Direct role for the Drosophila GIGYF protein in 4EHP-mediated mRNA repression

Abstract

The eIF4E-homologous protein (4EHP) is a translational repressor that competes with eIF4E for binding to the 5′-cap structure of specific mRNAs, to which it is recruited by protein factors such as the GRB10-interacting GYF (glycine-tyrosine-phenylalanine domain) proteins (GIGYF). Several experimental evidences suggest that GIGYF proteins are not merely facilitating 4EHP recruitment to transcripts but are actually required for the repressor activity of the complex. However, the underlying molecular mechanism is unknown. Here, we investigated the role of the uncharacterized Drosophila melanogaster (Dm) GIGYF protein in post-transcriptional mRNA regulation. We show that, when in complex with 4EHP, Dm GIGYF not only elicits translational repression but also promotes target mRNA decay via the recruitment of additional effector proteins. We identified the RNA helicase Me31B/DDX6, the decapping activator HPat and the CCR4–NOT deadenylase complex as binding partners of GIGYF proteins. Recruitment of Me31B and HPat via discrete binding motifs conserved among metazoan GIGYF proteins is required for downregulation of mRNA expression by the 4EHP–GIGYF complex. Our findings are consistent with a model in which GIGYF proteins additionally recruit decapping and deadenylation complexes to 4EHP-containing RNPs to induce translational repression and degradation of mRNA targets.

Molecular basis for GIGYF–Me31B complex assembly in 4EHP-mediated translational repression

“…1B;Supplemental Fig. S1B,C;Ruscica et al 2019). The MBM alone interacted with Me31B/DDX6 as efficiently as full-length (FL) GIGYF or the N-terminal fragment of GIGYF ( Fig.…”

“…The GIGYF orthologs contain a short conserved sequence motif with partial similarity to the CUP homology domain (CHD) present in 4E-T proteins ( Fig. 1A; Kamenska et al 2014;Ruscica et al 2019). Deletion of this Me31B/DDX6-binding motif (MBM) abrogated the interaction of Me31B/DDX6 with transiently expressed and tagged GIGYF (Dm GIGYF and Homo sapiens [Hs] GIGYF1/2) in Drosophila and human cells (Fig.…”

4EHP and GIGYF1/2 Mediate Translation-Coupled Messenger RNA Decay

“…GIGYF1/2-directed recruitment of 4EHP to the cap not only reduces translation initiation but also facilitates the activity of the decay machinery. The reduced cap affinity of 4EHP compared with that of eIF4E (Chapat et al, 2017;Peter et al, 2017;Rom et al, 1998;Zuberek et al, 2007) exposes the mRNA to decapping (Ruscica et al, 2019). A scenario in which the recruitment of deadenylation and decapping factors by GIGYF1/2 occurs co-translationally is in agreement with the ribosomal association and activity of decay factors, such as DDX6 (Sweet et al, 2012), the CCR4-NOT complex (Buschauer et al, 2020), and XRN1 (Pelechano et al, 2015;Tesina et al, 2019;Tuck et al, 2020), and would irreversibly prevent the translation of transcripts with impaired elongation.…”

The TUTase URT1 connects decapping activators and prevents the accumulation of excessively deadenylated mRNAs to avoid siRNA biogenesis

“…EXA1 is a GYF domain-containing protein, orthologous to human and Drosophila GRB10-interacting GYF domain proteins (GIGYF). GIGYF proteins interact with the deadenylation complex CCR4-NOT and several translational repressors or decapping activators such as the 5' cap-binding protein eIF4E-Homologous Protein (4EHP), the RNA helicase DDX6/Me31B and the decapping activator PAT1 20,21 . Interestingly, orthologs of all known GIGYF interactors are also significantly enriched in URT1 IPs alongside EXA1.…”

“…Those specificity factors include the RNA induced silencing complex (RISC) and several RNA binding proteins (RBPs), such as Tristetraprolin (TTP), Pumilio/fem-3 mRNA binding factor (PUF) proteins and the YT521-B homology (YTH) domain-containing family proteins YTHDF2 and Meiotic mRNA interception protein 1 (Mmi1) 13,[16][17][18][19] . In addition, the CCR4-NOT complex interacts with central regulators of translation and decapping, such as the GRB10-interacting GYF (glycine-tyrosinephenylalanine domain) proteins (GIGYF) 20,21 and the DExD/H-box RNA helicases DDX6, Dhh1 or Maternal expression at 31B (Me31B) in humans, yeast and Drosophila, respectively 13 . DDX6 also interacts with the decapping activators Enhancer of mRNA-decapping protein 3 (EDC3), DNA topoisomerase 2-associated protein (PAT1) and Like SM homolog 14 (LSM14), also called Suppressor of clathrin deficiency (Scd6) in yeast.…”