The RNA-binding protein Secisbp2 differentially modulates UGA codon reassignment and RNA decay.

Dual-assignment of codons as termination and elongation codons is used to expand the genetic code. In mammals, UGA can be reassigned to selenocysteine during translation of selenoproteins by a mechanism involving a 3΄ untranslated region (UTR) selenocysteine insertion sequence (SECIS) and the SECIS-binding protein Secisbp2. Here, we present data from ribosome profiling, RNA-Seq and mRNA half-life measurements that support distinct roles for Secisbp2 in UGA-redefinition and mRNA stability. Conditional deletions of the Secisbp2 and Trsp (tRNASec) genes in mouse liver were compared to determine if the effects of Secisbp2 loss on selenoprotein synthesis could be attributed entirely to the inability to incorporate Sec. As expected, tRNASec depletion resulted in loss of ribosome density downstream of all UGA-Sec codons. In contrast, the absence of Secisbp2 resulted in variable effects on ribosome density downstream of UGA-Sec codons that demonstrate gene-specific differences in Sec incorporation. For several selenoproteins in which loss of Secisbp2 resulted in greatly diminished mRNA levels, translational activity and Sec incorporation efficiency were shown to be unaffected on the remaining RNA. Collectively, these results demonstrate that Secisbp2 is not strictly required for Sec incorporation and has a distinct role in stabilizing mRNAs that can be separated from its effects on UGA-redefinition.

Translational redefinition of UGA codons is regulated by selenium availability.

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.

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.

Divergent regulation of the key enzymes of polyamine metabolism by chiral alpha-methylated polyamine analogues.

The natural polyamines are ubiquitous multifunctional organic cations which play important roles in regulating cellular proliferation and survival. Here we present a novel approach to investigating polyamine functions by using optical isomers of MeSpd (alpha-methylspermidine) and Me2Spm (alpha,omega-bismethylspermine), metabolically stable functional mimetics of natural polyamines. We studied the ability of MeSpd and Me2Spm to alter the normal polyamine regulation pathways at the level of polyamine uptake and the major control mechanisms known to affect the key polyamine metabolic enzymes. These include: (i) ODC (ornithine decarboxylase), which catalyses the rate-limiting step of polyamine synthesis; (ii) ODC antizyme, an inhibitor of ODC and polyamine uptake; (iii) SSAT (spermidine/spermine N1-acetyltransferase), the major polyamine catabolic enzyme; and (iv) AdoMetDC (S-adenosyl-L-methionine decarboxylase), which is required for the conversion of putrescine into spermidine, and spermidine into spermine. We show that the stereoisomers differ in their cellular uptake and ability to downregulate ODC and AdoMetDC, and to induce SSAT. These effects are mediated by the ability of the enantiomers to induce +1 ribosomal frameshifting on ODC antizyme mRNA, to suppress the translation of AdoMetDC uORF (upstream open reading frame) and to regulate the alternative splicing of SSAT pre-mRNA. The unique effects of chiral polyamine analogues on polyamine metabolism may offer novel possibilities for studying the physiological functions, control mechanisms, and targets of the natural polyamines, as well as advance therapeutic drug development in cancer and other human health-related issues.

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.

DMD pseudoexon mutations: splicing efficiency, phenotype, and potential therapy.

OBJECTIVE: 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. METHODS: We identified three pseudoexon insertion mutations in dystrophinopathy patients, two of whom had tissue available for further analysis. For these two out-of-frame pseudoexon mutations (one associated with Becker muscular dystrophy and one with DMD), mutation-induced splicing was tested by quantitative reverse transcription polymerase chain reaction; pseudoexon skipping was tested using AONs composed of 2'-O-methyl-modified bases on a phosphorothioate backbone to treat cultured primary myoblasts. RESULTS: Variable amounts of pseudoexon inclusion correlates with the severity of the dystrophinopathy phenotype in these two patients. AON treatment directed at the pseudoexon results in the expression of full-length dystrophin in a DMD myoblast line. INTERPRETATION: Both DMD and Becker muscular dystrophy can result from out-of-frame pseudoexons, with the difference in phenotype being due to variable efficiency of the newly generated splicing signal. AON-mediated pseudoexon skipping therapy is a viable approach to these patients and would be predicted to result in increased expression of wild-type dystrophin protein.

Antisense-induced ribosomal frameshifting.

Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1-ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem-loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce +1 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the +1 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5' of the shift site and to polyamine levels.

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

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.

Dietary Selenium Levels Affect Selenoprotein Expression and Support the Interferon-γ and IL-6 Immune Response Pathways in Mice.

Selenium is an essential element that is required to support a number of cellular functions and biochemical pathways. The objective of this study was to examine the effects of reduced dietary selenium levels on gene expression to assess changes in expression of non-selenoprotein genes that may contribute to the physiological consequences of selenium deficiency. Mice were fed diets that were either deficient in selenium or supplemented with selenium in the form of sodium selenite for six weeks. Differences in liver mRNA expression and translation were measured using a combination of ribosome profiling, RNA-Seq, microarrays, and qPCR. Expression levels and translation of mRNAs encoding stress-related selenoproteins were shown to be up-regulated by increased selenium status, as were genes involved in inflammation and response to interferon-γ. Changes in serum cytokine levels were measured which confirmed that interferon-γ, as well as IL-6, were increased in selenium adequate mice. Finally, microarray and qPCR analysis of lung tissue demonstrated that the selenium effects on immune function are not limited to liver. These data are consistent with previous reports indicating that adequate selenium levels can support beneficial immune responses, and further identify the IL-6 and interferon-γ pathways as being responsive to dietary selenium intake.

A recoding element that stimulates decoding of UGA codons by Sec tRNA[Ser]Sec.

Selenocysteine insertion during decoding of eukaryotic selenoprotein mRNA requires several trans-acting factors and a cis-acting selenocysteine insertion sequence (SECIS) usually located in the 3' UTR. A second cis-acting selenocysteine codon redefinition element (SRE) has recently been described that resides near the UGA-Sec codon of selenoprotein N (SEPN1). Similar phylogenetically conserved elements can be predicted in a subset of eukaryotic selenoprotein mRNAs. Previous experimental analysis of the SEPN1 SRE revealed it to have a stimulatory effect on readthrough of the UGA-Sec codon, which was not dependent upon the presence of a SECIS element in the 3' UTR; although, as expected, readthrough efficiency was further elevated by inclusion of a SECIS. In order to examine the nature of the redefinition event stimulated by the SEPN1 SRE, we have modified an experimentally tractable in vitro translation system that recapitulates efficient selenocysteine insertion. The results presented here illustrate that the SRE element has a stimulatory effect on decoding of the UGA-Sec codon by both the methylated and unmethylated isoforms of Sec tRNA([Ser]Sec), and confirm that efficient selenocysteine insertion is dependent on the presence of a 3'-UTR SECIS. The variation in recoding elements predicted near UGA-Sec codons implies that these elements may play a differential role in determining the amount of selenoprotein produced by acting as controllers of UGA decoding efficiency.

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.

Programmed ribosomal frameshifting in decoding the SARS-CoV genome.

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.

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 beta-catenin levels in colon tumor cells. APC and associated proteins thus form a beta-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: beta-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 beta-catenin. The observation of nuclear beta-catenin in fewer than half of carcinoma images and only rarely in polyps indicates that nuclear translocation of beta-catenin may not be an immediate consequence of the loss of APC.

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 Duchenne's and Becker's 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.