No abstract

Regulation of protein synthesis is fundamental for all aspects of eukaryotic biology by controlling development, homeostasis, and stress responses1,2. The 13-subunit, 800-kDa eukaryotic initiation factor 3 (eIF3) organizes initiation factor and ribosome interactions required for productive translation3. However, current understanding of eIF3 function does not explain genetic evidence correlating eIF3 deregulation with tissue-specific cancers and developmental defects4. Here we report the genome-wide discovery of human transcripts that interact with eIF3 using photo-activatable crosslinking and immunoprecipitation (PAR-CLIP)5. eIF3 binds to a highly specific programme of messenger RNAs (mRNAs) involved in cell growth control processes, including cell cycling, differentiation, and apoptosis, via the mRNA 5′ untranslated region (5′ UTR). Surprisingly, functional analysis of the interaction between eIF3 and two mRNAs encoding cell proliferation regulators, c-Jun and BTG1, reveals that eIF3 employs different modes of RNA stem loop binding to exert either translational activation or repression. Our findings illuminate a new role for eIF3 in governing a specialized repertoire of gene expression and suggest that binding of eIF3 to specific mRNAs could be targeted to control carcinogenesis.

Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1–Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1–Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit, whereas Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting A-T rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1–Cas2 complex to be the minimal machinery that catalyzes spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1–Cas2-mediated adaptive immunity.

Eukaryotic mRNAs contain a 5' cap structure critical for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression1 despite eIF4E inactivation. Here we describe a new cellular cap-dependent pathway of translation initiation that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eukaryotic initiation factor 3 (eIF3) complex. A 1.4 Å crystal structure of the eIF3d cap-binding domain reveals unexpected homology to endonucleases involved in RNA turnover, and allows modeling of cap recognition by eIF3d. eIF3d makes specific contacts to the cap, as exemplified by cap analog competition, and these interactions are essential for assembly of translation initiation complexes on eIF3-specialized mRNAs2 such as the cell proliferation regulator c-Jun. The c-Jun mRNA further encodes an inhibitory RNA element that blocks eIF4E recruitment, thus enforcing alternative cap recognition by eIF3d. Our results reveal a new mechanism of cap-dependent translation independent of eIF4E, and illustrate how modular RNA elements work in concert to direct specialized forms of translation initiation.