We have identified the DExH-box protein DHX29 as a factor that is required for efficient initiation on mammalian mRNAs with structured 5′-UTRs, which typically encode regulatory proteins. The extent of the requirement for DHX29 correlated with the stability of the secondary structure elements in the 5′-UTRs of mRNAs. Although isolated internal stems of ΔG=−13.1 kcal/mol or less could be efficiently overcome by scanning complexes in the presence of only eIF4A/4G/4B, efficient ribosomal movement through stems of ΔG>−19 kcal/mol required DHX29. Ribosomal scanning relies on the ATP-dependent helicase activity of eIF4A/4G/4B and is influenced by the conformation of scanning 43S complexes, which is modified by eIF1/1A. How does DHX29 stimulate 48S complex formation on mRNAs with structured 5′-UTRs? Does it participate directly in unwinding of mRNA or does it remodel 43S complexes to increase their scanning processivity? The answer to this question is linked to the mechanism by which eIF4A/4G/4B assist scanning, and elucidation of this requires knowledge of their location in ribosomal complexes. If eIF4A/4G/4B bind at the leading edge and unwind mRNA before it enters the 40S subunit, it is unlikely that another helicase, DHX29, would participate directly in the same process. In this case DHX29 could enhance the processivity of ribosomal movement by remodeling ribosomal complexes to ensure correct entry into and/or fixation of mRNA in the mRNA-binding cleft. But if eIF4A/4G/4B, as suggested (Siridechadilok et al., 2005
), bind at the trailing edge near the E-site and assist scanning by helicase-mediated “ratcheting” of mRNA through the mRNA-binding channel, then DHX29 might directly unwind mRNA before it enters the 40S subunit. Although this possibility seems unlikely because DHX29 is not a processive helicase, it cannot be strictly excluded that binding of DHX29 to ribosomal complexes might enhance its helicase activity. However, even if eIF4A/4G/4B act at the trailing edge, DHX29 could still assist scanning not by direct unwinding of mRNA, but by remodeling ribosomal complexes and influencing accommodation of mRNA in the mRNA-binding channel, in which case mRNA secondary structure would be unwound by the scanning 40S subunit itself. In this hypothetical situation, correct positioning of mRNA at the entrance to the mRNA-binding channel would be particularly important. We note that the bacterial ribosome has helicase activity, which involves ribosomal proteins S3, S4 and likely S5 (Takyar et al., 2005
Suppression of the aberrant +8-9 nt toe-print, which most likely represents a 48S complex with the 3′-portion of mRNA not firmly fixed in the mRNA-binding cleft of the 40S subunit, by DHX29 even on delayed addition to pre-assembled 48S complexes indicates that DHX29 does induce conformational changes in these complexes that influence ribosomal accommodation of the 3′-portion of mRNA. The appearance of aberrant +8-9 nt toe-prints depended on the presence of eIF1/1A. Binding of eIF1/1A to yeast 40S subunits causes the entry ‘latch’ between h18 in the body and h34/rpS5 in the neck to open and establishes a new connection between rpS3 and h16 (Passmore et al., 2007
). Such opening of the entry ‘latch’ might weaken fixation of the 3′-portion of mRNA in the mRNA-binding cleft that could account for appearance of the +8-9 nt toe-print. It is likely that the conformation of the 40S subunit with the open latch is more conducive to attachment of 43S complexes to mRNA, whereas processive scanning might require firm fixation of mRNA in the mRNA-binding cleft. In this case the conformation of ribosomal complexes would require further modification, which could be promoted by DHX29.
Another indication that DHX29 causes conformational changes in 40S subunits comes from its influence on ribosomal complexes assembled on viral IRESs. Thus, even on delayed addition, DHX29 affected 40S-ribosomal binding and proper fixation in the mRNA-binding cleft of CrPV and CSFV IRESs. The CrPV and CSFV-like HCV IRESs both induce similar conformational changes in 40S subunits, which were suggested to facilitate fixation of these IRESs in the mRNA-binding cleft (Spahn et al., 2001
). It is therefore likely that binding of DHX29 to 40S subunits either does not allow such IRES-induced changes to occur, and/or causes other conformational changes in 40S subunits that are not compatible with binding and proper positioning of the IRESs on 40S subunits. Moreover, DHX29 dissociated 48S complexes assembled on the CSFV IRES and influenced the ratio of 48S complexes assembled on AUG826
of the EMCV IRES. Although the dissociating effect of DHX29 on 48S complexes assembled on the CSFV IRES is similar to that reported for eIF1 (Pestova et al., 2008
), the conformational changes induced in 40S subunits by eIF1 (Passmore et al., 2007
) and the potential conformational changes induced by DHX29 are likely not identical because these factors have opposite effects on 40S/CrPV IRES complexes (Pestova et al., 2004
; this study) and on the ratio of 48S complexes assembled on two AUGs of the EMCV IRES (Pestova et al., 1998a
; this study).
Foot-printing experiments revealed that in 43S complexes, DHX29 protects h16 of 18S rRNA. We cannot conclude unambiguously whether such protection is caused by direct contact of DHX29 with h16 or reflects conformational changes in the 40S subunit induced by DHX29. If DHX29 indeed binds h16 near the mRNA entrance, this position of DHX29 would be consistent with both hypothetical modes of action (remodeling of 43S complexes or mRNA unwinding). However, if the observed protections correspond to conformational changes in ribosomal complexes, then the region of such changes is entirely consistent with remodeling of 40S subunits near the mRNA entrance, which would likely affect accommodation of the 3′-portion of mRNA in the mRNA-binding cleft. Moreover, it is exactly the area of the 40S subunit that undergoes conformational changes upon binding of eIF1 and eIF1A. It is not known whether stable secondary structures in 5′-UTRs only slow ribosomal scanning or also increase drop-off of 43S complexes. If drop-off can occur, then proper fixation of mRNA in the mRNA-binding cleft would also stabilize ribosomal association with mRNA and increase the processivity of scanning complexes. Moreover, the potential influence of the conformation of ribosomal complexes on the processivity of the ribosome-bound eIF4A/4G/4B helicase complex could strictly also not be excluded. Although we are not yet in a position to discriminate between the remodeling and unwinding mechanisms by which DHX29 might stimulate 48S complex formation, it is worth noting that it has become apparent that many DExH/D proteins function primarily in remodeling of RNA and RNP complexes rather than in processive unwinding of RNA duplexes (reviewed by Pyle, 2008
). Thus, many DExH/D proteins have additional RNA-binding domains that contribute to strong ATP-independent RNA annealing activity, which in conjunction with their ATP-dependent unwinding activity suggest that such proteins can induce alternating conformational rearrangements in RNA and RNP complexes upon their transition between ATP-bound and ATP-free states. In addition, DExH/D proteins can also function as RNPases, displacing proteins from RNA in ATP-dependent manner.
Interestingly, although DHX29 and eIF4F/4A/4B acted synergistically in 48S complex formation on mRNAs with 5′-UTRs containing stable hairpins, DHX29 alone also promoted relatively efficient 48S complex formation on mRNAs with 5′-UTRs containing less stable stems and unstructured 5′-terminal regions that could promote eIF4F/4A/4B-independent attachment of 43S complexes, and even mediated low-level 48S complex formation on mRNAs with 5′-UTRs containing stems of high stability. DHX29 might therefore be responsible for translation of at least a sub-class of mRNAs in conditions when eIF4G is depleted (Ramírez-Valle et al., 2008
In RRL, DHX29 was wholly associated with 40S-ribosomal complexes, but DHX29-bound 40S-ribosomal complexes nevertheless constitute only ~10% of all 40S-ribosomal complexes. In this respect, it is particularly important that DHX29 can participate in multiple rounds of 48S complex formation. Although it would be most logical to suggest that DHX29 remains associated with ribosomal complexes during the entire scanning process and dissociates from assembled 48S complexes as a result of conformational changes that likely occur upon establishment of codon-anticodon base-pairing, we cannot exclude that DHX29 might dissociate earlier, particularly if it functions by remodeling ribosomal complexes rather than by unwinding mRNA. Our experiments also indicate that a proportion of purified DHX29 might be inactive in stimulating 48S complex formation, even though it could still bind ribosomal complexes. Although DHX29 might have been partially inactivated during purification, phosphorylation of human DHX29 at Ser192, Ser200, Tyr811 and Tyr826 (www.phosphosite.org
) could also influence its activity.
The preceding discussion is based on the assumption that eIF4A is the only DEAD-box RNA helicase involved in initiation. However, biochemical and genetic analyses have implicated other DEAD/DExH-box proteins in initiation, including Ded1p and the homologous mammalian proteins DDX3/PL10, mammalian RNA helicase A (RHA) and Drosophila
Vasa (Chuang et al., 1997
; de la Cruz et al., 1997
; Hartman et al., 2006
; Johnstone and Lasko, 2004
; Lee et al., 2008
). The mechanism(s) by which Ded1p, DDX3, RHA and Vasa act in the initiation process are incompletely characterized but are likely distinct. Ded1p has been characterized in the greatest detail: it is a more processive helicase than eIF4A (Marsden et al., 2006
), its function is not redundant with that of eIF4A and mutations in Ded1p are synthetic-lethal with mutations in TIF1
(eIF4A) and cdc33
(eIF4E) and deletion of TIF4631
(eIF4G) or STM1/TIF3
(eIF4B) (Chuang et al., 1997
; de la Cruz et al., 1997
). This has led to suggestions that eIF4A may, as a subunit of eIF4F, function in promoting recruitment of 43S complexes to the cap-proximal region of mRNA, whereas Ded1p assists ribosomal complexes during scanning, particularly on mRNAs with long 5′UTRs (e.g. Marsden et al., 2006
). The molecular interactions that could couple Ded1p with scanning ribosomes are not known. It remains to be seen whether mammalian Ded1p homologues like DDX3 also function during scanning, after ribosomal loading, in which case they would likely unwind mRNA before it enters the mRNA-binding cleft, near its entrance. If this is indeed the case, it is even more likely that DHX29 functions in remodeling the 40S/mRNA/eIFs complex rather than in unwinding mRNA during initiation.