Two germ granule eIF4E isoforms reside in different mRNPs to hand off C elegans mRNAs from translational repression to activation.

We studied the function of translation factor eIF4E isoforms in regulating mRNAs in germ cell granules/condensates. Translational control of mRNAs plays an essential role in germ cell gene regulation. Messenger ribonucleoprotein (mRNP) complexes assemble on mRNAs as they move from the nucleus into perinuclear germ granules to exert both positive and negative post-transcriptional regulation in the cytoplasm. In C. elegans , germ granules are surprisingly dynamic mRNP condensates that remodel during development. Two eIF4E isoforms (called IFE-1 and IFE-3), eIF4E-Interacting Proteins (4EIPs), RBPs, DEAD-box helicases, polyadenylated mRNAs, Argonautes and miRNAs all occupy positions in germ granules. Affinity purification of IFE-1 and IFE-3 allowed mass spectrometry and mRNA-Seq to identify the proteins and mRNAs that populate stable eIF4E mRNPs. We find translationally controlled mRNAs (e.g. pos-1, mex-3, spn-4, etc.) enriched in IFE-3 mRNPs, but excluded from IFE-1 mRNPs. These mRNAs also require IFE-1 for efficient translation. The findings support a model in which oocytes and embryos utilize the two eIF4Es for opposite purposes on critically regulated germline mRNAs. Careful colocalization of the eIF4Es with other germ granule components suggests an architecture in which GLH-1, PGL-1 and the IFEs are stratified to facilitate sequential interactions for mRNAs. Biochemical characterization demonstrates opposing yet cooperative roles for IFE-3 and IFE-1 to hand-off of translationally controlled mRNAs from the repressed to the activated state, respectively. The model involves eIF4E mRNPs shuttling mRNAs through nuclear pore-associated granules/condensates to cytoplasmic ribosomes.

Silencing of Oncogenic KRAS by Mutant-Selective Small Interfering RNA.

Oncogenic mutations in the KRAS gene are well-established drivers of cancer. While the recently developed KRASG12C inhibitors offer a targeted KRAS therapy and have shown success in the clinic, KRASG12C represents only 11% of all KRAS mutations. Current therapeutic approaches for all other KRAS mutations are both indirect and nonmutant-selective, largely focusing on inhibition of downstream KRAS effectors such as MAP kinases. Inhibition of KRAS downstream signaling results in a system-wide down-modulation of the respective targets, raising concerns about systemic cell toxicity. Here, we describe a custom short interfering RNA oligonucleotide (EFTX-D1) designed to preferentially bind mRNA of the most commonly occurring KRAS missense mutations in codons 12 and 13. We determined that EFTX-D1 preferentially reduced the mutant KRAS sequence versus wild-type at the levels of both transcription and translation and reversed oncogenic KRAS-induced morphologic and growth transformation. Furthermore, EFTX-D1 significantly impaired the proliferation of several KRAS mutant cancer cell lines in 2-D as well as 3-D assays. Taken together, our data indicate a novel use of RNA interference to target oncogenic KRAS-driven cancers specifically.

Regulation of Germ Cell mRNPs by eIF4E:4EIP Complexes: Multiple Mechanisms, One Goal.

Translational regulation of mRNAs is critically important for proper gene expression in germ cells, gametes, and embryos. The ability of the nucleus to control gene expression in these systems may be limited due to spatial or temporal constraints, as well as the breadth of gene products they express to prepare for the rapid animal development that follows. During development germ granules are hubs of post-transcriptional regulation of mRNAs. They assemble and remodel messenger ribonucleoprotein (mRNP) complexes for translational repression or activation. Recently, mRNPs have been appreciated as discrete regulatory units, whose function is dictated by the many positive and negative acting factors within the complex. Repressed mRNPs must be activated for translation on ribosomes to introduce novel proteins into germ cells. The binding of eIF4E to interacting proteins (4EIPs) that sequester it represents a node that controls many aspects of mRNP fate including localization, stability, poly(A) elongation, deadenylation, and translational activation/repression. Furthermore, plants and animals have evolved to express multiple functionally distinct eIF4E and 4EIP variants within germ cells, giving rise to different modes of translational regulation.

Distinct roles of two eIF4E isoforms in the germline of Caenorhabditis elegans.

Germ cells use both positive and negative mRNA translational control to regulate gene expression that drives their differentiation into gametes. mRNA translational control is mediated by RNA-binding proteins, miRNAs and translation initiation factors. We have uncovered the discrete roles of two translation initiation factor eIF4E isoforms (IFE-1, IFE-3) that bind 7-methylguanosine (m7G) mRNA caps during Caenorhabditiselegans germline development. IFE-3 plays important roles in germline sex determination (GSD), where it promotes oocyte cell fate and is dispensable for spermatogenesis. IFE-3 is expressed throughout the germline and localizes to germ granules, but is distinct from IFE-1 and PGL-1, and facilitates oocyte growth and viability. This contrasts with the robust expression in spermatocytes of IFE-1, the isoform that resides within P granules in spermatocytes and oocytes, and promotes late spermatogenesis. Each eIF4E is localized by its cognate eIF4E-binding protein (IFE-1:PGL-1 and IFE-3:IFET-1). IFE-3 and IFET-1 regulate translation of several GSD mRNAs, but not those under control of IFE-1. Distinct mutant phenotypes, in vivo localization and differential mRNA translation suggest independent dormant and active periods for each eIF4E isoform in the germline.