William C. Merrick

MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F

MicroRNAs (miRNAs) play an important role in gene regulatory networks in animals. Yet, the mechanistic details of their function in translation inhibition or messenger RNA (mRNA) destabilization remain controversial. To directly examine the earliest events in this process, we have developed an in vitro translation system using mouse Krebs-2 ascites cell–free extract that exhibits an authentic miRNA response. We show here that translation initiation, specifically the 5′ cap recognition process, is repressed by endogenous let-7 miRNAs within the first 15 minutes of mRNA exposure to the extract when no destabilization of the transcript is observed. Our results indicate that inhibition of translation initiation is the earliest molecular event effected by miRNAs. Other mechanisms, such as mRNA degradation, may subsequently consolidate mRNA silencing.

2 The Pathway and Mechanism of Initiation of Protein Synthesis

Elucidation of the detailed molecular mechanism of protein synthesis is essential for understanding translational controls. This chapter is concerned with the prokaryotic and eukaryotic pathways of initiation, where most translational controls are found (Chapter 1). In general terms, it focuses on (1) how the initiation factors catalyze the binding of the initiator tRNA and mRNA to the small ribosomal subunit; (2) how the initiation codon is recognized; and (3) how the large ribosomal subunit joins to form an initiation complex capable of elongation. Considerable progress has been made during the past 5 years in refining our knowledge of the pathway and in determining the three-dimensional structures of some of the macromolecular components of initiation. Emphasis is placed on the structures of the initiation factors and how the factors function to promote and regulate the pathway. The reader is directed to other chapters in this volume for descriptions of elongation (Chapter 3) and termination (Chapter 11). The process of translation initiation was elucidated during the late 1960s through the 1970s primarily by biochemical studies that utilized radiolabeled amino acids and fractionated lysates derived from bacterial or mammalian cells. The major macromolecular components were identified by purifying proteins and nucleic acids required to reconstitute translation in vitro. The ribosome itself was viewed essentially as a “black box” that appeared to provide a rigid surface onto which the other translational components bind. Surprisingly, genetic approaches contributed only modestly to the identification of the 200 or more macromolecular components that comprise the translational apparatus.

Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin

Although the protein synthesis inhibitor cycloheximide (CHX) has been known for decades, its precise mechanism of action remains incompletely understood. The glutarimide portion of CHX is seen in a family of structurally related natural products including migrastatin, isomigrastatin and lactimidomycin (LTM). LTM, isomigrastatin and analogs were found to have a potent antiproliferative effect on tumor cell lines and selectively inhibit protein translation. A systematic comparative study of the effects of CHX and LTM on protein translation revealed both similarities and differences between the two inhibitors. Both LTM and CHX were found to block the translocation step in elongation. Footprinting experiments revealed protection of a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit, defining a common binding pocket for both inhibitors in the ribosome. These results shed new light on the molecular mechanism of inhibition of translation elongation by both CHX and LTM.

The Transformation Suppressor Pdcd4 Is a Novel Eukaryotic Translation Initiation Factor 4A Binding Protein That Inhibits Translation

ABSTRACT Pdcd4 is a novel transformation suppressor that inhibits tumor promoter-induced neoplastic transformation and the activation of AP-1-dependent transcription required for transformation. A yeast two-hybrid analysis revealed that Pdcd4 associates with the eukaryotic translation initiation factors eIF4AI and eIF4AII. Immunofluorescent confocal microscopy showed that Pdcd4 colocalizes with eIF4A in the cytoplasm. eIF4A is an ATP-dependent RNA helicase needed to unwind 5′ mRNA secondary structure. Recombinant Pdcd4 specifically inhibited the helicase activity of eIF4A and eIF4F. In vivo translation assays showed that Pdcd4 inhibited cap-dependent but not internal ribosome entry site (IRES)-dependent translation. In contrast, Pdcd4D418A, a mutant inactivated for binding to eIF4A, failed to inhibit cap-dependent or IRES-dependent translation or AP-1 transactivation. Recombinant Pdcd4 prevented eIF4A from binding to the C-terminal region of eIF4G (amino acids 1040 to 1560) but not to the middle region of eIF4G(amino acids 635 to 1039). In addition, both Pdcd4 and Pdcd4D418A bound to the middle region of eIF4G. The mechanism by which Pdcd4 inhibits translation thus appears to involve inhibition of eIF4A helicase, interference with eIF4A association-dissociation from eIF4G, and inhibition of eIF4A binding to the C-terminal domain of eIF4G. Pdcd4 binding to eIF4A is linked to its transformation-suppressing activity, as Pdcd4-eIF4A binding and consequent inhibition of translation are required for Pdcd4 transrepression of AP-1.

Dominant negative mutants of mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation.

Eukaryotic translation initiation factor‐4A (eIF‐4A) plays a critical role in binding of eukaryotic mRNAs to ribosomes. It has been biochemically characterized as an RNA‐dependent ATPase and RNA helicase and is a prototype for a growing family of putative RNA helicases termed the DEAD box family. It is required for mRNA‐ribosome binding both in its free form and as a subunit of the cap binding protein complex, eIF‐4F. To gain further understanding into the mechanism of action of eIF‐4A in mRNA‐ribosome binding, defective eIF‐4A mutants were tested for their abilities to function in a dominant negative manner in a rabbit reticulocyte translation system. Several mutants were demonstrated to be potent inhibitors of translation. Addition of mutant eIF‐4A to a rabbit reticulocyte translation system strongly inhibited translation of all mRNAs studied including those translated by a cap‐independent internal initiation mechanism. Addition of eIF‐4A or eIF‐4F relieved inhibition of translation, but eIF‐4F was six times more effective than eIF‐4A, whereas eIF‐4B or other translation factors failed to relieve the inhibition. Kinetic experiments demonstrated that mutant eIF‐4A is defective in recycling through eIF‐4F, thus explaining the dramatic inhibition of translation. Mutant eIF‐4A proteins also inhibited eIF‐4F‐dependent, but not eIF‐4A‐dependent RNA helicase activity. Taken together these results suggest that eIF‐4A functions primarily as a subunit of eIF‐4F, and that singular eIF‐4A is required to recycle through the complex during translation. Surprisingly, eIF‐4F, which binds to the cap structure, appears to be also required for the translation of naturally uncapped mRNAs.

Eukaryotic protein elongation factors.

In eukaryotes, peptide chain elongation is mediated by elongation factors EF-1 and EF-2. EF-1 is composed of a nucleotide-binding protein EF-1 alpha, and a nucleotide exchange protein complex, EF-1 beta gamma, while EF-2 catalyses the translocation of peptidyl-tRNA on the ribosome. Elongation factors are highly conserved among different species and may be involved in functions other than protein synthesis, such as organization of the mitotic apparatus, signal transduction, developmental regulation, ageing and transformation. Yeast contains a third factor, EF-3, whose structure and function is not yet well understood.

Biochemical and kinetic characterization of the RNA helicase activity of eukaryotic initiation factor 4A.

Eukaryotic initiation factor (eIF) 4A is the prototypic member of the DEAD box family of proteins and has been proposed to act as an RNA helicase to unwind secondary structure in the 5′-untranslated region of eukaryotic mRNAs. Previous studies have shown that the RNA helicase activity of eIF4A is dependent on the presence of a second initiation factor, eIF4B. In this report, eIF4A has been demonstrated to function independently of eIF4B as an ATP-dependent RNA helicase. The biochemical and kinetic properties of this activity were examined. By using a family of RNA duplexes with an unstructured single-stranded region followed by a duplex region of increasing length and stability, it was observed that the initial rate of duplex unwinding decreased with increasing stability of the duplex. Furthermore, the maximum amount of duplex unwound also decreased with increasing stability. Results suggest that eIF4A acts in a non-processive manner. eIF4B and eIF4H were shown to stimulate the helicase activity of eIF4A, allowing eIF4A to unwind longer, more stable duplexes with both an increase in initial rate and maximum amount of duplex unwound. A simple kinetic model is proposed to explain the mechanism by which eIF4A unwinds RNA duplex structures in an ATP-dependent manner.

Ribosomal RNA in Alzheimer disease is oxidized by bound redox-active iron.

Oxidative modification of cytoplasmic RNA in vulnerable neurons is an important, well documented feature of the pathophysiology of Alzheimer disease. Here we report that RNA-bound iron plays a pivotal role for RNA oxidation in vulnerable neurons in Alzheimer disease brain. The cytoplasm of hippocampal neurons showed significantly higher redox activity and iron(II) staining than age-matched controls. Notably, both were susceptible to RNase, suggesting a physical association of iron(II) with RNA. Ultrastructural analysis further suggested an endoplasmic reticulum association. Both rRNA and mRNA showed twice the iron binding as tRNA. rRNA, extremely abundant in neurons, was considered to provide the greatest number of iron binding sites among cytoplasmic RNA species. Interestingly, the difference of iron binding capacity disappeared after denaturation of RNA, suggesting that the higher order structure may contribute to the greater iron binding of rRNA. Reflecting the difference of iron binding capacity, oxidation of rRNA by the Fenton reaction formed 13 times more 8-hydroxyguanosine than tRNA. Consistent with in situ findings, ribosomes purified from Alzheimer hippocampus contained significantly higher levels of RNase-sensitive iron(II) and redox activity than control. Furthermore, only Alzheimer rRNA contains 8-hydroxyguanosine in reverse transcriptase-PCR. Addressing the biological significance of ribosome oxidation by redox-active iron, in vitro translation with oxidized ribosomes from rabbit reticulocyte showed a significant reduction of protein synthesis. In conclusion these results suggest that rRNA provides a binding site for redox-active iron and serves as a redox center within the cytoplasm of vulnerable neurons in Alzheimer disease in advance of the appearance of morphological change indicating neurodegeneration.

A new pathway of translational regulation mediated by eukaryotic initiation factor 3

We report a new pathway of translation regulation that may operate in interferon‐treated or virus‐infected mammalian cells. This pathway is activated by P56, a protein whose synthesis is strongly induced by interferons or double‐stranded RNA. Using a yeast two‐hybrid screen, we identified the P48 subunit of the mammalian translation initiation factor eIF‐3 as a protein that interacts with P56. The P56–P48 interaction was confirmed in human cells by co‐immunoprecipitation assays and confocal microscopy. Gel filtration assays revealed that P56 binds to the large eIF‐3 complex that contains P48. Purified recombinant P56 inhibited in vitro translation of reporter mRNAs in a dose‐dependent fashion, and that inhibition was reversed by the addition of purified eIF‐3. In vivo, expression of transfected P56 or induction of the endogenous P56 by interferon caused an inhibition of overall cellular protein synthesis and the synthesis of a transfected reporter protein. As expected, a P56 mutant that does not interact with P48 and eIF‐3 failed to inhibit protein synthesis in vitro and in vivo.

Structure of cDNAs Encoding Human Eukaryotic Initiation Factor 3 Subunits POSSIBLE ROLES IN RNA BINDING AND MACROMOLECULAR ASSEMBLY

The mammalian translation initiation factor 3 (eIF3), is a multiprotein complex of ∼600 kDa that binds to the 40 S ribosome and promotes the binding of methionyl-tRNA i and mRNA. cDNAs encoding 5 of the 10 subunits, namely eIF3-p170, -p116, -p110, -p48, and -p36, have been isolated previously. Here we report the cloning and characterization of human cDNAs encoding the major RNA binding subunit, eIF3-p66, and two additional subunits, eIF3-p47 and eIF3-p40. Each of these proteins is present in immunoprecipitates formed with affinity-purified anti-eIF3-p170 antibodies. Human eIF3-p66 shares 64% sequence identity with a hypothetical Caenorhabditis elegans protein, presumably the p66 homolog. Deletion analyses of recombinant derivatives of eIF3-p66 show that the RNA-binding domain lies within an N-terminal 71-amino acid region rich in lysine and arginine. The N-terminal regions of human eIF3-p40 and eIF3-p47 are related to each other and to 17 other eukaryotic proteins, including murine Mov-34, a subunit of the 26 S proteasome. Phylogenetic analyses of the 19 related protein sequences, called the Mov-34 family, distinguish five major subgroups, where eIF3-p40, eIF3-p47, and Mov-34 are each found in a different subgroup. The subunit composition of eIF3 appears to be highly conserved inDrosophila melanogaster, C. elegans, andArabidopsis thaliana, whereas only 5 homologs of the 10 subunits of mammalian eIF3 are encoded in S. cerevisiae.