Exploring the Role of AUG Triplets in Human Cap-Independent Translation Enhancing Elements

Abstract

Cap-independent translation is believed to play an important role in eukaryotic protein synthesis, but the mechanisms of ribosomal recruitment and translation initiation remain largely unknown. Messenger RNA display was previously used to profile the human genome for RNA leader sequences that can enhance cap-independent translation. Surprisingly, many of the isolated sequences contain AUG triplets, suggesting a possible functional role for these motifs during translation initiation. Herein, we examine the sequence determinants of AUG triplets within a set of human translation enhancing elements (TEEs). Functional analyses performed in vitro and in cultured cells indicate that AUGs have the capacity to modulate mRNA translation either by serving as part of a larger ribosomal recruitment site or by directing the ribosome to defined initiation sites. These observations help constrain the functional role of AUG triplets in human TEEs and advance our understanding of this specific mechanism of cap-independent translation initiation.

Figure 1

Functional analysis of a diverse set of human TEEs for translation initiation activity and early ribosomal initiation. (a) Schematic representation of the firefly luciferase reporter or maltose binding protein (MBP) constructs containing 5′ leader sequences that derive from human translation enhancing elements. (b) Translation initiation activity in HeLa cell lysate measured by luciferase expression: in-frame triplets (green), out-of-frame triplets (red), and the initiation codon at position +1 of the CDR (blue). Error bars designate the standard deviation. (c) Early ribosomal initiation on MBP constructs expressed in HeLa cell lysate and rabbit reticulocyte lysate. Protein expressed in the different lysate systems was purified, and recovery of radiolabeled MBP monitored by liquid scintillation counts. The amount of protein analyzed was normalized, based on the recovery counts, and gel electrophoretic mobility shifts indicate that human TEEs with AUG triplets in their sequence are prone to early ribosomal initiation. The slower-mobility bands correspond to MBP protein isoforms that contain N-terminal extensions. (d) Identification of authentic translation initiation sites by protein sequencing of in vitro-expressed MBP. The protein sequence is indicated below the nucleotide sequence of each TEE. Color scheme: MBP coding region (blue arrows), authentic translation initiation site (yellow arrows), and in-frame triplets relative to the MBP coding region (green).

Figure 2

Evaluating the local sequence context of AUG triplets in a human TEE with high translational activity. (a) Schematic of mutational analysis. In-frame (green) and out-of-frame (red) AUG triplets were removed from the 5′ leader sequence of a high-activity TEE (HGL6.877) and inserted into the 5′ leader sequence of an unselected genomic sequence (HGL0.53). Vectors were constructed with the luciferase or MBP coding regions (CDR). Initiation codon at position +1 of the CDR (blue). (b) Translation initiation activity of luciferase reporter vectors measured in HeLa cell lysate. The effect of insertion or deletion of in-frame (Δin), out-of-frame (Δout), or all (Δall) AUGs was determined. Error bars designate the standard deviation. (c) Early ribosomal initiation on MBP constructs expressed in HeLa cell lysate. Protein expressed in HeLa cell lysate was purified, and recovery monitored by liquid scintillation counts. On the basis of recovery counts, an equal amount of protein was analyzed by gel electrophoresis, with the resulting MBP protein from vectors containing mutations to HGL6.877 compared to those from vectors containing the wild-type sequence or a TEE that does not contain any AUGs (HGL6.738).

Figure 3

Identification of a short 13-mer motif with strong translation enhancing activity. (a) Ten closely related human translation enhancing elements were identified and ranked according to their luciferase activity in cultured HeLa cells. The predicted ribosomal recruitment site (black box) was identified by a comparative functional genomic sequence alignment: functional 13-mer motif (green), point mutations (red), and AUG triplets within the selected sequence family (green and red ovals for in-frame and out-of-frame, respectively). (b–d) Functional analysis of sequences that contain and lack the 13-mer motif in cultured mammalian cells. Panel b shows the translational activity of two low-activity TEEs (HGL6.646 and HGL6.347) unrelated to the 13-mer sequence family in cultured HeLa cells. The effect of the 13-mer motif was determined by the addition of the motif to the 5′ end of each TEE. Panels c and d show the translational activity in cultured cells for one high-activity TEE (HGL6.985) and one low-activity TEE (HGL6.499), respectively, from the 13-mer sequence family. The effect of removing the 13-mer motif from HGL6.985 or adding the motif to the 5′ end of HGL6.499 was determined. Luciferase expression in all cases was normalized to luciferase mRNA levels. Error bars designate the standard deviation. Mammalian cell lines: HeLa (human), BSC40 (monkey), RK13 (rabbit), BHK (hamster), and 129SV (mouse).

Figure 4

Characterization of sequence constraints for a 13-mer motif. (a) Identification of authentic translation initiation sites by protein sequencing of in vitro-expressed MBP from the high-activity TEE HGL6.985. The protein sequence is listed below the nucleotide sequence. Color scheme: 13-mer motif (black box), MBP coding region (blue arrow), authentic translation initiation site (yellow arrow), and AUG triplets in-frame and out-of-frame with the MBP coding region (green and red ovals, respectively). (b) Mutational analysis of HGL6.985. Modifications were made to increase the 5′ leader length through addition of both a half-repeat and a full repeat (+0.5 rpt and +1.0 rpt, respectively) of the HGL6.985 sequence. A decrease in sequence length was achieved by removal of 33 (Δ33), 50 (Δ50), and all of the nucleotides downstream of the 13-mer motif (13-mer only). The authentic initiation site (*) within the TEE sequence was also deleted through mutation (ΔAUG). The functional impact of each mutation was assessed by measuring luciferase expression levels in HeLa cells relative to the unmodified HGL6.985 sequence and normalized to luciferase mRNA levels. Error bars designate the standard deviation.