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Determinants of initiation codon selection during translation in mammalian cells.

Matsuda D, Mauro VP - PLoS ONE (2010)

Bottom Line: In addition, we observed that increased leader length by itself changed the ratio of the proteins and favored initiation at AUG1.These observations demonstrate that initiation codon selection is affected by various factors, including AUG codon-accessibility and 5' leader length, and is not necessarily determined by the order of AUG codons (5'→3').The modulation of AUG codon accessibility may provide a powerful means of translation regulation in eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, The Scripps Research Institute and The Skaggs Institute for Chemical Biology, La Jolla, California, USA.

ABSTRACT
Factors affecting translation of mRNA contribute to the complexity of eukaryotic proteomes. In some cases, translation of a particular mRNA can generate multiple proteins. However, the factors that determine whether ribosomes initiate translation from the first AUG codon in the transcript, from a downstream codon, or from multiple sites are not completely understood. Various mRNA properties, including AUG codon-accessibility and 5' leader length have been proposed as potential determinants that affect where ribosomes initiate translation. To explore this issue, we performed studies using synthetic mRNAs with two in-frame AUG codons-both in excellent context. Open reading frames initiating at AUG1 and AUG2 encode large and small isoforms of a reporter protein, respectively. Translation of such an mRNA in COS-7 cells was shown to be 5' cap-dependent and to occur efficiently from both AUG codons. AUG codon-accessibility was modified by using two different elements: an antisense locked nucleic acid oligonucleotide and an exon-junction complex. When either element was used to mask AUG1, the ratio of the proteins synthesized changed, favoring the smaller (AUG2-initiated) protein. In addition, we observed that increased leader length by itself changed the ratio of the proteins and favored initiation at AUG1. These observations demonstrate that initiation codon selection is affected by various factors, including AUG codon-accessibility and 5' leader length, and is not necessarily determined by the order of AUG codons (5'→3'). The modulation of AUG codon accessibility may provide a powerful means of translation regulation in eukaryotic cells.

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Related in: MedlinePlus

Ratio of AUG codon usage is affected by nucleotide composition of the 5′ leader.A. Schematic representation of CAT-FLAG mRNAs. The arrows (numbered 1-5) indicate the positions of individual LNA target sequences in the 5′ leaders of different constructs. Arrow 6 indicates the position of a mutated target sequence. The thick black bar at the 5′ end of the mRNA represents an AatII sequence; dashes in the 5′ leader represent CAA repeats. B. Relative expression of CAT-FLAG proteins. Expression from AUG1 is normalized to that from AUG2 in COS-7 cells that were transfected with in vitro transcribed capped and poly(A)-tailed mRNAs with 5′ leader sequences as depicted in (A). Transfections were performed in the presence (+; dark grey bar) and absence (-; light grey bar) of LNA oligonucleotide. The cells were harvested 5 hours post transfection. Three independent experiments were performed for final quantification of immunoblots with error bars indicating standard deviations. Cotransfection with LNA oligonucleotide did not significantly alter relative AUG usage (two-sided t-test). One-way ANOVA analysis of relative AUG codon usage in constructs 3–6 ((-)LNA) did not show significant differences. However, there were some significant differences between constructs 1 or 2 ((-)LNA) and various other constructs. One-sided t-tests of construct 1 compared to constructs 3–6 ((-)LNA) yielded p-values of 0.06, 0.04, 0.04, and 0.06, respectively. One-sided t-tests of construct 2 compared to constructs 3–6 ((-)LNA) yielded p-values of <0.01, <0.01, 0.02, and <0.01, respectively. C. Primer extension inhibition analysis on target mRNAs bound with LNA oligonucleotide. Total RNA extracted from cells 5 h post transfection was analyzed by primer extension to confirm the positions of LNA binding to target mRNAs, using a primer that anneals 67-nucleotides downstream of AUG2. The extension products were resolved using 6% denaturing PAGE along with a DNA size marker (M) and sequencing ladder from the plasmid with LNA-target site-AatII-(CAA)16. Primer extension reactions of RNA samples from COS-7 cells cotransfected with (+) or without (-) LNA oligonucleotide were compared in parallel. The results are representative of three experiments performed independently.
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pone-0015057-g005: Ratio of AUG codon usage is affected by nucleotide composition of the 5′ leader.A. Schematic representation of CAT-FLAG mRNAs. The arrows (numbered 1-5) indicate the positions of individual LNA target sequences in the 5′ leaders of different constructs. Arrow 6 indicates the position of a mutated target sequence. The thick black bar at the 5′ end of the mRNA represents an AatII sequence; dashes in the 5′ leader represent CAA repeats. B. Relative expression of CAT-FLAG proteins. Expression from AUG1 is normalized to that from AUG2 in COS-7 cells that were transfected with in vitro transcribed capped and poly(A)-tailed mRNAs with 5′ leader sequences as depicted in (A). Transfections were performed in the presence (+; dark grey bar) and absence (-; light grey bar) of LNA oligonucleotide. The cells were harvested 5 hours post transfection. Three independent experiments were performed for final quantification of immunoblots with error bars indicating standard deviations. Cotransfection with LNA oligonucleotide did not significantly alter relative AUG usage (two-sided t-test). One-way ANOVA analysis of relative AUG codon usage in constructs 3–6 ((-)LNA) did not show significant differences. However, there were some significant differences between constructs 1 or 2 ((-)LNA) and various other constructs. One-sided t-tests of construct 1 compared to constructs 3–6 ((-)LNA) yielded p-values of 0.06, 0.04, 0.04, and 0.06, respectively. One-sided t-tests of construct 2 compared to constructs 3–6 ((-)LNA) yielded p-values of <0.01, <0.01, 0.02, and <0.01, respectively. C. Primer extension inhibition analysis on target mRNAs bound with LNA oligonucleotide. Total RNA extracted from cells 5 h post transfection was analyzed by primer extension to confirm the positions of LNA binding to target mRNAs, using a primer that anneals 67-nucleotides downstream of AUG2. The extension products were resolved using 6% denaturing PAGE along with a DNA size marker (M) and sequencing ladder from the plasmid with LNA-target site-AatII-(CAA)16. Primer extension reactions of RNA samples from COS-7 cells cotransfected with (+) or without (-) LNA oligonucleotide were compared in parallel. The results are representative of three experiments performed independently.

Mentions: To determine whether the ratio of utilization of two AUG codons is altered by binding of an antisense LNA oligonucleotide to various locations in a 5′ leader other than that of AUG1, we generated a series of mRNA constructs with isosequential 5′ leaders by inserting an LNA-target sequence into different parts of the 5′ leader of the (CAA)16 CAT-FLAG mRNA (Figure 5A). The binding site used in this study is complementary to the LNA-C oligonucleotide, which was used as a control in our earlier studies. This set of studies was performed using RNA transfections as in Figure S3, because we found that it was easier to control the mRNA:LNA oligonucleotide ratio in cells using this approach, compared to plasmid cotransfections, which express the various recombinant mRNAs at different levels. For these studies, COS-7 cells were transfected with the various CAT-FLAG mRNAs and protein expression was quantified. In the absence of the LNA oligonucleotide, AUG1 is preferentially used in all of the constructs, and the AUG1:AUG2 ratio is higher when the LNA binding site is located at or near the 5′-end of the transcript (Figure 5B, see constructs 1 and 2). This result suggests that the sequence composition of the 5′ leader may be another variable that can influence the relative usage of AUG codons in an mRNA, and which may affect ribosomal interactions with AUG codons by other means (see Discussion).


Determinants of initiation codon selection during translation in mammalian cells.

Matsuda D, Mauro VP - PLoS ONE (2010)

Ratio of AUG codon usage is affected by nucleotide composition of the 5′ leader.A. Schematic representation of CAT-FLAG mRNAs. The arrows (numbered 1-5) indicate the positions of individual LNA target sequences in the 5′ leaders of different constructs. Arrow 6 indicates the position of a mutated target sequence. The thick black bar at the 5′ end of the mRNA represents an AatII sequence; dashes in the 5′ leader represent CAA repeats. B. Relative expression of CAT-FLAG proteins. Expression from AUG1 is normalized to that from AUG2 in COS-7 cells that were transfected with in vitro transcribed capped and poly(A)-tailed mRNAs with 5′ leader sequences as depicted in (A). Transfections were performed in the presence (+; dark grey bar) and absence (-; light grey bar) of LNA oligonucleotide. The cells were harvested 5 hours post transfection. Three independent experiments were performed for final quantification of immunoblots with error bars indicating standard deviations. Cotransfection with LNA oligonucleotide did not significantly alter relative AUG usage (two-sided t-test). One-way ANOVA analysis of relative AUG codon usage in constructs 3–6 ((-)LNA) did not show significant differences. However, there were some significant differences between constructs 1 or 2 ((-)LNA) and various other constructs. One-sided t-tests of construct 1 compared to constructs 3–6 ((-)LNA) yielded p-values of 0.06, 0.04, 0.04, and 0.06, respectively. One-sided t-tests of construct 2 compared to constructs 3–6 ((-)LNA) yielded p-values of <0.01, <0.01, 0.02, and <0.01, respectively. C. Primer extension inhibition analysis on target mRNAs bound with LNA oligonucleotide. Total RNA extracted from cells 5 h post transfection was analyzed by primer extension to confirm the positions of LNA binding to target mRNAs, using a primer that anneals 67-nucleotides downstream of AUG2. The extension products were resolved using 6% denaturing PAGE along with a DNA size marker (M) and sequencing ladder from the plasmid with LNA-target site-AatII-(CAA)16. Primer extension reactions of RNA samples from COS-7 cells cotransfected with (+) or without (-) LNA oligonucleotide were compared in parallel. The results are representative of three experiments performed independently.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991327&req=5

pone-0015057-g005: Ratio of AUG codon usage is affected by nucleotide composition of the 5′ leader.A. Schematic representation of CAT-FLAG mRNAs. The arrows (numbered 1-5) indicate the positions of individual LNA target sequences in the 5′ leaders of different constructs. Arrow 6 indicates the position of a mutated target sequence. The thick black bar at the 5′ end of the mRNA represents an AatII sequence; dashes in the 5′ leader represent CAA repeats. B. Relative expression of CAT-FLAG proteins. Expression from AUG1 is normalized to that from AUG2 in COS-7 cells that were transfected with in vitro transcribed capped and poly(A)-tailed mRNAs with 5′ leader sequences as depicted in (A). Transfections were performed in the presence (+; dark grey bar) and absence (-; light grey bar) of LNA oligonucleotide. The cells were harvested 5 hours post transfection. Three independent experiments were performed for final quantification of immunoblots with error bars indicating standard deviations. Cotransfection with LNA oligonucleotide did not significantly alter relative AUG usage (two-sided t-test). One-way ANOVA analysis of relative AUG codon usage in constructs 3–6 ((-)LNA) did not show significant differences. However, there were some significant differences between constructs 1 or 2 ((-)LNA) and various other constructs. One-sided t-tests of construct 1 compared to constructs 3–6 ((-)LNA) yielded p-values of 0.06, 0.04, 0.04, and 0.06, respectively. One-sided t-tests of construct 2 compared to constructs 3–6 ((-)LNA) yielded p-values of <0.01, <0.01, 0.02, and <0.01, respectively. C. Primer extension inhibition analysis on target mRNAs bound with LNA oligonucleotide. Total RNA extracted from cells 5 h post transfection was analyzed by primer extension to confirm the positions of LNA binding to target mRNAs, using a primer that anneals 67-nucleotides downstream of AUG2. The extension products were resolved using 6% denaturing PAGE along with a DNA size marker (M) and sequencing ladder from the plasmid with LNA-target site-AatII-(CAA)16. Primer extension reactions of RNA samples from COS-7 cells cotransfected with (+) or without (-) LNA oligonucleotide were compared in parallel. The results are representative of three experiments performed independently.
Mentions: To determine whether the ratio of utilization of two AUG codons is altered by binding of an antisense LNA oligonucleotide to various locations in a 5′ leader other than that of AUG1, we generated a series of mRNA constructs with isosequential 5′ leaders by inserting an LNA-target sequence into different parts of the 5′ leader of the (CAA)16 CAT-FLAG mRNA (Figure 5A). The binding site used in this study is complementary to the LNA-C oligonucleotide, which was used as a control in our earlier studies. This set of studies was performed using RNA transfections as in Figure S3, because we found that it was easier to control the mRNA:LNA oligonucleotide ratio in cells using this approach, compared to plasmid cotransfections, which express the various recombinant mRNAs at different levels. For these studies, COS-7 cells were transfected with the various CAT-FLAG mRNAs and protein expression was quantified. In the absence of the LNA oligonucleotide, AUG1 is preferentially used in all of the constructs, and the AUG1:AUG2 ratio is higher when the LNA binding site is located at or near the 5′-end of the transcript (Figure 5B, see constructs 1 and 2). This result suggests that the sequence composition of the 5′ leader may be another variable that can influence the relative usage of AUG codons in an mRNA, and which may affect ribosomal interactions with AUG codons by other means (see Discussion).

Bottom Line: In addition, we observed that increased leader length by itself changed the ratio of the proteins and favored initiation at AUG1.These observations demonstrate that initiation codon selection is affected by various factors, including AUG codon-accessibility and 5' leader length, and is not necessarily determined by the order of AUG codons (5'→3').The modulation of AUG codon accessibility may provide a powerful means of translation regulation in eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, The Scripps Research Institute and The Skaggs Institute for Chemical Biology, La Jolla, California, USA.

ABSTRACT
Factors affecting translation of mRNA contribute to the complexity of eukaryotic proteomes. In some cases, translation of a particular mRNA can generate multiple proteins. However, the factors that determine whether ribosomes initiate translation from the first AUG codon in the transcript, from a downstream codon, or from multiple sites are not completely understood. Various mRNA properties, including AUG codon-accessibility and 5' leader length have been proposed as potential determinants that affect where ribosomes initiate translation. To explore this issue, we performed studies using synthetic mRNAs with two in-frame AUG codons-both in excellent context. Open reading frames initiating at AUG1 and AUG2 encode large and small isoforms of a reporter protein, respectively. Translation of such an mRNA in COS-7 cells was shown to be 5' cap-dependent and to occur efficiently from both AUG codons. AUG codon-accessibility was modified by using two different elements: an antisense locked nucleic acid oligonucleotide and an exon-junction complex. When either element was used to mask AUG1, the ratio of the proteins synthesized changed, favoring the smaller (AUG2-initiated) protein. In addition, we observed that increased leader length by itself changed the ratio of the proteins and favored initiation at AUG1. These observations demonstrate that initiation codon selection is affected by various factors, including AUG codon-accessibility and 5' leader length, and is not necessarily determined by the order of AUG codons (5'→3'). The modulation of AUG codon accessibility may provide a powerful means of translation regulation in eukaryotic cells.

Show MeSH
Related in: MedlinePlus