Yuri V. Svitkin

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.

A NOVEL FUNCTIONAL HUMAN EUKARYOTIC TRANSLATION INITIATION FACTOR 4G

ABSTRACT Mammalian eukaryotic translation initiation factor 4F (eIF4F) is a cap-binding protein complex consisting of three subunits: eIF4E, eIF4A, and eIF4G. In yeast and plants, two related eIF4G species are encoded by two different genes. To date, however, only one functional eIF4G polypeptide, referred to here as eIF4GI, has been identified in mammals. Here we describe the discovery and functional characterization of a closely related homolog, referred to as eIF4GII. eIF4GI and eIF4GII share 46% identity at the amino acid level and possess an overall similarity of 56%. The homology is particularly high in certain regions of the central and carboxy portions, while the amino-terminal regions are more divergent. Far-Western analysis and coimmunoprecipitation experiments were used to demonstrate that eIF4GII directly interacts with eIF4E, eIF4A, and eIF3. eIF4GII, like eIF4GI, is also cleaved upon picornavirus infection. eIF4GII restores cap-dependent translation in a reticulocyte lysate which had been pretreated with rhinovirus 2A to cleave endogenous eIF4G. Finally, eIF4GII exists as a complex with eIF4E in HeLa cells, because eIF4GII and eIF4E can be purified together by cap affinity chromatography. Taken together, our findings indicate that eIF4GII is a functional homolog of eIF4GI. These results may have important implications for the understanding of the mechanism of shutoff of host protein synthesis following picornavirus infection.

Translational control of the innate immune response through IRF-7

Transcriptional activation of cytokines, such as type-I interferons (interferon (IFN)-α and IFN-β), constitutes the first line of antiviral defence. Here we show that translational control is critical for induction of type-I IFN production. In mouse embryonic fibroblasts lacking the translational repressors 4E-BP1 and 4E-BP2, the threshold for eliciting type-I IFN production is lowered. Consequently, replication of encephalomyocarditis virus, vesicular stomatitis virus, influenza virus and Sindbis virus is markedly suppressed. Furthermore, mice with both 4E- and 4E-BP2 genes (also known as Eif4ebp1 and Eif4ebp2, respectively) knocked out are resistant to vesicular stomatitis virus infection, and this correlates with an enhanced type-I IFN production in plasmacytoid dendritic cells and the expression of IFN-regulated genes in the lungs. The enhanced type-I IFN response in 4E-BP1-/- 4E-BP2-/- double knockout mouse embryonic fibroblasts is caused by upregulation of interferon regulatory factor 7 (Irf7) messenger RNA translation. These findings highlight the role of 4E-BPs as negative regulators of type-I IFN production, via translational repression of Irf7 mRNA.

Prokaryotic-like Cis Elements in the Cap-Independent Internal Initiation of Translation on Picornavirus RNA

Initiation of translation on picornavirus RNAs is accomplished through internal binding of ribosomes to a complex cis-acting element. Here we show that efficient function of this element involves two appropriately spaced smaller elements: UUUCC and an AUG. This conclusion emerged from analysis of the genome structures of spontaneous revertants of mutant polioviruses with extended insertions between the UUUCC and AUG motifs. It was confirmed by the results obtained with specially designed constructs. A similarity to the prokaryotic translation initiation mechanism, which involves the Shine-Dalgarno sequence, is emphasized, but in the picornavirus system the position of the UUUCC must be strictly fixed relative to upstream cis-acting elements, and the AUG may not necessarily serve as an initiation codon.

The major mRNA‐associated protein YB‐1 is a potent 5′ cap‐dependent mRNA stabilizer

mRNA silencing and storage play an important role in gene expression under diverse circumstances, such as throughout early metazoan development and in response to many types of environmental stress. Here we demonstrate that the major mRNA‐associated protein YB‐1, also termed p50, is a potent cap‐dependent mRNA stabilizer. YB‐1 addition or overexpression dramatically increases mRNA stability in vitro and in vivo, whereas YB‐1 depletion results in accelerated mRNA decay. The cold shock domain of YB‐1 is responsible for the mRNA stabilizing activity, and a blocked mRNA 5′ end is required for YB‐1‐mediated stabilization. Significantly, exogenously added YB‐1 destabilizes the interaction of the cap binding protein, eIF4E, with the mRNA cap structure. Conversely, sequestration of eIF4E from the cap increases the association of endogenous YB‐1 with mRNA at or near the cap, and significantly enhances mRNA stability. These data support a model whereby down‐regulation of eIF4E activity or increasing the YB‐1 mRNA binding activity or concentration in cells activates a general default pathway for mRNA stabilization.

Cap and cap-binding proteins in the control of gene expression

The 5′ mRNA cap structure is essential for efficient gene expression from yeast to human. It plays a critical role in all aspects of the life cycle of an mRNA molecule. Capping occurs co‐transcriptionally on the nascent pre‐mRNA as it emerges from the RNA exit channel of RNA polymerase II. The cap structure protects mRNAs from degradation by exonucleases and promotes transcription, polyadenylation, splicing, and nuclear export of mRNA and U‐rich, capped snRNAs. In addition, the cap structure is required for the optimal translation of the vast majority of cellular mRNAs, and it also plays a prominent role in the expression of eukaryotic, viral, and parasite mRNAs. Cap‐binding proteins specifically bind to the cap structure and mediate its functions in the cell. Two major cellular cap‐binding proteins have been described to date: eukaryotic translation initiation factor 4E (eIF4E) in the cytoplasm and nuclear cap binding complex (nCBC), a nuclear complex consisting of a cap‐binding subunit cap‐binding protein 20 (CBP 20) and an auxiliary protein cap‐binding protein 80 (CBP 80). nCBC plays an important role in various aspects of nuclear mRNA metabolism such as pre‐mRNA splicing and nuclear export, whereas eIF4E acts primarily as a facilitator of mRNA translation. In this review, we highlight recent findings on the role of the cap structure and cap‐binding proteins in the regulation of gene expression. We also describe emerging regulatory pathways that control mRNA capping and cap‐binding proteins in the cell. WIREs RNA 2011 2 277–298 DOI: 10.1002/wrna.52

Eukaryotic Translation Initiation Factor 4E (eIF4E) Binding Site and the Middle One-Third of eIF4GI Constitute the Core Domain for Cap-Dependent Translation, and the C-Terminal One-Third Functions as a Modulatory Region

ABSTRACT The mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three roughly equal regions; an amino-terminal one-third (amino acids [aa] 1 to 634), which contains the poly(A) binding protein (PABP) and eIF4E binding sites; a middle third (aa 635 to 1039), which binds eIF4A and eIF3; and a carboxy-terminal third (aa 1040 to 1560), which harbors a second eIF4A binding site and a docking sequence for the Ser/Thr kinase Mnk1. Previous reports demonstrated that the middle one-third of eIF4GI is sufficient for cap-independent translation. To delineate the eIF4GI core sequence required for cap-dependent translation, various truncated versions of eIF4GI were examined in an in vitro ribosome binding assay with β-globin mRNA. A sequence of 540 aa encompassing aa 550 to 1090, which contains the eIF4E binding site and the middle region of eIF4GI, is the minimal sequence required for cap-dependent translation. In agreement with this, a point mutation in eIF4GI which abolished eIF4A binding in the middle region completely inhibited ribosomal binding. However, the eIF4GI C-terminal third region, which does not have a counterpart in yeast, modulates the activity of the core sequence. When the eIF4A binding site in the C-terminal region of eIF4GI was mutated, ribosome binding was decreased three- to fourfold. These data indicate that the interaction of eIF4A with the middle region of eIF4GI is necessary for translation, whereas the interaction of eIF4A with the C-terminal region plays a modulatory role.

General RNA binding proteins render translation cap dependent.

Translation in rabbit reticulocyte lysate is relatively independent of the presence of the mRNA m7G cap structure and the cap binding protein, eIF‐4E. In addition, initiation occurs frequently at spurious internal sites. Here we show that a critical parameter which contributes to cap‐dependent translation is the amount of general RNA binding proteins in the extract. Addition of several general RNA binding proteins, such as hnRNP A1, La autoantigen, pyrimidine tract binding protein (hnRNP I/PTB) and the major core protein of cytoplasmic mRNP (p50), rendered translation in a rabbit reticulocyte lysate cap dependent. These proteins drastically inhibited the translation of an uncapped mRNA, but had no effect on translation of a capped mRNA. Based on these and other results, we suggest that one function of general mRNA binding proteins in the cytoplasm is to promote ribosome binding by a 5′ end, cap‐mediated mechanism, and prevent spurious initiations at aberrant translation start sites.

Eukaryotic Translation Initiation Factor 4E Availability Controls the Switch between Cap-Dependent and Internal Ribosomal Entry Site-Mediated Translation

ABSTRACT Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5′ end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.

Dual interactions of the translational repressor Paip2 with poly(A) binding protein.

ABSTRACT The cap structure and the poly(A) tail of eukaryotic mRNAs act synergistically to enhance translation. This effect is mediated by a direct interaction of eukaryotic initiation factor 4G and poly(A) binding protein (PABP), which brings about circularization of the mRNA. Of the two recently identified PABP-interacting proteins, one, Paip1, stimulates translation, and the other, Paip2, which competes with Paip1 for binding to PABP, represses translation. Here we studied the Paip2-PABP interaction. Biacore data and far-Western analysis revealed that Paip2 contains two binding sites for PABP, one encompassing a 16-amino-acid stretch located in the C terminus and a second encompassing a larger central region. PABP also contains two binding regions for Paip2, one located in the RNA recognition motif (RRM) region and the other in the carboxy-terminal region. A two-to-one stoichiometry for binding of Paip2 to PABP with two independentKd s of 0.66 and 74 nM was determined. Thus, our data demonstrate that PABP and Paip2 could form a trimeric complex containing one PABP molecule and two Paip2 molecules. Significantly, only the central Paip2 fragment, which binds with high affinity to the PABP RRM region, inhibits PABP binding to poly(A) RNA and translation.