Dataset Information

Conserved regional 3' grouping of rare codons in the coding sequence of ocular prosecretory mitogen lacritin.

ABSTRACT: Lacritin is a prosecretory mitogen in tears and, although a tear protein, it promotes basal tearing and lacrimal gland secretion. Since scale up is relevant to its potential use in the treatment of dry eye, we explored various mutagenic strategies to alter the stability, solubility, and translational efficiency of nascent lacritin, and discovered 3' clustering of rare human codons.Site-directed mutagenesis of lacritin coding cDNA "pLAC" generated 24 different nonsynonymous and 13 synonymous mutations. Nonsynonymous mutations altered amino acids with nonpolar, basic or acidic side chains to serine. Synonymous mutation progressively optimized human codons that are rare or uncommon in Escherichia coli without changing the amino acid specified. These changes were validated by sequencing and protein production, and analyzed via the "rare codon calculator" (RCC). Nonhuman primate and nonprimate lacritin coding sequences were extracted from Ensembl, and analyzed via RCC using codon usage appropriate for each species.Superior yields were obtained by modification of individual hydrophobic residues or a predicted salt bridge, suggesting that production was limited by lacritin stability. Accordingly, elimination of rare codons increased yields less effectively. Importantly, RCC analysis of human, nonhuman primate (mouse lemur) and nonprimate (cat, tree shrew) lacritin coding sequences revealed remarkable 3' clustering of rare codons, unlike human lipocalin-1 and 21 other widely expressed human tear genes.Lacritin protein yields were improved primarily by hydrophobic or salt bridge mutagenesis and less so by elimination of rare codons. The 3' clustering of rare codons is conserved in all lacritin orthologs examined.

SUBMITTER: McKown RL

PROVIDER: S-EPMC3604909 | biostudies-other | 2013 Mar

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Conserved regional 3' grouping of rare codons in the coding sequence of ocular prosecretory mitogen lacritin.

McKown Robert L RL Raab Ronald W RW Kachelries Patricia P Caldwell Sara S Laurie Gordon W GW

Investigative ophthalmology & visual science 20130319 3

<h4>Purpose</h4>Lacritin is a prosecretory mitogen in tears and, although a tear protein, it promotes basal tearing and lacrimal gland secretion. Since scale up is relevant to its potential use in the treatment of dry eye, we explored various mutagenic strategies to alter the stability, solubility, and translational efficiency of nascent lacritin, and discovered 3' clustering of rare human codons.<h4>Methods</h4>Site-directed mutagenesis of lacritin coding cDNA "pLAC" generated 24 different nons ...[more]

PMID: 23422824

Similar Datasets

Project description:Synonymous rare codons are considered to be sub-optimal for gene expression because they are translated more slowly than common codons. Yet surprisingly, many protein coding sequences include large clusters of synonymous rare codons. Rare codons at the 5' terminus of coding sequences have been shown to increase translational efficiency. Although a general functional role for synonymous rare codons farther within coding sequences has not yet been established, several recent reports have identified rare-to-common synonymous codon substitutions that impair folding of the encoded protein. Here we test the hypothesis that although the usage frequencies of synonymous codons change from organism to organism, codon rarity will be conserved at specific positions in a set of homologous coding sequences, for example to tune translation rate without altering a protein sequence. Such conservation of rarity-rather than specific codon identity-could coordinate co-translational folding of the encoded protein. We demonstrate that many rare codon cluster positions are indeed conserved within homologous coding sequences across diverse eukaryotic, bacterial, and archaeal species, suggesting they result from positive selection and have a functional role. Most conserved rare codon clusters occur within rather than between conserved protein domains, challenging the view that their primary function is to facilitate co-translational folding after synthesis of an autonomous structural unit. Instead, many conserved rare codon clusters separate smaller protein structural motifs within structural domains. These smaller motifs typically fold faster than an entire domain, on a time scale more consistent with translation rate modulation by synonymous codon usage. While proteins with conserved rare codon clusters are structurally and functionally diverse, they are enriched in functions associated with organism growth and development, suggesting an important role for synonymous codon usage in organism physiology. The identification of conserved rare codon clusters advances our understanding of distinct, functional roles for otherwise synonymous codons and enables experimental testing of the impact of synonymous codon usage on the production of functional proteins.

Project description:Neurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs) in its >700-nucleotide (nt) 5' leader, and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4, respectively, in controlling translation. We also found that the 5' region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5' conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs) in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitroIn vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscores the emerging significance of noncanonical initiation events in controlling gene expression.IMPORTANCE There is a deepening and widening appreciation of the diverse roles of translation in controlling gene expression. A central fungal transcription factor, the best-studied example of which is Saccharomyces cerevisiae GCN4, is crucial for the response to amino acid limitation. Two upstream open reading frames (uORFs) in the GCN4 mRNA are critical for controlling GCN4 synthesis. We observed that two uORFs in the corresponding Neurospora crassa cpc-1 mRNA appear functionally analogous to the GCN4 uORFs. We also discovered that, surprisingly, unlike GCN4, the CPC1 coding sequence extends far upstream from the presumed AUG start codon with no other in-frame AUG codons. Similar extensions were seen in homologs from many filamentous fungi. We observed that multiple non-AUG near-cognate codons (NCCs) in this extended reading frame, some conserved, initiated translation to produce longer forms of CPC1, underscoring the significance of noncanonical initiation in controlling gene expression.

Project description:BackgroundA substantial fraction of non-coding DNA sequences of multicellular eukaryotes is under selective constraint. In particular, approximately 5% of the human genome consists of conserved non-coding sequences (CNSs). CNSs differ from other genomic sequences in their nucleotide composition and must play important functional roles, which mostly remain obscure.ResultsWe investigated relative abundances of short sequence motifs in all human CNSs present in the human/mouse whole-genome alignments vs. three background sets of sequences: (i) weakly conserved or unconserved non-coding sequences (non-CNSs); (ii) near-promoter sequences (located between nucleotides -500 and -1500, relative to a start of transcription); and (iii) random sequences with the same nucleotide composition as that of CNSs. When compared to non-CNSs and near-promoter sequences, CNSs possess an excess of AT-rich motifs, often containing runs of identical nucleotides. In contrast, when compared to random sequences, CNSs contain an excess of GC-rich motifs which, however, lack CpG dinucleotides. Thus, abundance of short sequence motifs in human CNSs, taken as a whole, is mostly determined by their overall compositional properties and not by overrepresentation of any specific short motifs. These properties are: (i) high AT-content of CNSs, (ii) a tendency, probably due to context-dependent mutation, of A's and T's to clump, (iii) presence of short GC-rich regions, and (iv) avoidance of CpG contexts, due to their hypermutability. Only a small number of short motifs, overrepresented in all human CNSs are similar to binding sites of transcription factors from the FOX family.ConclusionHuman CNSs as a whole appear to be too broad a class of sequences to possess strong footprints of any short sequence-specific functions. Such footprints should be studied at the level of functional subclasses of CNSs, such as those which flank genes with a particular pattern of expression. Overall properties of CNSs are affected by patterns in mutation, suggesting that selection which causes their conservation is not always very strong.

Dataset Information

Conserved regional 3' grouping of rare codons in the coding sequence of ocular prosecretory mitogen lacritin.

Publications

Conserved regional 3' grouping of rare codons in the coding sequence of ocular prosecretory mitogen lacritin.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets