Authentication of Hippuristanol-eIF4A1 Target Engagement Facilitates Identification of eIF4A1 Helicase Dependencies within 5’ Leader Regions
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA636959 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11915236.fastq.gz | Fastqsanger.gz | |||
| SRR11915237.fastq.gz | Fastqsanger.gz | |||
| SRR11915238.fastq.gz | Fastqsanger.gz | |||
| SRR11915239.fastq.gz | Fastqsanger.gz | |||
| SRR11915240.fastq.gz | Fastqsanger.gz |
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Project description:Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitmentt o mRNA templates. Using a CRISPR/Cas9-based variomics screen, we identify functional eIF4A1 Hipp-resistant alleles, which in turn allow us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target-engagement. Genome-wide translational profiling in the absence or presence of Hipp (~EC50) were undertaken and our validation studies provided insight into structure-activity relationships of eIF4A-dependent mRNAs.
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Project description:Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5' leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5' leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.
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Project description:EIF4A1 and cofactors EIF4B and EIF4H have been well characterised in cancers, including B cell malignancies, for their ability to promote the translation of oncogenes with structured 5’ untranslated regions but very little is known of their roles in non-malignant cells. Using mouse models to delete Eif4a1, Eif4b or Eif4h in B cells we show that EIF4A1, but not EIF4B or EIF4H, is essential for B cell development and the germinal centre response. Following activation, EIF4A1 facilitates an increased rate of protein synthesis, MYC expression and expression of cell cycle regulators. However, EIF4A1-deficient cells remain viable whereas Hippuristanol treatment induces cell death.
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Project description:Eukaryotic translation initiation factor (eIF) 4A — a DEAD-box RNA-binding protein — plays an essential role in translation initiation. Recent reports have suggested helicase-dependent and helicase-independent functions for eIF4A, but the multifaceted roles of eIF4A have not been fully explored. Here, we show that eIF4A1 enhances translational repression during the inhibition of mechanistic target of rapamycin complex 1 (mTORC1), an essential kinase complex controlling cell proliferation. RNA pulldown followed by sequencing revealed that eIF4A1 preferentially binds to mRNAs containing terminal oligopyrimidine (TOP) motifs (TOP mRNAs), whose translation is rapidly repressed upon mTORC1 inhibition. This selective interaction depends on a La-related RNA-binding protein, LARP1. Ribosome profiling revealed that deletion of EIF4A1 attenuated the translational repression of TOP mRNAs upon mTORC1 inactivation. Moreover, eIF4A1 increases the affinity between TOP mRNAs and LARP1 and thus ensures stronger translational repression upon mTORC1 inhibition. Our data show the multimodality of eIF4A1 in modulating protein synthesis through an inhibitory binding partner and provide a unique example of the repressive role of a universal translational activator.
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Project description:The PIWI-interacting RNA (piRNA) pathway guides the DNA methylation of young active transposons during male mouse germline development. piRNAs tether the PIWI protein MIWI2 (PIWIL4) to the nascent transposon transcript that results in DNA methylation through SPOCD1. Transposon methylation requires exacting precision: all copies need to be methylated yet, at the same time, off-target methylation must be avoided. However, the underlying mechanisms that ensure this precision remain unknown. Here, we show that SPOCD1 directly interacts with SPIN1 (SPINDLIN1), a chromatin reader that primarily binds H3K4me3 K9me3. The prevailing assumption is that all molecular events required for piRNA-directed DNA methylation occur after the engagement of MIWI2. Interestingly, we find that SPIN1 expression precedes that of both SPOCD1 and MIWI2. Furthermore, we demonstrate that young LINE1s, but not old copies are marked by H3K4me3, H3K9me3 and SPIN1 prior to the initiation of piRNA-directed DNA methylation. We generated a Spocd1 separation-of-function allele in the mouse that encodes a SPOCD1 variant that no longer interacts with SPIN1. We found that the SPOCD1-SPIN1 interaction is essential for spermatogenesis and piRNA-directed DNA methylation of young LINE1 elements. We propose that young LINE1 elements require a two-factor authentication process for DNA methylation, the first being the recruitment of SPIN1-SPOCD1 to licence the locus and the second is MIWI2 engagement with the nascent transcript, which is also the trigger for methylation. In summary, independent events that licence, and trigger methylation underpin the basis of precision.
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Project description:The PIWI-interacting RNA (piRNA) pathway guides the DNA methylation of young active transposons during male mouse germline development. piRNAs tether the PIWI protein MIWI2 (PIWIL4) to the nascent transposon transcript that results in DNA methylation through SPOCD1. Transposon methylation requires exacting precision: all copies need to be methylated yet, at the same time, off-target methylation must be avoided. However, the underlying mechanisms that ensure this precision remain unknown. Here, we show that SPOCD1 directly interacts with SPIN1, a chromatin reader that primarily binds H3K4me3 and this association is augmented by H3K9me3. The prevailing assumption is that all molecular events required for piRNA-directed DNA methylation occur after the engagement of MIWI2. Interestingly, we find that SPIN1 expression precedes that of both SPOCD1 and MIWI2. Furthermore, we demonstrate that young LINE1s, but not old copies are marked by H3K4me3 and H3K9me3 prior to the initiation of piRNA-directed DNA methylation. We generated a Spocd1 separation-of-function allele in the mouse that encodes a SPOCD1 variant that no longer interacts with SPIN1. We found that the SPOCD1-SPIN1 interaction is essential for spermatogenesis and piRNA-directed DNA methylation of young LINE1 elements. We propose that young LINE1 elements require a two-factor authentication process for DNA methylation, the first being the recruitment of SPIN1-SPOCD1 to licence the locus and the second is MIWI2 engagement with the nascent transcript, which is the trigger for methylation. In summary, independent events that licence, and trigger methylation underpin the basis of precision.
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