Edward Darzynkiewicz

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 nuclear cap binding protein complex involved in pre-mRNA splicing

A cap-binding protein complex (CBC) present in the nuclei of HeLa cells has been characterized. Purified CBC consists of two previously identified proteins, CBP80 and CBP20. These proteins are shown to cofractionate to apparent homogeneity and to be coimmunoprecipitable with anti-CBP80 antibodies. Analysis of the inhibition of pre-mRNA splicing in vitro and in vivo by chemically modified analogs of the cap structure, and of the binding of these analogs to CBC in vitro, suggests a role for the complex in splicing. Extracts immunodepleted of CBC do not efficiently splice an adenoviral pre-mRNA owing to blockage of an early step in splicing complex formation. CBC may therefore play a role in pre-mRNA recognition.

The trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear targeting signal

The ability of series of U1 snRNAs and U6 snRNAs to migrate into the nucleus of Xenopus oocytes after injection into the cytoplasm was analyzed. The U snRNAs were made either by injecting U snRNA genes into the nucleus of oocytes or, synthetically, by T7 RNA polymerase, incorporating a variety of cap structures. The results indicate that nuclear targeting of U1 snRNA requires both a trimethylguanosine cap structure and binding of at least one common U snRNP protein. Using synthetic U6 snRNAs, it is further demonstrated that the trimethylguanosine cap structure can act in nuclear targeting in the absence of the common U snRNP proteins. These results imply that U snRNP nuclear targeting signals are of a modular nature.

Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo

Vaccination with in vitro transcribed RNA coding for tumor antigens is considered a promising approach for cancer immunotherapy and has already entered human clinical testing. One of the basic objectives for development of RNA as a drug is the optimization of immunobioavailability of the encoded antigen in vivo. By analyzing the effect of different synthetic 5′ mRNA cap analogs on the kinetics of the encoded protein, we found that m27,2′−OGppSpG (β-S-ARCA) phosphorothioate caps, in particular the D1 diastereoisomer, profoundly enhance RNA stability and translational efficiency in immature but not mature dendritic cells. Moreover, in vivo delivery of the antigen as β-S-ARCA(D1)-capped RNA species is superior for protein expression and for efficient priming and expansion of naïve antigen-specific T cells in mice. Our findings establish 5′ mRNA cap analogs as yet another module for tuning immunopharmacological properties of recombinant antigen-encoding RNA for vaccination purposes.

Multiple Isoforms of Eukaryotic Protein Synthesis Initiation Factor 4E in Caenorhabditis elegans Can Distinguish between Mono- and Trimethylated mRNA Cap Structures

The rate-limiting step for cap-dependent translation initiation in eukaryotes is recruitment of mRNA to the ribosome. An early event in this process is recognition of the m7GTP-containing cap structure at the 5′-end of the mRNA by initiation factor eIF4E. In the nematode Caenorhabditis elegans, mRNAs from 70% of the genes contain a different cap structure, m3 2,2,7GTP. This cap structure is poorly recognized by mammalian elF4E, suggesting that C. elegansmay possess a specialized form of elF4E that can recognize m3 2,2,7GTP. Analysis of the C. elegans genomic sequence data base revealed the presence of three elF4E-like genes, here named ife-1, ife-2, andife-3. cDNAs for these three eIF4E isoforms were cloned and sequenced. Isoform-specific antibodies were prepared from synthetic peptides based on nonhomologous regions of the three proteins. All three eIF4E isoforms were detected in extracts of C. elegans and were retained on m7GTP-Sepharose. One eIF4E isoform, IFE-1, was also retained on m3 2,2,7GTP-Sepharose. Furthermore, binding of IFE-1 and IFE-2 to m7GTP-Sepharose was inhibited by m3 2,2,7GTP. These results suggest that IFE-1 and IFE-2 bind both m7GTP- and m3 2,2,7GTP-containing mRNA cap structures, although with different affinities. In conjunction with IFE-3, these eIF4E isoforms would permit cap-dependent recruitment of all C. elegans mRNAs to the ribosome.

Differential Inhibition of mRNA Degradation Pathways by Novel Cap Analogs

mRNA degradation predominantly proceeds through two alternative routes: the 5′→3′ pathway, which requires deadenylation followed by decapping and 5′→3′ hydrolysis; and the 3′→5′ pathway, which involves deadenylation followed by 3′→5′ hydrolysis and finally decapping. The mechanisms and relative contributions of each pathway are not fully understood. We investigated the effects of different cap structure (Gp3G, m7Gp3G, or m27,3′-O Gp3G) and 3′ termini (A31,A60, or G16) on both translation and mRNA degradation in mammalian cells. The results indicated that cap structures that bind eIF4E with higher affinity stabilize mRNA to degradation in vivo. mRNA stability depends on the ability of the 5′ terminus to bind eIF4E, not merely the presence of a blocking group at the 5′-end. Introducing a stem-loop in the 5′-UTR that dramatically reduces translation, but keeping the cap structure the same, does not alter the rate of mRNA degradation. To test the relative contributions of the 5′→3′ versus 3′→5′ pathways, we designed and synthesized two new cap analogs, in which a methylene group was substituted between the α- and β-phosphate moieties, m27,3′-OGppCH2pG and m27,3′-OGpCH2ppG, that are predicted to be resistant to cleavage by Dcp1/Dcp2 and DcpS, respectively. These cap analogs were recognized by eIF4E and conferred cap-dependent translation to mRNA both in vitro and in vivo. Oligonucleotides capped with m27,3′-OGppCH2pG were resistant to hydrolysis by recombinant human Dcp2 in vitro. mRNAs capped with m27,3′-OGppCH2pG, but not m27,3′-OGpCH2ppG, were more stable in vivo, indicating that the 5′→3′ pathway makes a major contribution to overall degradation. Luciferase mRNA containing a 5′-terminal m27,3′-OGppCH2pG and 3′-terminal poly(G) had the greatest stability of all mRNAs tested.

Stopped-flow Kinetic Analysis of eIF4E and Phosphorylated eIF4E Binding to Cap Analogs and Capped Oligoribonucleotides EVIDENCE FOR A ONE-STEP BINDING MECHANISM

Recruitment of eukaryotic mRNA to the 48 S initiation complex is rate-limiting for protein synthesis under normal conditions. Binding of the 5′ -terminal cap structure of mRNA to eIF4E is a critical event during this process. Mammalian eIF4E is phosphorylated at Ser-209 by Mnk1 and Mnk2 kinases. We investigated the interaction of both eIF4E and phosphorylated eIF4E (eIF4E(P)) with cap analogs and capped oligoribonucleotides by stopped-flow kinetics. For m7GpppG, the rate constant of association, kon, was dependent on ionic strength, decreasing progressively up to 350 mm KCl, but the rate constant of dissociation, koff, was independent of ionic strength. Phosphorylation of eIF4E decreased kon by 2.1-2.3-fold at 50-100 mm KCl but had progressively less effect at higher ionic strengths, being negligible at 350 mm. Contrary to published evidence, eIF4E phosphorylation had no effect on koff. Several observations supported a simple one-step binding mechanism, in contrast to published reports of a two-step mechanism. The kinetic function that best fit the data changed from single- to double-exponential as the eIF4E concentration was increased. However, measuring koff for dissociation of a pre-formed eIF4E·m7GpppG complex suggested that the double-exponential kinetics were caused by dissociation of eIF4E dimers, not a two-step mechanism. Addition of a 12-nucleotide chain to the cap structure increased affinity at high ionic strength for both eIF4E (24-fold) and eIF4E(P) (7-fold), primarily due to a decrease in koff. This suggests that additional stabilizing interactions between capped oligoribonucleotides and eIF4E, which do not occur with cap analogs alone, act to slow dissociation.

Binding Specificities and Potential Roles of Isoforms of Eukaryotic Initiation Factor 4E in Leishmania

ABSTRACT The 5′ cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m7GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m7GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3′ untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m7GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.

Synthetic mRNA cap analogs with a modified triphosphate bridge – synthesis, applications and prospects

The cap structure was discovered at the 5′-end of the eukaryotic mRNA over three and a half decades ago. Since then, hundreds of chemically modified cap analogs have been synthesized and applied in numerous studies on the elucidation of cap-related physiological processes in the cell, and became important members of the biophosphate family. In this Perspective, we present recent developments in the synthesis of cap analogs modified within 5′,5′-triphosphate bridges and their utility for interdisciplinary studies on cap-dependent processes in gene expression and its regulation, as well as biotechnological applications and perspectives in medicine.

Synthesis, Conformation and Hydrolytic Stability of p1,p3−Dinucleoside Triphosphates Related to mRNA 5′-cap, and Comparative Kinetic Studies on their Nucleoside and Nucleoside Monophosphate Analogs

P1,P3−Dinucleoside triphosphates, N(5′)G(5′)G, have been prepared in which N is 7-Me-, 7-Et-, 7-Bn, N2, 7-diMe- or N2,N2, 7-triMe-guanosine. Conformations of the nucleoside moieties have been deter...