The requirement for eukaryotic initiation factor 4A (eIF4A) in translation is in direct proportion to the degree of mRNA 5′ secondary structure

Svitkin, Yuri V.; Pause, Arnim; Haghighat, Ashkan; Pyronnet, Stéphane; Witherell, Gary W.; Belsham, Graham J.; Sonenberg, Nahum

doi:10.1017/s135583820100108x

Cited by 423 publications

(382 citation statements)

References 72 publications

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“…The 5′ UTR plays a critical role in protein translation efficiency as it represents the site of binding for the preinitiation complex initiation of protein translation [15,16]. For example, the eukaryotic initiation factor-4A (eIF4A) binding to the 5′ UTR is important in the unwinding initiation of protein translation [17] and the secondary structure of the 5′ UTR plays a critical role in binding of eIF1A to mRNA [18]. Although the secondary structure of these 5′ UTR is difficult to predict based on the presence of modified nucleotides, it is interesting to speculate that these UTRs may share a common secondary structure which can impact protein translation.…”

Section: Discussionmentioning

confidence: 99%

Optimization of mRNA untranslated regions for improved expression of therapeutic mRNA

et al. 2018

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ABSTRACTmRNA based therapies hold great promise for the treatment of genetic diseases. However, this therapeutic approach suffers from multiple challenges including the short half-life of exogenously administered mRNA and subsequent protein production. Modulation of untranslated regions (UTR) represents one approach to enhance both mRNA stability and translation efficiency. The current studies describe and validate screening methods using a diverse set of 5′UTR and 3′UTR combinations for improved expression of the Arginase 1 (ARG1) protein, a potential therapeutic mRNA target. Data revealed a number of critical aspects which need to be considered when developing a screening approach for engineering mRNA improvements. First, plasmid-based screening methods do not correlate with protein expression driven by exogenously expressed mRNA. Second, improved ARG1 protein production was driven by increased translation and not improved mRNA stability. Finally, the 5′ UTR appears to be the key driver in protein expression for exogenously delivered mRNA. From the testing of the combinatorial library, the 5′UTR for complement factor 3 (C3) and cytochrome p4502E1 (CYP2E1) showed the largest and most consistent increase in protein expression relative to a reference UTR. Collectively, these data provide important information for the development and optimization of therapeutic mRNAs.

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Section: Discussionmentioning

confidence: 99%

Optimization of mRNA untranslated regions for improved expression of therapeutic mRNA

et al. 2018

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“…Extrapolating from its unwinding activity 80 , and the correlation of an increased need for eIF4A during translation of mRNAs with structured 5′ untranslated regions (UTRs), eIF4A is often suggested to unwind RNA secondary structures in the 5′ UTR that would other wise inhibit ribosome scanning 93,94 . But the unwinding of such structures by eIF4A during translation initiation has not been shown directly, and eIF4A-independent scanning has been seen 95 .…”

Section: Nuclear Specklesmentioning

confidence: 99%

From unwinding to clamping — the DEAD box RNA helicase family

Linder

Jankowsky

2011

Nat Rev Mol Cell Biol

949

1,069

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Nearly all aspects of RNA metabolism, from transcription and translation to mRNA decay, involve RNA helicases, which are enzymes that use ATP to bind or remodel RNA and RNA-protein complexes (ribonucleoprotein (RNP) complexes) 1 . RNA helicases are found in all three domains of life, and many viruses also encode one or more of these proteins 2,3 . Together with the structurally related DNA helicases that function in replication, recombination and repair, the RNA helicases are classified into superfamilies and families, based on sequence and structural features 3,4 . DEAD box proteins form the largest helicase family, with 37 members in humans and 26 in Saccharomyces cerevisiae 3 , and are characterized by the presence of an Asp-Glu-Ala-Asp (DEAD) motif.DEAD box helicases have central and, in many cases, essential physiological roles in cellular RNA metabolism 5 (FIG. 1). The proteins generally function as part of larger multicomponent assemblies, such as the spliceosom e or the eukaryotic translation initiation machinery 6 . Mutations and deregulation of several DEAD box proteins have been linked to disease states, including cancer 7 . Although all DEAD box proteins contain a structurally highly conserved core with conserved ATP-binding and RNA-binding sites, different proteins have been associated with diverse and seemingly unrelated functions, including the disassembly of RNPs, chaperoning during RNA folding and even stabil ization of protein complexes on RNA 5,6 . How these highly conserved proteins fulfil such an array of different functions has been a long-standing question.Research has now started to illuminate the molecular basis for this functional diversity. In this Review, we summarize how structural data, together with biochemical and biophysical studies, have revealed unexpected modes by which these proteins function. We also discuss how novel molecular and cell biological approaches have better defined the cellular roles of DEAD box helicases. We outline our current view on common structural and mechan istic themes that have emerged, and on physical models of how these 'unusual' RNA helicases work.Structures of DEAD box RNA helicases A number of structural studies have universally shown that members of the DEAD box family contain a highly conserved helicase core that harbours the binding sites for ATP and RNA 3,4,8 . The core is surrounded by variable auxiliary domains, which are thought to be critical for the diverse functions of these enzymes.Structure of the helicase core. DEAD box proteins belong to helicase superfamily 2 (SF2) 2 . Similarly to all SF2 helicases, DEAD box proteins are built around a highly conserved helicase core of two virtually identical domains that resemble the bacterial recombination protein recombinase A (RecA) 3,4,8 (FIG. 2a,b). Within this helicase core, at least 12 characteristic sequence motifs are located at conserved positions (FIG. 2a,b). Some of these motifs are conserved across the entire SF2 family, whereas others are found only in the DEAD box family 3 ....

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“…The initiation factors eIF4G, eIF4A, eIF2 and eIF3 are required for 48S complex formation in a reconstituted 40S ribosome assay with representative members of type II IRES elements (Table 1) EMCV, TMEV and FMDV (Pestova et al, 1996;Kolupaeva et al, 1998;Pilipenko et al, 2000Pilipenko et al, , 2001. Accordingly, translation initiation promoted by the EMCV IRES, and to lesser extent PV IRES elements, is sensitive to a dominant-negative mutant of eIF4A (Pause et al, 1994; Svitkin et al, 2001). In contrast to these findings, the teschovirus IRES and the HCV IRES elements are not inhibited by the dominant-negative mutant of eIF4A (Chard et al, 2006b;Pestova et al, 1998), indicating that its function is independent of eIF4F (Table 1).…”

Section: Picornavirus Ires-driven Protein Synthesismentioning

confidence: 99%

New insights into internal ribosome entry site elements relevant for viral gene expression

Martínez‐Salas

Pacheco

Serrano

et al. 2008

Journal of General Virology

119

106

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A distinctive feature of positive-strand RNA viruses is the presence of high-order structural elements at the untranslated regions (UTR) of the genome that are essential for viral RNA replication. The RNA of all members of the family Picornaviridae initiate translation internally, via an internal ribosome entry site (IRES) element present in the 59 UTR. IRES elements consist of cis-acting RNA structures that usually require specific RNA-binding proteins for translational machinery recruitment. This specialized mechanism of translation initiation is shared with other viral RNAs, e.g. from hepatitis C virus and pestivirus, and represents an alternative to the cap-dependent mechanism. In cells infected with many picornaviruses, proteolysis or changes in phosphorylation of key host factors induces shut off of cellular protein synthesis. This event occurs simultaneously with the synthesis of viral gene products since IRES activity is resistant to the modifications of the host factors. Viral gene expression and RNA replication in positive-strand viruses is further stimulated by viral RNA circularization, involving direct RNA-RNA contacts between the 59 and 39 ends as well as RNA-binding protein bridges. In this review, we discuss novel insights into the mechanisms that control picornavirus gene expression and compare them to those operating in other positive-strand RNA viruses.

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The requirement for eukaryotic initiation factor 4A (eIF4A) in translation is in direct proportion to the degree of mRNA 5′ secondary structure

Cited by 423 publications

References 72 publications

Optimization of mRNA untranslated regions for improved expression of therapeutic mRNA

Optimization of mRNA untranslated regions for improved expression of therapeutic mRNA

From unwinding to clamping — the DEAD box RNA helicase family

New insights into internal ribosome entry site elements relevant for viral gene expression

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