Recapitulation of the IRE-IRP position effect in a reticulocyte cell translation extract.
To examine the molecular basis of the IRE-IRP position effect by biochemical means, we aimed to recapitulate this effect in a cell-free translation extract. To this end, we designed the CAT reporter mRNAs IRE.34, IRE.66, and IRE.100, bearing IREs 34, 66, and 100 nucleotides, respectively, downstream from the cap structure (Fig. A). The sequences of the spacer inserts between the cap structure and the IRE were predicted not to form extensive secondary structure nor to disrupt the structure of the IRE itself. To examine the translation of these mRNAs in the absence of bound IRPs, short IRE competitor transcripts were added to sequester endogenous IRPs present in the extract. While all three CAT mRNAs are efficiently translated in the presence of the IRE competitor RNA (Fig. A, lanes 2, 4, and 6), IRE.34 mRNA is profoundly repressed by the addition of recombinant IRP-1 (lane 3). Increasing the distance between the cap structure and the IRE in IRE.66 and IRE.100 mRNAs leads to partial derepression of IRP-mediated translational control (compare lanes 5 and 7 with lane 3). Since the magnesium concentration can affect cell-free translation reactions (4
), the experiment was performed at low (0.7 mM [Fig. A]) and high (2.1 mM [data not shown]) magnesium concentrations. The position effect is clearly reflected at both magnesium concentrations. Importantly, the quantitative difference in translational repression between mRNAs bearing a cap-proximal IRE (IRE.34) and a more cap-distal IRE (IRE.66 [2.3-fold]) in vitro accurately reflects the quantitative difference seen in vivo (2.05-fold) (10
) when similar reporter mRNAs are assayed (see Fig. and Discussion). Further increases in the distance between the IRE and the cap structure (IRE.100) cause a further loss in translational repression by IRP binding (3.4-fold [Fig. ]).
FIG. 2 Translation of IRE.34, IRE.66, and IRE.100 mRNAs in RRL. (A) NOP1 (2 ng) (lanes 2 to 7) and 2.5 ng of IRE.34 (lanes 2 and 3), IRE.66 (lanes 4 and 5), or IRE.100 mRNA (lanes 6 and 7) were translated in vitro. Where indicated (+), 15 ng of IRE competitor (more ...) The position of the IRE critically affects the recruitment of the small ribosomal subunit.
Sucrose gradient analysis of initiation reactions had previously shown that cap-proximal IRE–IRP-1 complexes prevent the recruitment of 43S preinitiation complexes to the mRNA (13
). To examine the effect of a cap-distal IRE–IRP-1 complex on the association of the small ribosomal subunit with the IRE.100 mRNA, we employed sucrose gradient analysis of initiation assays containing guanylyl-imidodiphosphate (GMP-PNP). This nonhydrolyzable GTP analog permits the recruitment of the small ribosomal subunit to the mRNA and its migration toward the translation initiation codon, but inhibits the GTP-dependent subsequent joining of the large ribosomal subunit leading to the accumulation of 48S complexes (43S preinitiation complex plus mRNA) (2
). In the absence of available IRP (i.e., when IRE competitor transcripts are added), both mRNAs accumulate in 48S and 66S complexes (Fig. ), indicating the association of one or two small ribosomal subunits, respectively, with the mRNAs (13
). The identity of these complexes was confirmed by parallel reactions with mixtures containing either cycloheximide or cap analog and by RNase treatment of GMP-PNP-induced complexes which resulted in a single peak at 48S (reference 13
and data not shown). Upon addition of IRP-1, the recruitment of 43S preinitiation complexes to IRE.34 mRNA is prevented, and the mRNA accumulates in the top fractions of the gradient in mRNPs (Fig. A). In contrast, IRE.100 mRNA redistributes into the 48S region of the gradient, indicating that single 43S complexes associate with the IRE.100 mRNA in the presence of IRP-1 (compare Fig. B and A). This result shows that the inhibition of 43S complex recruitment by a cap-proximal IRE-IRP complex is position dependent. Interestingly, the presence of a single 43S preinitiation complex (rather than multiple complexes) on IRE.100 mRNA may indicate that the IRE–IRP-1 complex stalls scanning along the 5′ UTR, leaving insufficient room for a second 43S complex to bind.
FIG. 3 Recruitment of 43S complexes to IRE.34 and IRE.100 mRNAs in RRL. Shortened mRNA transcripts of IRE.34 (A) and IRE.100 (B) were incubated in RRL for 5 min in the presence of either 45 ng of IRE competitor RNA (open squares) or 125 ng of recombinant IRP-1 (more ...)
To further explore 43S complex recruitment to these mRNAs, kinetic experiments in the presence of GMP-PNP were undertaken (Fig. ). These experiments were performed at high magnesium concentrations, where initiation proceeds more slowly (2
), to increase the kinetic resolution. Analysis of IRE.34 mRNA revealed the expected large difference between ribosomal subunit recruitment in the presence and absence of IRP (Fig. A to C). The pattern of small ribosomal subunit recruitment in the absence of IRP does not significantly change over time, although a minor shift from 48S to 66S complexes occurs (Fig. A to C). Even at the longest time points, in the presence of IRP, there is not a significant accumulation of IRE.34 mRNA in 48S or 66S complexes (Fig. C). A greater proportion of IRE.100 mRNA is associated with 66S rather than 48S complexes compared to IRE.34 mRNA in the absence of IRP (Fig. G to I). This can be explained in terms of the longer 5′ UTR, which favors preloading (28
). In the presence of IRP, small ribosomal subunits are already efficiently recruited to IRE.100 mRNA at 5 min (Fig. G), and there is not a noticeable difference in binding at 2 min (data not shown). However, even at 30 min, the majority of IRE.100 mRNAs are present in 48S rather than 66S complexes (Fig. I), strongly supporting the notion that the small ribosomal subunits have not migrated sufficiently far from the 5′ end of the mRNA to provide unimpeded access for subsequent subunits.
FIG. 4 Kinetic analysis of the recruitment of small ribosomal subunits. Shortened transcripts of IRE.34 (A to C), IRE.66 (D to F), and IRE.100 (G to I) were assayed in the presence of either 45 ng of IRE competitor mRNA (open squares) or 125 ng of recombinant (more ...)
The kinetics of small ribosomal subunit association with IRE.66 mRNA was also examined (Fig. D to F). When IRP is not available, IRE.66 mRNA is present in a higher proportion of 66S rather than 48S complexes compared to IRE.34 mRNA (Fig. D to F). At the shortest time point, IRE.66 mRNA association with small ribosomal subunits is strongly inhibited by IRP-1 and appears similar to IRE.34 mRNA (Fig. D). However, the effect of IRP-1 on small ribosomal subunit recruitment is much less profound after 30 min than for IRE.34 mRNA (Fig. F). It therefore seems that the recruitment of small ribosomal subunits to IRE.66 mRNA is kinetically delayed by IRP-1.
We next analyzed the kinetics of 80S ribosome formation on IRE.34 and IRE.100 mRNAs. In agreement with the previous analysis, Fig. D to F show that the presence of IRP-1 does not retard 48S complex formation with IRE.100 mRNA compared to in its absence. Furthermore, comparison of Fig. D to F with Fig. A to C reveals the temporary accumulation of 48S complexes on IRE.100 mRNA even in the absence of GTP analogs, indicative of stalled scanning. In contrast to analysis in the presence of GMP-PNP, in its absence, small ribosomal subunits eventually overcome the cap-distal IRE-IRP complexes, as the formation of 80S ribosomes can be observed (Fig. E to F). The formation of 80S complexes on IRE.100 mRNA at the later time points does not reflect an inactivation or passive dissociation of IRP-1, because IRE.34 mRNA remains repressed even at the latest time point (Fig. C). Compatible results were obtained at low (0.7 mM) magnesium concentrations (percent mRNA in 80S or heavier complexes in the presence of IRP: IRE.34, 6%; IRE.100, 20%). Thus the sucrose gradient analysis of IRE.100 mRNA suggests that its partial translational repression cannot be attributed to a slower recruitment of 43S complexes to the mRNA, but more likely is due to the kinetic effect of pausing scanning ribosomal subunits.
FIG. 5 Kinetic analysis of 80S ribosome assembly on IRE.34 and IRE.100 mRNAs. Shortened transcripts of IRE.34 (A to C) and IRE.100 (D to F) were assayed in the presence of either 45 ng of IRE competitor RNA (open squares) or 125 ng of recombinant IRP-1 (solid (more ...)
In conclusion, this analysis reveals that two mechanisms exist to impede translation by RNA-protein complexes. In the first, cap-proximal RNA-protein complexes inhibit ribosomal association with the mRNA (IRE.34). In the second, cap-distal RNA-protein complexes delay productive scanning towards 80S complex formation (IRE.100).
Elongating ribosomes linearly transgress IRE–IRP-1 complexes located within the open reading frame.
To examine if an IRE–IRP-1 complex located within the open reading frame of an mRNA can affect translation, an IRE was inserted into the CAT coding region, 32 nucleotides downstream of the A of the AUG codon, to create IRE.ORF (Fig. B). This insertion preserved and extended the CAT open reading frame. IRE.34 mRNA and IRE.ORF mRNA were translated in the RRL cell-free system (Fig. ), including NOP1 mRNA as an internal control and IRE-mut mRNA as a negative control with a point-mutated IRE that cannot bind IRP-1. In the presence of competitor IRE transcripts to titrate the endogenous IRPs, all mRNAs are translated with similar efficiencies (lanes 2, 5, and 8). The translation product of ORF.CAT mRNA displays a slower mobility (labelled CAT+ [lane 5]), consistent with the insertion of the IRE sequence. When the competitor IRE is omitted (lanes 3 and 6) or when saturating quantities of recombinant IRP-1 are added (lanes 4, 7, and 9), only IRE.34 mRNA translation is repressed, while the translation of IRE.ORF mRNA is entirely unaffected and yields the extended CAT+ translation product (lanes 6 and 7). Thus, elongating ribosomes possess the capacity to linearly translate regions of the mRNA that are bound by high-affinity RNA binding proteins. The impediment imposed by such RNA-protein complexes is not sufficient to significantly impinge on the yield of translation products.
FIG. 6 IRE–IRP-1 complexes within the open reading frame do not affect mRNA translation. NOP1 (5 ng) (lanes 2 to 9) and 2.5 ng of IRE.34 (lanes 2 to 4), IRE.ORF (lanes 5 to 7), or IRE-mut (lanes 8 and 9) were translated in RRL. Where indicated, 15 ng (more ...) Linear scanning through cap-distal IRE-IRP complexes during translation initiation.
To overcome cap-distal IRE-IRP complexes subsequent to the recruitment of the small ribosomal subunit to IRE.100 mRNA, the initiation apparatus might either traverse the entire 5′ UTR in a linear fashion or bypass the IRE–IRP-1 complex by a jumping or shunting mechanism, as shown for the initiation of translation of cauliflower mosaic virus 35S RNA (7
) or adenovirus late mRNAs (44
). To distinguish between these possibilities, an in-frame AUG codon was introduced into the IREs of IRE.34, IRE.66, and IRE.100 mRNAs to create IRE/AUG.34, IRE/AUG.66, and IRE/AUG.100 mRNAs, respectively (Fig. C). If the translation machinery were to hop or shunt past the IRE–IRP-1 complex, initiation should occur at the downstream CAT initiation codon. However, if the entire 5′ UTR is linearly scanned, protein synthesis should start from the AUG within the IRE and yield an extended protein product. When endogenous IRPs are titrated by competitor IREs, translation of the three different IRE/AUG mRNAs predominantly yields the N-terminally extended CAT polypeptide, indicating that the AUG codon within the IRE is efficiently recognized in the absence of IRP-1 (Fig. , lanes 5, 9, and 13). Interestingly, leaky scanning, which leads to the synthesis of the smaller CAT polypeptide, decreases as the distance between the 5′ end of the mRNA and the initiation codon is increased (compare IRE/AUG.34 with IRE/AUG.100). All three IRE/AUG mRNAs display the same response to IRP-1 as their non-IRE/AUG counterparts (compare lanes 5 and 6 with lanes 3 and 4, lanes 9 and 10 with lanes 7 and 8, and lanes 13 and 14 with lanes 11 and 12, respectively), demonstrating that the position effect is preserved following the insertion of the AUGs. Importantly, IRE/AUG.66 and IRE/AUG.100 mRNAs yield the N-terminally extended CAT polypeptides, even in the presence of IRP-1. Thus, the entire 5′ UTR including the IRE is scanned in a linear fashion during initiation of translation, inconsistent with the notion that the IRE-IRP complex is bypassed by a hopping or shunting mechanism.
FIG. 7 Cap-distal IRE-IRP complexes are not bypassed during initiation of translation. NOP1 (5 ng) (lanes 2 to 14) and 2.5 ng of IRE.34 (lanes 3 and 4), IRE/AUG.34 (lanes 5 and 6), IRE.66 (lanes 7 and 8), IRE/AUG.66 (lanes 9 and 10), IRE.100 (lanes 11 and 12), (more ...) Active IRP-1 displacement from cap-distal IREs by the mammalian translation apparatus.
The possibility that the observed IRE position effect could result from different affinities of the respective IREs for IRP-1 was excluded by competition gel retardation assays (data not shown). This is consistent with previous results (10
Another series of competition gel retardation assays were then performed to estimate the passive dissociation rate of IRP-1 in the cell-free-translation extract (Fig. ). The RRL was pretreated with an m7GpppG cap analog to prevent the recruitment of the translation apparatus to the mRNAs, which might cause active IRP-1 displacement. Capped radiolabelled IRE.34, IRE.100, and IRE.ORF mRNAs were subsequently incubated in this extract with 50 ng of recombinant IRP-1 to allow formation of IRE–IRP-1 complexes. After 10 min, 1 μg of IRE competitor or rRNA as a nonspecific competitor was added, and the incubation was continued to assess the passive dissociation of IRP-1 from the mRNAs. Aliquots taken at 0, 10, 20, and 60 min after the addition of competitor RNA were loaded on a running, native polyacrylamide gel, resulting in the shorter migration distances of IRE-IRP complexes of samples loaded at later time points (Fig. ). Addition of the IRE competitor simultaneously with the probe completely abolishes complex formation (lanes 9 to 12), demonstrating that the competitor is in excess. Complexes formed between the IRE.34, IRE.100, and IRE.ORF IREs and IRP-1 appear to be equally stable, and the majority of the mRNAs are still associated with IRP-1 after 60 min. Due to the stability of the IRE–IRP-1 complexes, their half-lives (t1/2s) exceed the duration of the assay; the t1/2s were thus extrapolated to range between 66 and 73 min (Fig. ). The slow passive dissociation of IRP-1 from IRE.34, IRE.100, and IRE.ORF RNAs suggests that both the initiation apparatus and the elongation translation apparatus play an active role in displacing IRP-1 from cap-distal IREs.
FIG. 8 Stability of the IRE–IRP-1 complexes. 32P-labelled IRE.34, IRE.100, or IRE.ORF probes (2 ng) were incubated with 100 ng of recombinant IRP-1 in 40% RRL for 10 min at 30°C, to allow formation of IRE-IRP complexes, before addition (more ...) The position effect is species restricted.
IRP-1-mediated repression has previously been reconstituted in WGE (13
), which, unlike mammalian cells and extracts, does not contain endogenous IRP (16
). To test whether IRP-1-mediated repression in this system is position dependent, the translation of IRE.34 and IRE.100 was examined in WGE by using IRE-mut and NOP mRNAs as negative and internal controls, respectively. In the absence of exogenous IRP-1, all three test transcripts are translated with comparable efficiencies (Fig. , lanes 2, 4, and 6). The translation of both IRE.34 and IRE.100 mRNAs is repressed specifically and to a comparable extent with saturating (lanes 3 and 5) and subsaturating (data not shown) amounts of exogenously added IRP-1. We also evaluated the effect of IRP-1 in a yeast-free-cell translation extract, which, like wheat germ, is devoid of endogenous IRE-binding activity (34
). As in the wheat germ system, IRP-mediated translational repression does not exhibit a significant position dependence in yeast (25
) or in a yeast-free-cell translation extract (data not shown). This apparent difference between RRL and the wheat germ and yeast extracts was further investigated with the wheat germ system. Control experiments showed that the observed differences were not due to different buffer conditions or incubation temperatures (data not shown). Direct comparison of ribosome assembly on IRE.100 mRNA in the presence of IRP-1 shows that both 48S and 80S complexes assemble in RRL after 30 min, whereas only 48S complexes assemble in WGE (compare Fig. B and C [Fig. A is a control for IRP-1 activity]). This result is best explained by extended stalling of the scanning subunits by the cap-distal IRE–IRP-1 complexes in WGE. These results also show that the position effect observed in mammalian cells and in the cell-free-translation extract derived from mammalian cells is not universal, but species restricted. They suggest that mammalian cells possess an activity with which they can overcome cap-distal high-affinity RNA-protein complexes.
FIG. 9 Position-independent translational repression by IRP-1 in WGE. NOP1 (10 ng) (lanes 2 to 7) and 5 ng of IRE.34 (lanes 2 and 3), IRE.100 (lanes 4 and 5), or IRE-mut (lanes 6 and 7) mRNAs were translated in WGE in the absence (−) (lanes 2, 4, and (more ...)