Assembly of 48S Translation Initiation Complexesfrom Purified Components with mRNAs That Have Some Base Pairingwithin Their 5′ UntranslatedRegions

Dmitriev, Sergey E.; Terenin, Ilya M.; Dunaevsky, Yan E.; Merrick, William C.; Shatsky, Ivan N.

doi:10.1128/mcb.23.24.8925-8933.2003

Cited by 84 publications

(93 citation statements)

References 35 publications

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“…As seen from Fig. 3 (lanes 1 to 4), such complexes did form, and in the presence of initiation factors 1A, 2, 3, 4A, and 4F (eIF4B was not used in the assembly, since it is required only for mRNAs with structured 5Ј-UTRs [6]), their assembly occurred more efficiently than with 80S ternary complexes (compare lanes 2 and 8). The stimulatory effect of eIF4F (in the absence of eIF1) on the binding of uncapped, leaderless, and single-stranded cI mRNAs with the 40S ribosomal subunit was surprising.…”

Section: Resultsmentioning

confidence: 86%

“…The reconstitution of 48S complexes from purified components and their analysis by toeprinting were performed as described previously (5,6,16,17). For the assembly and toeprinting analysis of 80S translation initiation complexes, the reaction mixture (20 l) contained 80S reassociated ribosomes (2.5 pmol), an mRNA (0.5 pmol), MettRNA i Met (5 pmol), and, where indicated, the initiation factors at the same molar ratio as for the assembly and analysis of 48S translation initiation complexes (5).…”

Section: Methodsmentioning

confidence: 99%

“…To study pathways of recruitment of leaderless mRNAs onto mammalian 80S ribosomes, we have decided to use our system of assembly of mammalian translation complexes from totally purified components combined with toeprinting (16). This approach has proven to be very fruitful in elucidating initiation factor requirements for several viral internal ribosome entry site elements (for a review, see reference 9) and standard cap-dependent mRNAs (6,17). Here, using this technique, we show for the first time that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis.…”

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A Leaderless mRNA Can Bind to Mammalian 80S Ribosomes and Direct Polypeptide Synthesis in the Absence of Translation Initiation Factors

Andreev

Terenin

Dunaevsky

et al. 2006

Molecular and Cellular Biology

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Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis. We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred. The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such an mRNA was supplied with a 5 leader, irrespective of whether it was in a totally single-stranded conformation or was prone to base pairing. The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial 70S ribosomes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents a relic from early steps in the evolution of the translation apparatus.More than a decade ago, we showed using the toeprint assay that the prokaryotic leaderless mRNA coding for the cI repressor of lambda phage assisted by initiator Met-tRNA was able to bind directly to undissociated 70S ribosomes in the absence of initiation factor 1 (IF1), IF2, and IF3 (1). Under the same conditions, no initiation complex was observed with the 30S ribosomal subunit. In the presence of initiation factors, the 30S subunit invariably bound to internal sites on the mRNA immediately downstream of the closest Shine-Dalgarno (SD) sequence, whereas undissociated 70S ribosome strongly preferred the initiation triplet as close as possible to the 5Ј end of the mRNA. These studies were substantially extended and confirmed in other laboratories both in vitro and in vivo (12,13,15,28) and resulted in similar conclusions. Natural leaderless mRNAs have been identified in all kingdoms of life (see references 10 and 11). This class of mRNAs is well represented in archaea and mammalian mitochondria. Although they have not yet been found among cellular mammalian mRNAs, leaderless monocistronic N and dicistronic N-P RNAs of measles virus were found to be associated with polysomes in infected mammalian cells (4). In addition, it is unlikely that a systematic search for such mRNAs in mammalian cells has ever been undertaken. There is one well-documented example of a translatable leaderless mRNA in unicellular eukaryotes. It encodes a variant specific surface protein of Giardia lamblia and is certainly translated in these cells (14). Leaderless mRNAs are efficiently translated in mammalian cell-free systems (see reference 12). However, it is not known whether they use a similar (80S) pathway for translation initiation in eukaryotes, given the remarkable quantitative and qualitative differences in the organization of eukaryotic and prokaryotic t...

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Section: Resultsmentioning

confidence: 86%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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A Leaderless mRNA Can Bind to Mammalian 80S Ribosomes and Direct Polypeptide Synthesis in the Absence of Translation Initiation Factors

Andreev

Terenin

Dunaevsky

et al. 2006

Molecular and Cellular Biology

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“…Translational repression via the catalytic activity of eIF4A would selectively target mRNAs with 5Ј UTR structural content that rely on unwinding for efficient initiation (18,20,43,61). Using differential IRES analysis, it has previously been shown that BC1 RNA is not effective in repressing translation initiation of mRNAs that do not require unwinding of 5Ј UTR secondary structure by eIF4A (65,66).…”

Section: Discussionmentioning

confidence: 99%

Translational Control by a Small RNA: Dendritic BC1 RNA Targets the Eukaryotic Initiation Factor 4A Helicase Mechanism

Lin¹,

Pestova²,

Hellen³

et al. 2008

Molecular and Cellular Biology

162

152

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Translational repressors, increasing evidence suggests, participate in the regulation of protein synthesis at the synapse, thus providing a basis for the long-term plastic modulation of synaptic strength. Dendritic BC1 RNA is a non-protein-coding RNA that represses translation at the level of initiation. However, the molecular mechanism of BC1 repression has remained unknown. Here we identify the catalytic activity of eukaryotic initiation factor 4A (eIF4A), an ATP-dependent RNA helicase, as a target of BC1-mediated translational control. BC1 RNA specifically blocks the RNA duplex unwinding activity of eIF4A but, at the same time, stimulates its ATPase activity. BC200 RNA, the primate-specific BC1 counterpart, targets eIF4A activity in identical fashion, as a result decoupling ATP hydrolysis from RNA duplex unwinding. In vivo, BC1 RNA represses translation of a reporter mRNA with 5 secondary structure. The eIF4A mechanism places BC RNAs in a central position to modulate protein synthesis in neurons.Local protein synthesis at the synapse, increasing evidence suggests, is one of the key molecular mechanisms that underlie input-specific modulations of synaptic strength and consequently higher brain functions that rely on such synaptic plasticity. Translational control mechanisms in synapto-dendritic domains are thought to be essential for the long-term spatiotemporal modulation of mosaic local protein repertoires. Such mechanisms are therefore attracting increasing interest among neuroscientists and molecular and cellular biologists (reviewed in references 3, 11, 24, 26, 27, 51, 60, and 67).At the same time, there has been growing awareness of the biological significance of small, functional RNAs in eukaryotic cells in general and in neurons in particular (2, 7, 11). Small, non-protein-coding RNAs (npcRNAs) (also known as untranslated RNAs) (9) may be particularly well suited as posttranscriptional regulators of gene expression that enhance brainenvironment interactions (11). Neuronal BC1 RNA is a small dendritic npcRNA (64) that operates as a translational repressor (30,65,66). BC1 RNA, which is targeted to dendrites (38,39) and is abundant at the synapse (13), represses translation at the level of initiation (65, 66).In eukaryotes, translation initiation proceeds in three stages, each of which is mediated by a number of eukaryotic initiation factors (eIFs) (reviewed in references 25 and 43). An eukaryotic initiation factor 2 (eIF2) ⅐ GTP ⅐ GMet-tRNA i ternary complex first binds to the 40S ribosomal subunit to form a 43S preinitiation complex. This complex is subsequently recruited to the mRNA, typically to the 5Ј cap structure, and scans to the initiator codon to form a 48S initiation complex. In the final step, this complex is joined by the large ribosomal subunit to form an 80S monoribosome complex ready for elongation. It is frequently the second step, recruitment of the 43S complex and 48S complex assembly, that is rate limiting and the target for regulation. This step is mediated by the eIF4 family of facto...

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“…24 Yeast eIF4B preferentially stimulates translation from mRNAs with moderately stable secondary structure in the 5 0 UTR while it stimulates the higher basal level of translation from mRNAs that lack secondary structure only moderately. 54 Formation of a 48 S translation initiation complex (i.e., the binding of a 40 S subunit to an mRNA) with animal mRNAs containing a structured 5 0 UTR was dependent on eIF4B, 55 suggesting that, although the ability of eIF4B to promote eIF4A helicase activity is likely involved in the translation of mRNAs in general, it may be of particular importance for certain types of mRNAs.…”

Section: Plants Express a Novel Eif4g Isoform Not Found In Other Eukamentioning

confidence: 99%

The Role of the Poly(A) binding protein in the assembly of the Cap-binding complex during translation initiation in plants

Gallie¹

2014

translation

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Translation initiation in eukaryotes requires the involvement of multiple initiation factors (eIFs) that facilitate the binding of the 40 S ribosomal subunit to an mRNA and assemble the 80 S ribosome at the correct initiation codon. eIF4F, composed of eIF4E, eIF4A, and eIF4G, binds to the 5 0 -cap structure of an mRNA and prepares an mRNA for recruitment of a 40 S subunit. eIF4B promotes the ATPdependent RNA helicase activity of eIF4A and eIF4F needed to unwind secondary structure present in a 5 0 -leader that would otherwise impede scanning of the 40 S subunit during initiation. The poly(A) binding protein (PABP), which binds the poly(A) tail, interacts with eIF4G and eIF4B to promote circularization of an mRNA and stimulates translation by promoting 40 S subunit recruitment. Thus, these factors serve essential functions in the early steps of protein synthesis. Their assembly and function requires multiple interactions that are competitive in nature and determine the nature of interactions between the termini of an mRNA. In this review, the domain organization and partner protein interactions are presented for the factors in plants which share similarities with those in animals and yeast but differ in several important respects. The functional consequences of their interactions on factor activity are also discussed.

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Assembly of 48S Translation Initiation Complexesfrom Purified Components with mRNAs That Have Some Base Pairingwithin Their 5′ UntranslatedRegions

Cited by 84 publications

References 35 publications

A Leaderless mRNA Can Bind to Mammalian 80S Ribosomes and Direct Polypeptide Synthesis in the Absence of Translation Initiation Factors

A Leaderless mRNA Can Bind to Mammalian 80S Ribosomes and Direct Polypeptide Synthesis in the Absence of Translation Initiation Factors

Translational Control by a Small RNA: Dendritic BC1 RNA Targets the Eukaryotic Initiation Factor 4A Helicase Mechanism

The Role of the Poly(A) binding protein in the assembly of the Cap-binding complex during translation initiation in plants

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