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The Silent Sway of Splicing by Synonymous Substitutions.

ABSTRACT: Alternative splicing diversifies mRNA transcripts in human cells. This sequence-driven process can be influenced greatly by mutations, even those that do not change the protein coding potential of the transcript. Synonymous mutations have been shown to alter gene expression through modulation of splicing, mRNA stability, and translation. Using a synonymous position mutation library in SMN1 exon 7, we show that 23% of synonymous mutations across the exon decrease exon inclusion, suggesting that nucleotide identity across the entire exon has been evolutionarily optimized to support a particular exon inclusion level. Although phylogenetic conservation scores are insufficient to identify synonymous positions important for exon inclusion, an alignment of organisms filtered based on similar exon/intron architecture is highly successful. Although many of the splicing neutral mutations are observed to occur, none of the exon inclusion reducing mutants was found in the filtered alignment. Using the modified phylogenetic comparison as an approach to evaluate the impact on pre-mRNA splicing suggests that up to 45% of synonymous SNPs are likely to alter pre-mRNA splicing. These results demonstrate that coding and pre-mRNA splicing pressures co-evolve and that a modified phylogenetic comparison based on the exon/intron architecture is a useful tool in identifying splice altering SNPs.

SUBMITTER: Mueller WF

PROVIDER: S-EPMC4646019 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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The Silent Sway of Splicing by Synonymous Substitutions.

Mueller William F WF Larsen Liza S Z LS Garibaldi Angela A Hatfield G Wesley GW Hertel Klemens J KJ

The Journal of biological chemistry 20150930 46

Alternative splicing diversifies mRNA transcripts in human cells. This sequence-driven process can be influenced greatly by mutations, even those that do not change the protein coding potential of the transcript. Synonymous mutations have been shown to alter gene expression through modulation of splicing, mRNA stability, and translation. Using a synonymous position mutation library in SMN1 exon 7, we show that 23% of synonymous mutations across the exon decrease exon inclusion, suggesting that n ...[more]

PMID: 26424794

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Project description:Synonymous genome recoding has been widely used to study different aspects of virus biology. Codon usage affects the temporal regulation of viral gene expression. In this study, we performed synonymous codon mutagenesis to investigate whether codon usage affected HIV-1 Env protein expression and virus viability. We replaced the codons AGG, GAG, CCU, ACU, CUC, and GGG of the HIV-1 env gene with the synonymous codons CGU, GAA, CCG, ACG, UUA, and GGA, respectively. We found that recoding the Env protein gp120 coding region (excluding the Rev response element [RRE]) did not significantly affect virus replication capacity, even though we introduced 15 new CpG dinucleotides. In contrast, changing a single codon (AGG to CGU) located in the gp41 coding region (HXB2 env position 2125 to 2127), which was included in the intronic splicing silencer (ISS), completely abolished virus replication and Env expression. Computational analyses of this mutant revealed a severe disruption in the ISS RNA secondary structure. A variant that restored ISS secondary RNA structure also reestablished Env production and virus viability. Interestingly, this codon variant prevented both virus replication and Env translation in a eukaryotic expression system. These findings suggested that disrupting mRNA splicing was not the only means of inhibiting translation. Our findings indicated that synonymous gp120 recoding was not always deleterious to HIV-1 replication. Importantly¸ we found that disrupting an external ISS loop strongly affected HIV-1 replication and Env translation.IMPORTANCE Synonymous substitutions can influence virus phenotype, replication capacity, and virulence. In this study, we explored how synonymous codon mutations impacted HIV-1 Env protein expression and virus replication capacity. We changed a single codon, AGG to CGU, which was located in the gp41 coding region (env nucleotide residues 2125 to 2127) and was included in the HIV-1 intronic splicing silencer. This change completely abolished virus replication and Env expression. We also found that changing codon usage in the gp120 region by including an increased number of CpG dinucleotides did not significantly affect Env expression or virus viability. Our findings showed that synonymous recoding was useful for altering viral phenotype and exploring virus biology.

Dataset Information

The Silent Sway of Splicing by Synonymous Substitutions.

Publications

The Silent Sway of Splicing by Synonymous Substitutions.

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