The IRES within the complex IGF1R 5’-UTR
In their original characterization of the human IGF1R
mRNA, Cooke et al determined by primer extension and RNase protection methods that the transcription start site was 1,038 base-pairs upstream of the coding sequence [Cooke et al., 1991
]. Data generated more recently by high-throughput genome-scale methodologies such as CAGE tag analysis allow us to re-examine this question (). Although there is evidence for a minor population of IGF1R
mRNA molecules that begin ~700 or ~900 nucleotides upstream of the initiation codon, and an additional small cluster of IGF1R
transcription start sites ~50,000 base pairs further upstream (not shown), this new data confirms that the predominantly utilized transcription start site imparts an ~1,043 nucleotide long 5’-untranslated region to the IGF1R
The core functional IRES within the complex IGF1R 5’-UTR
The long, G-C-rich 5’-untranslated region of the IGF1R
mRNA is projected to adopt a secondary structure with extensive internal base-pairing () and high thermodynamic stability (dG=−515.2 kCal/mol). The core functional IRES, which had been localized to the 3’-terminal 90 nucleotides (951–1040) of the 5’-UTR [Meng et al., 2008
], is projected to adopt a relatively simple structure composed of one central multi-loop and two terminal stem-loops (see bracket in ). Although the mfold algorithm suggested multiple base-pairing alternatives with similar thermodynamic stabilities available to the 5’-UTR as a whole, the core functional IRES itself appears to represent an independently-folding domain, highly favored within the context of the full-length 5’-UTR. In fact, within the 20 highest scoring structures generated by mfold 3.2 for the 1,040 nucleotide 5’-UTR, 19 exhibited precisely the same 3-Loop folding pattern for the core functional IRES sequence. We were concerned however that the projected structures for the IRES could be influenced by its position so near the end of the sequence entered into the program. Therefore, we repeated the mfold analysis after adding the natural exon 1 and 2 sequences to the 5’-UTR (). Although the structure projected for the 5’-UTR was dramatically altered by the presence of exons 1 and 2, the functional core IRES, now positioned near the middle of the sequence being analyzed, still preferentially adopted the same 3-Loop structure that had been highly favored in the original analysis.
A magnified image of the 3-Loop structure anticipated for the core functional IRES is presented as it might exist in the context of the full-length 5’-UTR (), or as an isolated 90 nucleotide fragment () which displays even higher IRES activity than the full-length 5’-UTR [Meng et al., 2008
]. We have designated the asymmetrical central multi-loop as Loop1, the six nucleotide hairpin loop appended to a four-base pair helix as Loop2, and the large poly(U) sequence appended on a 6-base-pair stem as Loop3. RNase T1 / V1 sensitivity data provide experimental support for the natural existence of this structure both in the intact 5'-UTR () as well as the isolated IRES (). A symmetrical pattern of susceptibility to RNase V1 (which cleaves within double stranded RNA) is observed for residues expected to lie within Stem 2. G-residues within Stem2 (3 out of 4 base pairs are G.C) were partially or completely protected from RNase T1 digestion (which cleaves after single stranded G residues) even in the absence of added magnesium, while G-residues within Stem 3 (of which only 2 out of 6 base pairs are G.C) were more dependent on magnesium for base-pairing and protection from RNase T1 digestion.
We had previously noted that progressive 5’-deletions of the IGF1R
5’-UTR sequence were associated with progressive loss and then return of IRES activity [Meng et al., 2008
]. We suspected that the loss of IRES activity observed with the intermediate deletions could be attributed to altered base-pairing patterns negatively impacting the core functional IRES. Indeed, we observe a strong correlation between measured IRES activity and the potential of the RNA to adopt the 3-Loop structure ().
Correlation between IRES function and capacity to adopt the 3-loop IRES structure
Site-directed mutagenesis of critical residues within the IGF1R IRES
To further dissect the IRES, we introduced site-directed mutations within the core IGF1R IRES sequence, to identify individual sequence elements important for IRES operation and regulation. The initial series of mutations were introduced and tested within the context of the full-length 1,040 nucleotide 5’-UTR (). We focused our attention on two features: Stem2/Loop2 and the homopolymeric Loop3.
Site-directed mutagenesis implicates Stem2/Loop2 and Loop3 as critical sequence elements for operation and regulation of the IGF1R IRES
The Stem 2/Loop2 sequence was of particular interest because the hexaloop (AUUUCA) is very similar to a sequence element (UUUCC) thought to contribute to the function of several viral IRESs [Pilipenko et al., 1992
; Scheper et al., 1994
], and also very similar to the consensus (AUUUA) for the A-U-rich element binding protein HuR [Antic and Keene, 1997
], which we had already determined binds to the IRES and potently regulates its activity [Meng et al., 2005
]. We found that mutation of two adjacent nucleotides within either Loop2 or Stem2 resulted in ~50% loss of IRES activity. Five additional mutants altering from one to four residues within Loop2 were also associated with ~25–50% decrease in IRES activity (data not shown). A compensatory mutation in Stem2, modifying 4 out of 1,040 nucleotides and with no anticipated change in secondary structure, was associated with >80% loss of IRES activity, suggesting that the primary sequence of this region may be critical for internal ribosome entry.
To determine what effect the large poly(U)-tract comprising Loop3 might exert on IRES function, the (U19) sequence was minimized to a tetraloop. Based on mfold analysis of the mutated sequence, no additional changes to the core IRES structure were anticipated (). (In fact, the identical 3-Loop structure was projected for each of the top 20 scoring structures generated for both the Stem2 compensatory mutation and the Loop 3 minimization mutation.) Minimization of the Loop3 poly(U)-tract was associated with a dramatic (~2.5X) increase in IRES activity (), suggesting that, when intact, this long homopolymeric sequence serves as a negative regulator of IGF1R IRES-mediated translation initiation.
To further establish the importance of Stem2/Loop2 and Loop3 to IRES function, we tested the mutant constructs in a series of representative human tumor cell lines in which the IGF1R IRES is active. Very similar results were obtained in T47D (breast carcinoma in which the IGF1R IRES was originally characterized, very high IRES activity), T98G (glioblastoma, moderately high IRES activity), and Saos-2 (osteosarcoma, modest but readily detectable IRES activity) (). Note that the compensatory Stem2 mutation was severely debilitating to IRES activity, while the Loop3 minimization mutant exhibited a marked increase in IRES activity, in all three cell lines.
To confirm these results, a second series of mutations was designed within the context of the 90 nucleotide core functional IRES. A compensatory substitution within Stem2 retaining the natural G-C-rich sequence composition resulted in near 50% decrease in IRES activity, while a similar mutation which replaced the three G-C base pairs with A-U base pairs exhibited >80% loss of IRES activity (). This result suggests that, in addition to the changes in primary sequence, weakening of this stem may also have severe consequences for IRES function. To further assess the effect of the size of the homopolymeric Loop3 sequence on IRES activity, a more modest diminution of the polyU-tract (from U21 to U13, representing the naturally-occurring allelic variant of this sequence, derived by PCR amplification from T47D genomic DNA, see below) was tested. The decrease in size of Loop3 was again associated with a modest but significant (~20%) increase in IRES activity.
Together, these findings suggest that Stem2/Loop2 may be the key sequence element facilitating internal ribosomal entry, while Loop 3 may serve to modulate the activity of the IRES.
Kinetic analysis of the IGF1R IRES
We extended our assays to further assess the effects of the Stem2 and Loop3 mutations on function of the IGF1R IRES over time (). The full-length 1,040 nucleotide IGF1R 5’-UTR and the 90 nucleotide isolated core IRES exhibit nearly identical kinetic profiles, with the isolated IRES exhibiting ~25% higher activity. By the 48 and 72 hour time points, the activity of the IGF1R IRES exceeds that of the well-characterized encephalomyocarditis virus (EMCV) IRES by a considerable margin, although the viral IRES is activated much more rapidly in the cells (peaking at the 4 to 8 hour time points). The Loop3 minimization mutant has lost this intrinsic dampening effect, allowing the IGF1R IRES to accelerate at a level that approaches that of the viral IRES, and ultimately displaying a higher activity than any of the other IRES constructs. The near complete loss of IRES function of the stem 2 compensatory mutant is obvious.
Kinetic analysis of IGF1R IRES activity
These results further support the conclusion that Stem2/Loop2 contains the sequence most critical for operation of the IRES (recruitment of the 40S ribosomal subunit), while the poly(U) tract of Loop3 may serve to limit the maximal rate of translation initiation mediated through the IRES.
Possible Shine-Dalgarno-like (mRNA-rRNA base-pairing) interaction between the IGF1R IRES and the human 18S rRNA
The fundamental purpose of the IRES is to recruit the 40S ribosomal subunit. A direct mRNA-rRNA base-pairing interaction between the IGF1R
IRES and the 18S rRNA could conceivably facilitate ribosome recruitment. Upon careful examination, we noted that there exists a near-perfect Watson-Crick complementarity between the Stem2/Loop2 sequence of the IGF1R
IRES and the G961 loop / helix 23b of the human 18S rRNA (). The 959–964 region of the human 18S rRNA is known to be accessible to chemical probes, and therefore potentially available for direct base-pairing interactions with the mRNA [Demeshkina et al., 2003
; Demeshkina et al., 2000
]. Our mutational analyses indicate that alterations to the Stem2Loop2 sequence which disrupt this complementarity are associated with a substantial loss of IRES activity. Furthermore, the G961 loop is projected to occupy an ideal position in three-dimensional space, on the platform of the 40S ribosomal subunit, from which to interact with the mRNA and facilitate translation initiation (). Based on the relative positions of the mRNA and rRNA deduced from the crystal structure of the T. thermophilus
30S ribosomal subunit [Yusupova et al., 2001
], it appears that the G693 loop of the prokaryotic 16S rRNA (homologous to the eukaryotic G961 loop of the 18S rRNA) lies in the immediate vicinity of both the anti-Shine-Dalgarno sequence (which is not present in eukaryotes) as well as the Shine-Dalgarno segment of the proximal 5’-untranslated region of the mRNA at the E-site. Together these findings suggest that the IGF1R
IRES may recruit the 40S ribosome by a eukaryotic equivalent of the Shine-Dalgarno interaction, with the G961 loop of the 18S rRNA substituting for the anti-Shine-Dalgarno segment and base-pairing directly with the Stem2/Loop2 sequence of the IRES. The parallels between the mRNA-rRNA base-pairing interaction potentially involved in the operation of the IGF1R
IRES in humans and the classical Shine-Dalgarno interaction of prokaryotes are summarized in .
Potential Watson-Crick base-pairing between the IGF1R IRES and the 18S rRNA
mRNA - rRNA Interactions Facilitating Translation Initiation1
Allelic variation of the Loop 3 sequence of the IGF1R IRES
Examination of multiple GenBank entries containing the human IGF1R 5’-untranslated sequence reveals evidence of natural variation in the size of the Loop3 poly(U)-tract, ranging from 14 to 24 contiguous U residues, with a bimodal distribution centered around U16 and U24 (). To explore this issue further, we PCR-amplified the core IGF1R IRES sequence from several different sources of human genomic DNA (). In most cases a doublet (two distinct bands) was observed on agarose gel electrophoresis of the PCR product. Direct sequencing of these PCR products generated results that became challenging to interpret upon reaching the Loop3 sequence from either direction, however, we were able to definitively establish that the results were a composite of two distinct alleles (most commonly U21 and U13) differing by 8 in the number of residues in the Loop3 poly(U)-tract, with additional minor variants generated by polymerase error (most commonly +1, +2, or −1 U residues). However, for several of the human tumor cell lines tested, the PCR-amplified core IGF1R IRES yielded only 1 band on agarose gel, apparently representing only the smaller of the two alleles. To confirm these findings, PCR products generated from two representative cell lines: T47D (biallelic) and MDA-MB-231 (monoallelic) were cloned and multiple individual clones sequenced. The results were indicative of the existence of two distinct alleles, clustered around U13 and U21, in T47D cells, but only 1 allele, clustered around U13, in MDA-MB-231 ().
Evidence for natural allelic variation of the Loop3 poly(U) sequence in human genomic DNA
Differential modulation of mutant IRES constructs by RNA-binding IRES-regulatory proteins HuR and hnRNP C
We have previously characterized the RNA-binding protein HuR as a potent repressor of the IGF1R
IRES [Meng et al., 2005
], while hnRNP C appears to activate the IRES [Meng et al., 2008
]. We were interested to determine what consequences the representative Stem2 compensatory (decrease in function) and Loop3 minimization (gain in function) IRES mutations would have on the IRES-regulatory capabilities of these RNA-binding proteins. Experiments were performed in which the various IRES reporter constructs were cotransfected with expression vectors for HuR or hnRNP C, as had been done originally to establish the IRES-regulatory activities of these RNA-binding proteins (). We found that each mutant IRES construct remained sensitive to the positive or negative modulatory effects of each of the RNA-binding proteins, with one exception: the severely debilitated Stem2 compensatory mutant IRES could not be activated by hnRNP C.
Effect of IRES-regulatory proteins HuR and hnRNP C on activity of mutant IRES constructs
Mutations in the core IRES sequence alter protein binding to the IGF1R 5’-UTR
We have developed a high resolution northwestern protocol to analyze sequence-specific interactions between putative IRES trans-acting factors (ITAFs) and the IGF1R 5’-UTR. The northwestern is a functional assay of RNA-binding activity performed under highly stringent conditions. Utilizing the wild-type sequence as probe, a reproducible pattern of bands representing a series of proteins capable of interacting specifically with the IGF1R 5’-UTR is observed (). To test whether the Stem2 or Loop3 IRES mutations might alter the pattern of protein binding to the IGF1R 5’-UTR, the northwestern assay was repeated using the mutant RNA sequences as probes. A marked change in affinity of multiple individual proteins for binding the mutant 5’-UTR RNA sequences was observed. The compensatory mutation to Stem2, which dramatically decreases IRES activity, was associated with a near complete loss of binding of a number of the putative ITAFs (particularly Bands 2, 3, 4, 4b, 5, 8a, and 8d) detected by the northwestern assay. A distinct subset of bands (7, 9, and 10) remained relatively unchanged or even increased in intensity, confirming that the effect of the Stem2 mutation on protein binding to the IGF1R 5’-UTR was selective. Interestingly, the minimization of Loop3, which dramatically increases IRES activity, was associated with an equally dramatic increase in affinity for the same ITAFs which had been negatively impacted by the Stem2 mutation. Importantly, we have definitively characterized bands 2, 4, and 5 as “internal” ITAFs, binding within the 90 nucleotide core functional IRES sequence, thus their altered affinity for the mutant IRES probes can be rationalized on this basis. Band 8d, which we have determined to be hnRNP C (also an internal ITAF) exhibits the same trend as the other internal ITAFs (decreased affinity with the Stem 2 mutation, increased affinity with minimization of Loop 3). In contrast, we have determined that bands 7, 9, and 10 are “external” ITAFs, binding primarily outside of the core IRES sequence, and indeed these bands show very little variation in intensity accompanying mutation of the core IRES sequence.
Mutations to critical sequence elements of the IGF1R IRES alter the pattern of protein binding to the IGF1R 5’-UTR