafarnejad

Matta‐Camacho

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

111

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“…We generated stable HEK293 cell lines for doxycycline-inducible expression of FLAG/HA-tagged human LIN28A and LIN28B (Spitzer et al 2011). Recombinant human LIN28A and LIN28B proteins were obtained by bacterial expression (Supplemental Fig.…”

Section: Lin28a and B Are Predominantly Cytoplasmic Mrna-binding Protmentioning

confidence: 99%

“…HEK293 cells expressing FLAG/HA-tagged LIN28A and LIN28B were prepared as described (Landthaler et al 2008;Spitzer et al 2011). Cells were maintained in DMEM containing 10% FBS, 2 mM glutamine, 15 μg/mL blasticidin, and 100 μg/mL hygromycin.…”

Section: Cell Culturementioning

confidence: 99%

Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition

Hafner

Max

Bandaru

et al. 2013

RNA

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152

179

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Human LIN28A and LIN28B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ∼3000 mRNAs at ∼9500 sites located in the 3 ′ UTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors.

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“…Stable cell lines inducibly and constitutively expressing FLAG-HAtagged RBPMS and RBPMS2 were generated using the Gateway Recombination Cloning Technology and Flp-In T-REx HEK293 cell lines (Invitrogen) as previously described (for detailed protocol see Spitzer et al 2013). Plasmids for generation of these cell lines are available from Addgene.…”

Section: Stable Cell Lines and Their Culturementioning

confidence: 99%

Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets

Farazi

Leonhardt

Mukherjee

et al. 2014

RNA

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Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. We defined transcriptome-wide RNA targets using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing exonic mature and intronic pre-mRNA binding sites, in agreement with the nuclear and cytoplasmic localization of the proteins. Computational and biochemical approaches defined the RNA recognition element (RRE) as a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to other mRNA-binding proteins, RBPMS family of proteins relocalized to cytoplasmic stress granules under oxidative stress conditions suggestive of a support function for mRNA localization in large and/or multinucleated cells where it is preferentially expressed.

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Rapid Creation of Stable Mammalian Cell Lines for Regulated Expression of Proteins Using the Gateway® Recombination Cloning Technology and Flp-In T-REx® Lines

Cited by 25 publications

References 6 publications

Cap-binding protein 4EHP effects translation silencing by microRNAs

Cap-binding protein 4EHP effects translation silencing by microRNAs

Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition

Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets

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