Gene structure for the alpha 1 chain of a human short-chain collagen (type XIII) with alternatively spliced transcripts and translation termination codon at the 5' end of the last exon.

The mature 109-amino-acid human platelet-derived growth factor B (PDGF-B) peptide is derived by intracellular processing from a 241-amino-acid precursor synthesized in mammalian cells, with removal of 81 N-terminal and 51 C-terminal amino acids. In order to produce directly the mature 109-amino acid PDGF-B peptide as a recombinant protein in Escherichia coli, a CGA codon at position 110 of a DNA sequence encoding the full-length precursor form of PDGF-B was converted into the translation termination codon TGA by in vitro mutagenesis. Expression of this DNA via a plasmid vector in E. coli resulted in production of two distinct PDGF-B proteins having apparent molecular masses of 15 and 19 kDa, with the latter species predominating. Structural characterization employing N- and C-terminal amino acid sequencing and MS analyses indicated that the 15 kDa protein is the expected 109-amino-acid PDGF-B, and that the 19 kDa protein represents a C-terminal extended PDGF-B containing 160 amino acids. Characterization of a unique tryptic peptide derived from the 19 kDa protein revealed that this longer form of PDGF-B results from mistranslation of the introduced TGA termination codon at position 110 as tryptophan, with translation subsequently proceeding to the naturally occurring TAG termination codon at position 161. Owing to the high rate of translation readthrough of TGA codons in this and occasionally other proteins, it appears that the use of TGA as a translation termination codon for proteins to be expressed in E. coli should be avoided when possible.

Download Full-text

Translation Termination Is Involved in Histone mRNA Degradation when DNA Replication Is Inhibited

Molecular and Cellular Biology ◽

10.1128/mcb.25.16.6879-6888.2005 ◽

2005 ◽

Vol 25 (16) ◽

pp. 6879-6888 ◽

Cited By ~ 43

Author(s):

Handan Kaygun ◽

William F. Marzluff

Keyword(s):

Dna Synthesis ◽

Translation Termination ◽

Termination Codon ◽

Histone Mrna ◽

Stem Loop ◽

Mrna Metabolism ◽

Histone Mrnas ◽

Iron Response Element ◽

Translation Termination Codon ◽

The Stability

ABSTRACT The levels of replication-dependent histone mRNAs are coordinately regulated with DNA synthesis. A major regulatory step in histone mRNA metabolism is regulation of the half-life of histone mRNAs. Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated. Instead, they end with a conserved stem-loop structure, which is recognized by the stem-loop binding protein (SLBP). SLBP is required for histone mRNA processing, as well as translation. We show here, using histone mRNAs whose translation can be regulated by the iron response element, that histone mRNAs need to be actively translated for their rapid degradation following the inhibition of DNA synthesis. We also demonstrate the requirement for translation using a mutant SLBP which is inactive in translation. Histone mRNAs are not rapidly degraded when DNA synthesis is inhibited or at the end of S phase in cells expressing this mutant SLBP. Replication-dependent histone mRNAs have very short 3′ untranslated regions, with the stem-loop located 30 to 70 nucleotides downstream of the translation termination codon. We show here that the stability of histone mRNAs can be modified by altering the position of the stem-loop, thereby changing the distance from the translation termination codon.

Download Full-text

Nucleotide sequence of a macronuclear DNA molecule coding for α-tubulin from the ciliateStylonychia lemnae. Special codon usage: TAA is not a translation termination codon

Nucleic Acids Research ◽

10.1093/nar/13.2.415 ◽

1985 ◽

Vol 13 (2) ◽

pp. 415-433 ◽

Cited By ~ 114

Author(s):

E. Helftenbem

Keyword(s):

Nucleotide Sequence ◽

Codon Usage ◽

Translation Termination ◽

Termination Codon ◽

Dna Molecule ◽

Translation Termination Codon

Download Full-text

Biochemical properties, tissue expression, and gene structure of a short chain dehydrogenase/reductase able to catalyze cis-retinol oxidation

The Journal of Lipid Research ◽

10.1016/s0022-2275(20)32103-9 ◽

1999 ◽

Vol 40 (12) ◽

pp. 2279-2292 ◽

Cited By ~ 2

Author(s):

Mary V. Gamble ◽

Enyuan Shang ◽

Roseann Piantedosi Zott ◽

James R. Mertz ◽

Debra J. Wolgemuth ◽

...

Keyword(s):

Gene Structure ◽

Tissue Expression ◽

Biochemical Properties ◽

Short Chain ◽

Short Chain Dehydrogenase

Download Full-text

Caenorhabditis elegans SMG-2 Selectively Marks mRNAs Containing Premature Translation Termination Codons

Molecular and Cellular Biology ◽

10.1128/mcb.00410-07 ◽

2007 ◽

Vol 27 (16) ◽

pp. 5630-5638 ◽

Cited By ~ 39

Author(s):

Lisa Johns ◽

Andrew Grimson ◽

Sherry L. Kuchma ◽

Carrie Loushin Newman ◽

Philip Anderson

Keyword(s):

Caenorhabditis Elegans ◽

Mammalian Cells ◽

Translation Termination ◽

Yeast Two Hybrid ◽

Eukaryotic Mrnas ◽

Nonsense Mediated Mrna Decay ◽

Endogenous Genes ◽

Alternatively Spliced ◽

Two Hybrid ◽

Premature Translation Termination

ABSTRACT Eukaryotic mRNAs containing premature translation termination codons (PTCs) are rapidly degraded by a process termed “nonsense-mediated mRNA decay” (NMD). We examined protein-protein and protein-RNA interactions among Caenorhabditis elegans proteins required for NMD. SMG-2, SMG-3, and SMG-4 are orthologs of yeast (Saccharomyces cerevisiae) and mammalian Upf1, Upf2, and Upf3, respectively. A combination of immunoprecipitation and yeast two-hybrid experiments indicated that SMG-2 interacts with SMG-3, SMG-3 interacts with SMG-4, and SMG-2 interacts indirectly with SMG-4 via shared interactions with SMG-3. Such interactions are similar to those observed in yeast and mammalian cells. SMG-2-SMG-3-SMG-4 interactions require neither SMG-2 phosphorylation, which is abolished in smg-1 mutants, nor SMG-2 dephosphorylation, which is reduced or eliminated in smg-5 mutants. SMG-2 preferentially associates with PTC-containing mRNAs. We monitored the association of SMG-2, SMG-3, and SMG-4 with mRNAs of five endogenous genes whose mRNAs are alternatively spliced to either contain or not contain PTCs. SMG-2 associates with both PTC-free and PTC-containing mRNPs, but it strongly and preferentially associates with (“marks”) those containing PTCs. SMG-2 marking of PTC-mRNPs is enhanced by SMG-3 and SMG-4, but SMG-3 and SMG-4 are not detectably associated with the same mRNPs. Neither SMG-2 phosphorylation nor dephosphorylation is required for selective association of SMG-2 with PTC-containing mRNPs, indicating that SMG-2 is phosphorylated only after premature terminations have been discriminated from normal terminations. We discuss these observations with regard to the functions of SMG-2 and its phosphorylation during NMD.

Download Full-text

The gene structure and hypervariability of the complete Penaeus monodon Dscam gene

Scientific Reports ◽

10.1038/s41598-019-52656-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

Author(s):

Kantamas Apitanyasai ◽

Shiao-Wei Huang ◽

Tze Hann Ng ◽

Shu-Ting He ◽

Yu-Hsun Huang ◽

...

Keyword(s):

Alternative Splicing ◽

Gene Structure ◽

Transmembrane Domain ◽

Stop Codon ◽

Penaeus Monodon ◽

Cytoplasmic Tail ◽

Extracellular Region ◽

Alternatively Spliced ◽

Alternatively Spliced Exons ◽

Selection Of

Abstract Using two advanced sequencing approaches, Illumina and PacBio, we derive the entire Dscam gene from an M2 assembly of the complete Penaeus monodon genome. The P. monodon Dscam (PmDscam) gene is ~266 kbp, with a total of 44 exons, 5 of which are subject to alternative splicing. PmDscam has a conserved architectural structure consisting of an extracellular region with hypervariable Ig domains, a transmembrane domain, and a cytoplasmic tail. We show that, contrary to a previous report, there are in fact 26, 81 and 26 alternative exons in N-terminal Ig2, N-terminal Ig3 and the entirety of Ig7, respectively. We also identified two alternatively spliced exons in the cytoplasmic tail, with transmembrane domains in exon variants 32.1 and 32.2, and stop codons in exon variants 44.1 and 44.2. This means that alternative splicing is involved in the selection of the stop codon. There are also 7 non-constitutive cytoplasmic tail exons that can either be included or skipped. Alternative splicing and the non-constitutive exons together produce more than 21 million isoform combinations from one PmDscam locus in the P. monodon gene. A public-facing database that allows BLAST searches of all 175 exons in the PmDscam gene has been established at http://pmdscam.dbbs.ncku.edu.tw/.

Download Full-text

Structural Characterization of the Rous Sarcoma Virus RNA Stability Element

Journal of Virology ◽

10.1128/jvi.02113-08 ◽

2008 ◽

Vol 83 (5) ◽

pp. 2119-2129 ◽

Cited By ~ 23

Author(s):

Jason E. Weil ◽

Michalis Hadjithomas ◽

Karen L. Beemon

Keyword(s):

Secondary Structure ◽

Rous Sarcoma Virus ◽

Translation Termination ◽

Rna Stability ◽

Premature Termination Codon ◽

Termination Codon ◽

Stem Loop ◽

Future Design ◽

Rous Sarcoma ◽

Sarcoma Virus

ABSTRACT In eukaryotic cells, an mRNA bearing a premature termination codon (PTC) or an abnormally long 3′ untranslated region (UTR) is often degraded by the nonsense-mediated mRNA decay (NMD) pathway. Despite the presence of a 5- to 7-kb 3′ UTR, unspliced retroviral RNA escapes this degradation. We previously identified the Rous sarcoma virus (RSV) stability element (RSE), an RNA element downstream of the gag natural translation termination codon that prevents degradation of the unspliced viral RNA. Insertion of this element downstream of a PTC in the RSV gag gene also inhibits NMD. Using partial RNase digestion and selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, we determined the secondary structure of this element. Incorporating RNase and SHAPE data into structural prediction programs definitively shows that the RSE contains an AU-rich stretch of about 30 single-stranded nucleotides near the 5′ end and two substantial stem-loop structures. The overall secondary structure of the RSE appears to be conserved among 20 different avian retroviruses. The structural aspects of this element will serve as a tool in the future design of cis mutants in addressing the mechanism of stabilization.

Download Full-text

A Premature Termination Codon Interferes with the Nuclear Function of an Exon Splicing Enhancer in an Open Reading Frame-Dependent Manner

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.1640 ◽

1999 ◽

Vol 19 (3) ◽

pp. 1640-1650 ◽

Cited By ~ 17

Author(s):

Anand Gersappe ◽

David J. Pintel

Keyword(s):

Translation Termination ◽

Rna Stability ◽

Open Reading Frame ◽

Termination Codon ◽

Dependent Manner ◽

Enhancer Element ◽

Reading Frame ◽

Exon Splicing ◽

Nuclear Rna ◽

Splicing Enhancer

ABSTRACT Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5′ element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5′ and 3′ elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3′ enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3′ splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus.

Download Full-text