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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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LNA-AUG1 modulates translation of the target mRNA.A. The top panel is a Western blot analysis of COS-7 cells transfected with plasmid constructs expressing (CAA)4 CAT-FLAG and FLAG-Luc2 mRNAs, together with various amounts of LNA-AUG1 oligonucleotide. The lower panel is a semi-quantitative RT-PCR analysis of (CAA)4 CAT-FLAG mRNA expression in cells exposed to different amounts of LNA oligonucleotides. Duplex RT-PCR reactions were performed to analyze the levels of the control FLAG-luc2 mRNA in each sample. B. Quantification of the effects of LNA-AUG1. An indication of relative AUG codon usage (left ordinate) is indicated by black squares and solid line and is obtained by normalizing the expression of the 31 kDa protein from AUG1 to that of the 28 kDa protein from AUG2. This ratio is plotted against concentration of LNA-AUG1 oligonucleotide. Relative expression of normalized CAT-FLAG mRNA levels (right ordinate; expressed relative to the sample with no LNA cotransfection) are indicated by open circles and dashed line. C. Target mRNA remains intact in cells cotransfected with LNA-AUG1. 1.0X β-globin CAT-FLAG mRNA from COS-7 cells cotransfected with LNA-AUG1 in independent triplicates was analyzed by Northern blot using probes that hybridize to regions upstream or downstream of the LNA-AUG1 target site. The diagram shows the relative positions of the probes. The control FLAG-luc2 mRNA was detected by a subsequent hybridization using probes specific to this control mRNA. The five-fold dilutions were of an equivalent in vitro transcribed RNA. D. Primer extension analysis of RNA samples tested by Northern analysis using a primer that anneals 23-nucleotides downstream of AUG2. An in vitro transcribed RNA was included as a control for the position of primer extension inhibition by LNA-AUG1 binding. This RNA was incubated with (+) or without (-) LNA-AUG1. The sequencing ladder is from the corresponding plasmid; the marker is DNA. RNA samples from cells transfected with plasmid (TC; -), with plasmid and LNA-AUG1 (TC; +), and from untransfected cells (UTC) were analyzed in parallel. The positions of the mRNA 5′ ends and LNA stop sites are indicated by arrows. The position of AUG1 is indicated by asterisks in the sequencing ladder.
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pone-0015057-g002: LNA-AUG1 modulates translation of the target mRNA.A. The top panel is a Western blot analysis of COS-7 cells transfected with plasmid constructs expressing (CAA)4 CAT-FLAG and FLAG-Luc2 mRNAs, together with various amounts of LNA-AUG1 oligonucleotide. The lower panel is a semi-quantitative RT-PCR analysis of (CAA)4 CAT-FLAG mRNA expression in cells exposed to different amounts of LNA oligonucleotides. Duplex RT-PCR reactions were performed to analyze the levels of the control FLAG-luc2 mRNA in each sample. B. Quantification of the effects of LNA-AUG1. An indication of relative AUG codon usage (left ordinate) is indicated by black squares and solid line and is obtained by normalizing the expression of the 31 kDa protein from AUG1 to that of the 28 kDa protein from AUG2. This ratio is plotted against concentration of LNA-AUG1 oligonucleotide. Relative expression of normalized CAT-FLAG mRNA levels (right ordinate; expressed relative to the sample with no LNA cotransfection) are indicated by open circles and dashed line. C. Target mRNA remains intact in cells cotransfected with LNA-AUG1. 1.0X β-globin CAT-FLAG mRNA from COS-7 cells cotransfected with LNA-AUG1 in independent triplicates was analyzed by Northern blot using probes that hybridize to regions upstream or downstream of the LNA-AUG1 target site. The diagram shows the relative positions of the probes. The control FLAG-luc2 mRNA was detected by a subsequent hybridization using probes specific to this control mRNA. The five-fold dilutions were of an equivalent in vitro transcribed RNA. D. Primer extension analysis of RNA samples tested by Northern analysis using a primer that anneals 23-nucleotides downstream of AUG2. An in vitro transcribed RNA was included as a control for the position of primer extension inhibition by LNA-AUG1 binding. This RNA was incubated with (+) or without (-) LNA-AUG1. The sequencing ladder is from the corresponding plasmid; the marker is DNA. RNA samples from cells transfected with plasmid (TC; -), with plasmid and LNA-AUG1 (TC; +), and from untransfected cells (UTC) were analyzed in parallel. The positions of the mRNA 5′ ends and LNA stop sites are indicated by arrows. The position of AUG1 is indicated by asterisks in the sequencing ladder.

Mentions: An LNA antisense oligonucleotide (LNA-AUG1) was designed to target AUG1 in the (CAA)4 CAT-FLAG mRNA. This oligonucleotide was cotransfected into cells along with plasmid constructs expressing the (CAA)4 CAT-FLAG mRNA and a cotransfection control mRNA (FLAG-Luc2). The LNA-AUG1 oligonucleotide was tested at different dilutions while keeping the total amount of oligonucleotide constant in each transfection reaction by using a non-specific isosequential LNA oligonucleotide (LNA-C) as filler. The results showed that expression of both the large and small CAT-FLAG proteins was differentially inhibited in a manner dependent on the amount of cotransfected LNA-AUG1 (Figure 2A, Western blot). Although translation from both AUG codons was decreased, translation from AUG1, which is targeted by LNA-AUG1, was decreased substantially more (>3-fold) than translation from AUG2. This decrease was most pronounced at oligonucleotide concentrations above 33 nM (Figure 2B). The relative expression levels of CAT-FLAG mRNAs were not significantly affected by cotransfection of cells with LNA-AUG1, as measured by semi-quantitative RT-PCR (Figure 2A, RT-PCR; and 2B). This result suggests that the effect of the LNA-AUG1 oligonucleotide on the (CAA)4 CAT-FLAG mRNA is post-transcriptional and is not due to degradation of the target mRNA.


Determinants of initiation codon selection during translation in mammalian cells.

Matsuda D, Mauro VP - PLoS ONE (2010)

LNA-AUG1 modulates translation of the target mRNA.A. The top panel is a Western blot analysis of COS-7 cells transfected with plasmid constructs expressing (CAA)4 CAT-FLAG and FLAG-Luc2 mRNAs, together with various amounts of LNA-AUG1 oligonucleotide. The lower panel is a semi-quantitative RT-PCR analysis of (CAA)4 CAT-FLAG mRNA expression in cells exposed to different amounts of LNA oligonucleotides. Duplex RT-PCR reactions were performed to analyze the levels of the control FLAG-luc2 mRNA in each sample. B. Quantification of the effects of LNA-AUG1. An indication of relative AUG codon usage (left ordinate) is indicated by black squares and solid line and is obtained by normalizing the expression of the 31 kDa protein from AUG1 to that of the 28 kDa protein from AUG2. This ratio is plotted against concentration of LNA-AUG1 oligonucleotide. Relative expression of normalized CAT-FLAG mRNA levels (right ordinate; expressed relative to the sample with no LNA cotransfection) are indicated by open circles and dashed line. C. Target mRNA remains intact in cells cotransfected with LNA-AUG1. 1.0X β-globin CAT-FLAG mRNA from COS-7 cells cotransfected with LNA-AUG1 in independent triplicates was analyzed by Northern blot using probes that hybridize to regions upstream or downstream of the LNA-AUG1 target site. The diagram shows the relative positions of the probes. The control FLAG-luc2 mRNA was detected by a subsequent hybridization using probes specific to this control mRNA. The five-fold dilutions were of an equivalent in vitro transcribed RNA. D. Primer extension analysis of RNA samples tested by Northern analysis using a primer that anneals 23-nucleotides downstream of AUG2. An in vitro transcribed RNA was included as a control for the position of primer extension inhibition by LNA-AUG1 binding. This RNA was incubated with (+) or without (-) LNA-AUG1. The sequencing ladder is from the corresponding plasmid; the marker is DNA. RNA samples from cells transfected with plasmid (TC; -), with plasmid and LNA-AUG1 (TC; +), and from untransfected cells (UTC) were analyzed in parallel. The positions of the mRNA 5′ ends and LNA stop sites are indicated by arrows. The position of AUG1 is indicated by asterisks in the sequencing ladder.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991327&req=5

pone-0015057-g002: LNA-AUG1 modulates translation of the target mRNA.A. The top panel is a Western blot analysis of COS-7 cells transfected with plasmid constructs expressing (CAA)4 CAT-FLAG and FLAG-Luc2 mRNAs, together with various amounts of LNA-AUG1 oligonucleotide. The lower panel is a semi-quantitative RT-PCR analysis of (CAA)4 CAT-FLAG mRNA expression in cells exposed to different amounts of LNA oligonucleotides. Duplex RT-PCR reactions were performed to analyze the levels of the control FLAG-luc2 mRNA in each sample. B. Quantification of the effects of LNA-AUG1. An indication of relative AUG codon usage (left ordinate) is indicated by black squares and solid line and is obtained by normalizing the expression of the 31 kDa protein from AUG1 to that of the 28 kDa protein from AUG2. This ratio is plotted against concentration of LNA-AUG1 oligonucleotide. Relative expression of normalized CAT-FLAG mRNA levels (right ordinate; expressed relative to the sample with no LNA cotransfection) are indicated by open circles and dashed line. C. Target mRNA remains intact in cells cotransfected with LNA-AUG1. 1.0X β-globin CAT-FLAG mRNA from COS-7 cells cotransfected with LNA-AUG1 in independent triplicates was analyzed by Northern blot using probes that hybridize to regions upstream or downstream of the LNA-AUG1 target site. The diagram shows the relative positions of the probes. The control FLAG-luc2 mRNA was detected by a subsequent hybridization using probes specific to this control mRNA. The five-fold dilutions were of an equivalent in vitro transcribed RNA. D. Primer extension analysis of RNA samples tested by Northern analysis using a primer that anneals 23-nucleotides downstream of AUG2. An in vitro transcribed RNA was included as a control for the position of primer extension inhibition by LNA-AUG1 binding. This RNA was incubated with (+) or without (-) LNA-AUG1. The sequencing ladder is from the corresponding plasmid; the marker is DNA. RNA samples from cells transfected with plasmid (TC; -), with plasmid and LNA-AUG1 (TC; +), and from untransfected cells (UTC) were analyzed in parallel. The positions of the mRNA 5′ ends and LNA stop sites are indicated by arrows. The position of AUG1 is indicated by asterisks in the sequencing ladder.
Mentions: An LNA antisense oligonucleotide (LNA-AUG1) was designed to target AUG1 in the (CAA)4 CAT-FLAG mRNA. This oligonucleotide was cotransfected into cells along with plasmid constructs expressing the (CAA)4 CAT-FLAG mRNA and a cotransfection control mRNA (FLAG-Luc2). The LNA-AUG1 oligonucleotide was tested at different dilutions while keeping the total amount of oligonucleotide constant in each transfection reaction by using a non-specific isosequential LNA oligonucleotide (LNA-C) as filler. The results showed that expression of both the large and small CAT-FLAG proteins was differentially inhibited in a manner dependent on the amount of cotransfected LNA-AUG1 (Figure 2A, Western blot). Although translation from both AUG codons was decreased, translation from AUG1, which is targeted by LNA-AUG1, was decreased substantially more (>3-fold) than translation from AUG2. This decrease was most pronounced at oligonucleotide concentrations above 33 nM (Figure 2B). The relative expression levels of CAT-FLAG mRNAs were not significantly affected by cotransfection of cells with LNA-AUG1, as measured by semi-quantitative RT-PCR (Figure 2A, RT-PCR; and 2B). This result suggests that the effect of the LNA-AUG1 oligonucleotide on the (CAA)4 CAT-FLAG mRNA is post-transcriptional and is not due to degradation of the target mRNA.

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