Christine B. Anderson

Readthrough of dystrophin stop codon mutations induced by aminoglycosides

We report the translational readthrough levels induced by the aminoglycosides gentamicin, amikacin, tobramycin, and paromomycin for eight premature stop codon mutations identified in Duchennes and Beckers muscular dystrophy patients. In a transient transfection reporter assay, aminoglycoside treatment results show that one stop codon mutation is suppressed significantly better (up to 10% stop codon readthrough) than the others; five show lower but statistically significant suppression (<2% stop codon readthrough); and two appear refractory to aminoglycoside treatment. Readthrough levels do not substantially vary between different sources of gentamicin, and, for this set of mutations, the efficiency of termination at the premature stop codon mutation does not appear to correlate with disease severity.

Subcellular distribution of Wnt pathway proteins in normal and neoplastic colon.

Mutations in the APC tumor suppressor gene are present in approximately 85% of colorectal tumors and are thought to contribute early in the process of tumorigenesis. The truncated protein resulting from most APC mutations can lead to elevated β-catenin levels in colon tumor cells. APC and associated proteins thus form a β-catenin regulatory complex, with axin playing a key role. Although cell culture studies have revealed intriguing aspects of this complex, little characterization has been done in human colonocytes, the target tissue of colon carcinogenesis. The present study of intact human colon crypts, adenomatous polyps, and adenocarcinomas focuses on subcellular localization of some key elements of the complex: β-catenin, APC, axin, and axin2. We examined endogenous protein localization within the framework of three-dimensional tissue architecture by using laser scanning confocal microscopy, and immunofluorescence staining of whole-mount fixed tissue from more than 50 patients. Expression patterns suggest that APC and axin colocalize in the nucleus and at lateral cell borders, and show that axin2 is limited to the nucleus. Altered nuclear expression of axin seen in colon polyps and carcinomas may be a consequence of the loss of full-length APC and the advent of nuclear β-catenin. The observation of nuclear β-catenin in fewer than half of carcinoma images and only rarely in polyps indicates that nuclear translocation of β-catenin may not be an immediate consequence of the loss of APC.

Recoding elements located adjacent to a subset of eukaryal selenocysteine-specifying UGA codons.

Incorporation of the 21st amino acid, selenocysteine, into proteins is specified in all three domains of life by dynamic translational redefinition of UGA codons. In eukarya and archaea, selenocysteine insertion requires a cis‐acting selenocysteine insertion sequence (SECIS) usually located in the 3′UTR of selenoprotein mRNAs. Here we present comparative sequence analysis and experimental data supporting the presence of a second stop codon redefinition element located adjacent to a selenocysteine‐encoding UGA codon in the eukaryal gene, SEPN1. This element is sufficient to stimulate high‐level (6%) translational redefinition of the SEPN1 UGA codon in human cells. Readthrough levels further increased to 12% when tested in the presence of the SEPN1 3′UTR SECIS. Directed mutagenesis and phylogeny of the sequence context strongly supports the importance of a stem loop starting six nucleotides 3′ of the UGA codon. Sequences capable of forming strong RNA structures were also identified 3′ adjacent to, or near, selenocysteine‐encoding UGA codons in the Sps2, SelH, SelO, and SelT selenoprotein genes.

DMD pseudoexon mutations: Splicing efficiency, phenotype, and potential therapy

The degenerative muscle diseases Duchenne (DMD) and Becker muscular dystrophy result from mutations in the DMD gene, which encodes the dystrophin protein. Recent improvements in mutational analysis techniques have resulted in the increasing identification of deep intronic point mutations, which alter splicing such that intronic sequences are included in the messenger RNA as “pseudoexons.” We sought to test the hypothesis that the clinical phenotype correlates with splicing efficiency of these mutations, and to test the feasibility of antisense oligonucleotide (AON)–mediated pseudoexon skipping.

Programmed ribosomal frameshifting in decoding the SARS-CoV genome

Abstract Programmed ribosomal frameshifting is an essential mechanism used for the expression of orf1b in coronaviruses. Comparative analysis of the frameshift region reveals a universal shift site U_UUA_AAC, followed by a predicted downstream RNA structure in the form of either a pseudoknot or kissing stem loops. Frameshifting in SARS-CoV has been characterized in cultured mammalian cells using a dual luciferase reporter system and mass spectrometry. Mutagenic analysis of the SARS-CoV shift site and mass spectrometry of an affinity tagged frameshift product confirmed tandem tRNA slippage on the sequence U_UUA_AAC. Analysis of the downstream pseudoknot stimulator of frameshifting in SARS-CoV shows that a proposed RNA secondary structure in loop II and two unpaired nucleotides at the stem I–stem II junction in SARS-CoV are important for frameshift stimulation. These results demonstrate key sequences required for efficient frameshifting, and the utility of mass spectrometry to study ribosomal frameshifting.

Nonsense mutation-associated Becker muscular dystrophy: interplay between exon definition and splicing regulatory elements within the DMD gene†

Nonsense mutations are usually predicted to function as null alleles due to premature termination of protein translation. However, nonsense mutations in the DMD gene, encoding the dystrophin protein, have been associated with both the severe Duchenne Muscular Dystrophy (DMD) and milder Becker Muscular Dystrophy (BMD) phenotypes. In a large survey, we identified 243 unique nonsense mutations in the DMD gene, and for 210 of these we could establish definitive phenotypes. We analyzed the reading frame predicted by exons flanking those in which nonsense mutations were found, and present evidence that nonsense mutations resulting in BMD likely do so by inducing exon skipping, confirming that exonic point mutations affecting exon definition have played a significant role in determining phenotype. We present a new model based on the combination of exon definition and intronic splicing regulatory elements for the selective association of BMD nonsense mutations with a subset of DMD exons prone to mutation‐induced exon skipping. Hum Mutat 32:299–308, 2011.

Translational Redefinition of UGA Codons Is Regulated by Selenium Availability

Background: Selenium is incorporated into selenoproteins as the amino acid, selenocysteine. Results: Dietary selenium supplementation increases ribosome density downstream of selenocysteine-encoding UGA codons. Conclusion: Dietary selenium levels differentially regulate selenoprotein expression by controlling the rate-limiting step of selenocysteine incorporation. Significance: The mechanisms by which dietary selenium can affect the readout of the genetic code and selenoprotein expression have been illuminated. Incorporation of selenium into ∼25 mammalian selenoproteins occurs by translational recoding whereby in-frame UGA codons are redefined to encode the selenium containing amino acid, selenocysteine (Sec). Here we applied ribosome profiling to examine the effect of dietary selenium levels on the translational mechanisms controlling selenoprotein synthesis in mouse liver. Dietary selenium levels were shown to control gene-specific selenoprotein expression primarily at the translation level by differential regulation of UGA redefinition and Sec incorporation efficiency, although effects on translation initiation and mRNA abundance were also observed. Direct evidence is presented that increasing dietary selenium causes a vast increase in ribosome density downstream of UGA-Sec codons for a subset of selenoprotein mRNAs and that the selenium-dependent effects on Sec incorporation efficiency are mediated in part by the degree of Sec-tRNA[Ser]Sec Um34 methylation. Furthermore, we find evidence for translation in the 5′-UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability. These data illustrate how dietary levels of the trace element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins.

A mutation in the SEPN1 selenocysteine redefinition element (SRE) reduces selenocysteine incorporation and leads to SEPN1‐related myopathy

Mutations in SEPN1 result in a spectrum of early‐onset muscle disorders referred to as SEPN1‐related myopathy. The SEPN1 gene encodes selenoprotein N (SelN), which contains the amino acid selenocysteine (Sec). Incorporation of Sec occurs due to redefinition of a UGA codon during translation. Efficient insertion requires a Sec insertion sequence (SECIS) in the 3′UTR and, for at least a subset of selenoprotein genes, a Sec redefinition element (SRE) located adjacent to the UGA codon. We report the effect of three novel and one previously reported point mutation in the SelN SRE element on Sec insertion efficiency. Notably, the previously reported mutation c.1397G>A (p.R466Q), which weakens the secondary structure of the SRE element, reduces Sec insertion efficiency and SelN RNA levels. Muscle from patients with this mutation have negligible levels of SelN protein. This data highlights the importance of the SRE element during SelN expression and illustrates a novel molecular mechanism by which point mutations may lead to SEPN1‐related myopathy. Hum Mutat 0, 1–6, 2008.

Attenuation of an amino-terminal premature stop codon mutation in the ATRX gene by an alternative mode of translational initiation

Nonsense mutations located in the 5′ end of the coding sequence of a gene are commonly considered to be null alleles. Not only do such mutations result in the production of a truncated and usually inactive protein product, but premature stop codon mutations that occur upstream of the last exon–exon junction are also known to activate nonsense mediated decay (NMD) which results in the specific degradation of the affected mRNA.1,2 However, several mechanisms may allow either fully or partially functional protein to be produced from alleles containing a premature stop codon mutation and this phenomenon may lead to considerable amelioration of the resulting phenotype. First, translational decoding (miscoding) of the stop codon as a sense codon, which can occur naturally due to the context of the stop codon,3 may result in low level “leaky” expression of full length protein. Although no examples of stop codon readthrough leading to disease amelioration have been clearly documented in humans, this mechanism has been implied for several premature stop codons, most notably for a premature stop codon mutation in the cystic fibrosis transmembrane conductance regulator gene (MIM 602421) leading to mild pulmonary presentation.4,5 In this case, the equivalent mutation in a yeast gene, Ste6, has been shown to be suppressed at levels as high as 10%.3 Second, an internally truncated form of the protein may be expressed due to altered processing of the mRNA in which the stop codon containing exon (and sometimes adjacent exons) are removed from the mature mRNA.6 A correlation between exon skipping and suppression of disease symptoms has been documented in several cases of Becker muscular dystrophy (MIM 300376), a milder form of Duchenne muscular dystrophy.7,8 Third, protein expression in some genes can be rescued by translational initiation at internal start …

Identification of a new antizyme mRNA +1 frameshifting stimulatory pseudoknot in a subset of diverse invertebrates and its apparent absence in intermediate species.

Abstract The expression of eukaryotic antizyme genes requires +1 translational frameshifting. The frameshift in decoding most vertebrate antizyme mRNAs is stimulated by an RNA pseudoknot 3′ of the frameshift site. Although the frameshifting event itself is conserved in a wide variety of organisms from yeast to mammals, until recently no corresponding 3′ RNA pseudoknot was known in invertebrate antizyme mRNAs. A pseudoknot, different in structure and origin from its vertebrate counterparts, is now shown to be encoded by the antizyme genes of distantly related invertebrates. Identification of the 3′ frameshifting stimulator in intermediate species or other invertebrates remains unresolved.