Pushing the limits of the scanning mechanism for initiation of translation

Abstract

Selection of the translational initiation site in most eukaryotic mRNAs appears to occur via a scanning mechanism which predicts that proximity to the 5' end plays a dominant role in identifying the start codon. This "position effect" is seen in cases where a mutation creates an AUG codon upstream from the normal start site and translation shifts to the upstream site. The position effect is evident also in cases where a silent internal AUG codon is activated upon being relocated closer to the 5' end. Two mechanisms for escaping the first-AUG rule--reinitiation and context-dependent leaky scanning--enable downstream AUG codons to be accessed in some mRNAs. Although these mechanisms are not new, many new examples of their use have emerged. Via these escape pathways, the scanning mechanism operates even in extreme cases, such as a plant virus mRNA in which translation initiates from three start sites over a distance of 900 nt. This depends on careful structural arrangements, however, which are rarely present in cellular mRNAs. Understanding the rules for initiation of translation enables understanding of human diseases in which the expression of a critical gene is reduced by mutations that add upstream AUG codons or change the context around the AUG(START) codon. The opposite problem occurs in the case of hereditary thrombocythemia: translational efficiency is increased by mutations that remove or restructure a small upstream open reading frame in thrombopoietin mRNA, and the resulting overproduction of the cytokine causes the disease. This and other examples support the idea that 5' leader sequences are sometimes structured deliberately in a way that constrains scanning in order to prevent harmful overproduction of potent regulatory proteins. The accumulated evidence reveals how the scanning mechanism dictates the pattern of transcription--forcing production of monocistronic mRNAs--and the pattern of translation of eukaryotic cellular and viral genes.

Fig. 1

Examples of ‘maximally leaky’ scanning wherein one mRNA produces three independently initiated proteins. Major (thick arrow) and minor (thin arrow) translation products are identified below their respective start codons. Sequences that cause the initiation site to be weak, and thus promote leaky scanning, are highlighted in red. Offset rectangles represent ORFs in different reading frames. (A) With c-myc mRNA, a leaky scanning mechanism was inferred from experiments in which optimizing the context around the first AUG codon suppressed production of the 50 kDa isoform, while changing the upstream CUG codon to AUG suppressed production of both the 65 and 50 kDa isoforms (Spotts et al., 1997). Access to the downstream start site might be more complicated than here depicted, as there is a small out-of-frame ORF between the 65 and 50 kDa start sites. (B) With C/EBPβ mRNA, a mutation that strengthens the first start codon (UUCaugC→ACCaugG) blocked production of all shorter isoforms, implicating a leaky scanning mechanism (Calkhoven et al., 2000). A small upORF (blue) superimposes another level of control, causing more ribosomes to bypass the start site for isoform B1 than would be expected from leaky scanning alone. Presumably because the AUG^START codon for isoform B1 is positioned close to the termination site of the upORF, reinitiation at site B1 is inefficient and some ribosomes thus reach the far downstream start site for the 20 kDa isoform (LIP). As evidence for this reinitiation shunt, Calkhoven et al. (2000) showed that eliminating the AUG codon of the upORF abolished production of LIP and that strengthening or weakening the context around the upORF start codon caused corresponding changes in the yield of LIP. Although the smallest form of C/EBPβ can be generated in some situations by proteolysis (Dearth et al., 2001), the effects of the aforementioned mutations clearly implicate a translational mechanism. The LAP/LIP ratio shows tissue and stage specific variation (Dearth et al., 2001, Descombes and Schibler, 1991). (C) Whereas leaky scanning allows initiation at multiple sites within a single ORF in C/EBPβ and c-myc mRNAs, leaky scanning allows translation of three separate ORFs in the pregenomic mRNA of rice tungro bacilliform virus. These ORFs (not drawn to scale) have overlapping start and stop codons of the form AUGA. Translation via leaky scanning was inferred from the strong reduction (>13-fold) in translation of ORF2 and ORF3 when the start codon of ORF1 was changed from AUU to AUG (Fütterer et al., 1997) and from the inhibitory effect on expression of ORF3 when an adventitious AUG codon was inserted into ORF2. The 5′ leader sequence that precedes ORF1 has ten small upORFs which are not depicted here because that peculiar leader sequence, postulated to be translated by ribosome hopping (Fütterer et al., 1996), is not required for the leaky scanning mechanism that underlies translation of ORFs 1, 2 and 3. (D) The avian reovirus S1 mRNA supports translation of one structural and two nonstructural proteins (Bodelón et al., 2001). The depicted mechanism postulates that ORF1 has a dual function, encoding its own polypeptide (p10) and facilitating translation of ORF3 by shunting some ribosomes past the strong AUG^START codon for ORF2. The absence of extraneous AUG codons in the 310 nt region between the end of ORF1 and the start of ORF3 is consistent with the idea that ORF3 might be translated by reinitiation. Some ribosomes would be expected to translate p17 (ORF2) by leaky scanning, engendered by the poor context at the start of ORF1. Improving the context at the start of ORF1 indeed increased production of p10 (Shmulevitz et al., 2002); unfortunately, the yield of p17, which would be expected to decrease, was not monitored. The observation that strengthening the context at the start of ORF1 had no effect on the yield of σC is not surprising because the reinitiation mechanism postulated to underlie translation of ORF3 would probably be limited by other features, such as the relatively large size of ORF1.

Fig. 2

Examples of minimally leaky scanning in which a strong, but not quite perfect, context at AUG#1 causes most ribosomes to initiate there while allowing a low level of initiation downstream. With the depicted viral mRNAs (A,B), the predominant product of translation is the capsid protein initiated from AUG#1. Low-level leaky scanning generates a small but adequate amount of the indicated second protein. With bovine coronavirus, a mutation in position +4 (U→G, indicated in red) flanking AUG#1 strongly reduced translation from the downstream site (Senanayake and Brian, 1997), supporting the interpretation that the natural mRNA is slightly leaky because the context flanking AUG#1 is not a perfect match to the consensus sequence. With hepatitis B virus, ribosomes en route to the P start site (AUG#5) apparently bypass the weak AUG#2 by leaky scanning, while translation of the small ORF initiated at AUG#3 enables some ribosomes to miss the inhibitory AUG#4 (inhibitory because it resides in a strong context and overlaps the P ORF) and thus to reach AUG#5. Whereas the core protein start codon (AUG#1) here depicted resides in a context which allows a low level of leaky scanning, a slightly longer mRNA which encodes the pre-core protein has a stronger start codon (A in position −3) and polymerase cannot be translated from that form of mRNA (Fouillot and Rossignol, 1996). The publications on which the scheme shown here is based (Fouillot et al., 1993, Hwang and Su, 1998) also discuss some alternative possibilities vis-à-vis translation of polymerase. (C) The first AUG codon in rat histone H4 mRNA initiates translation of the full-length protein. The second AUG, 85 codons downstream and in the same reading frame, initiates production of a peptide which has growth-regulatory properties (Bab et al., 1999). (D) With rat A_2AR adenosine receptor mRNA, an overlapping upORF that initiates at an AUG codon in a strong context is used to minimize production of A_2AR protein. The overlapping arrangement precludes reinitiation but the not-quite-perfect context at the upstream start site allows low-level leaky scanning. This interpretation is supported by the observed ten-fold increase in translation of A_2AR in vivo when the start codon of the upORF was eliminated (Lee et al., 1999). Via a second promoter, the rat A_2A-R gene produces some transcripts with additional upORFs, but no transcript has yet been found that lacks the inhibitory upORF discussed here. Here and in Fig. 3, the major coding domain is shaded gray. Small regulatory ORFs (blue rectangles) are not drawn to scale.

Fig. 3

Small upstream ORFs in eukaryotic mRNAs function in various ways to modulate translation. Only the 5′ end of each mRNA is depicted. (A) The presence of upORFs forces translation of the major ORF to occur by a reinitiation mechanism, which is usually inefficient. The extent of inhibition depends on the number and arrangement of upORFs and whether the context flanking the upstream start codon(s) allows some escape via leaky scanning. (B) Because reinitiation can occur only in the forward direction, an overlapping upORF strongly impairs translation of the major ORF. (C) Whereas type B mRNAs have a single in-frame start codon which is bypassed due to the overlapping upORF, type C mRNAs initiate from two in-frame start codons; the upORF serves to divert some ribosomes to the downstream start site. The depicted sequence is a simplified representation of GlyRS mRNA (Mudge et al., 1998). Translation of Bag-1 mRNA can also be fitted to this pattern: the first start site is an in-frame CUG codon which produces the 50 kDa form of Bag-1; the next start site (AUG#1, out-of-frame) initiates a small upORF within which the first in-frame AUG codon (AUG#2) resides, and that AUG is thereby skipped; the 36 kDa form of Bag-1 is produced from AUG#3 which is accessed by reinitiation following translation of the small upORF (Packham et al., 1997). Some other mRNAs that use an upORF to dodge one AUG codon in favor of another are described elsewhere (Mittag et al., 1997, Sarrazin et al., 2000). Note that the reinitiation shunt as here defined adheres to the linear scanning mechanism, unlike a shunt postulated to operate with cauliflower mosaic virus mRNA (Ryabova et al., 2000). (D) The common feature of mRNAs that use mechanism D is inhibition of translation in cis by a peptide encoded within the upORF. The amino acid sequence of the inhibitory peptide is different in each case (Morris and Geballe, 2000). In the column at the far right, asterisks indicate examples in which the translational control mechanism is regulated, e.g. via a change in concentration of eIF2 (GCN4) or arginine (CPA1) or polyamines (AdoMetDC) or, more commonly, via an alternative promoter that generates a simpler form of mRNA devoid of upORFs (c-mos, MDM2, IL-12; see text for other examples).

Fig. 4

A low-level reinitiation mechanism normally prevents overproduction of TPO. Translational yields from various forms of TPO mRNA in transfected COS cells (far right column) are expressed relative to a control transcript that has a short, unencumbered 5′ UTR. P1 and P2 are alternative promoters; a cluster of arrows indicates that P2 produces staggered start sites. The TPO coding domain (horizontal black bar) begins at an AUG codon which is labeled #8 because, in the longest form of mRNA (line 1), it is preceded by seven AUG codons that initiate small upORFs. Superscript letters indicate whether each upstream AUG resides in a strong (S) or weak (W) context and horizontal blue lines depict the approximate length and arrangement of the upORFs. Vertical lines demarcate the boundaries of exons; carets depict the introns in alternatively spliced transcripts. Only the beginning of the TPO coding domain (exons 3–7) is shown. The key point is that the normal set of transcripts supports translation poorly because upORF7 overlaps the TPO start site. Various mutations (shown in red) that relieve this constraint elevate the translation of TPO, and this overproduction causes hereditary thrombocythemia. Among the normal set of mRNAs, the ‘rare’ transcript from promoter P1 (line 2) supports translation slightly better than the others, perhaps because the short distance between upORF2 and AUG#7 enables some reinitiating ribosomes to bypass AUG#7 and thus reach AUG#8. Because of the strong context at AUGs #1 and #2, upORFs 1 and 2 would be more effective than upORFs 5 and 6 in setting up this reinitiation shunt. The depicted scheme is based on experiments described by Ghilardi et al. (1998) and Wiestner et al. (1998). Additional mutations diagrammed near the bottom of the figure were described by Ghilardi and Skoda (1999), Ghilardi et al. (1999), and Kondo et al. (1998).