Effect of the [CCTG]n repeat expansion on ZNF9 expression in myotonic dystrophy type II (DM2)

Myotonic dystrophy is caused by two different mutations: a (CTG)n expansion in 3' UTR region of the DMPK gene (DM1) and a (CCTG)n expansion in intron 1 of the ZNF9 gene (DM2). The most accredited mechanism for DM pathogenesis is an RNA gain-of-function. Other findings suggest a contributory role of DMPK-insufficiency in DM1. To address the issue of ZNF9 role in DM2, we have analyzed the effects of (CCTG)n expansion on ZNF9 expression in lymphoblastoid cell lines (n=4) from DM2 patients. We did not observe any significant alteration in ZNF9 mRNA and protein levels, as shown by QRT-PCR and Western blot analyses. Additional RT-PCR experiments demonstrated that ZNF9 pre-mRNA splicing pattern, which includes two isoforms, is unmodified in DM2 cells. Our results indicate that the (CCTG)n expansion in the ZNF9 intron does not appear to have a direct consequence on the expression of the gene itself.     

The double-stranded RNA-binding protein,Staufen1, is an IRES-transacting factor regulating HIV-1 cap-independent translation initiation

Translation initiation of the viral genomic mRNA (vRNA) of human immunodeficiency virus-type 1 (HIV-1) can be mediated by a cap- or an internal ribosome entry site (IRES)-dependent mechanism. A previous report shows that Staufen1, a cellular double-stranded (ds) RNA-binding protein (RBP), binds to the 5’untranslated region (5′UTR) of the HIV-1 vRNA and promotes its cap-dependent translation. In this study, we now evaluate the role of Staufen1 as an HIV-1 IRES-transacting factor (ITAF). We first confirm that Staufen1 associates with both the HIV-1 vRNA and the Gag protein during HIV-1 replication. We found that in HIV-1-expressing cells, siRNA-mediated depletion of Staufen1 reduces HIV-1 vRNA translation. Using dual-luciferase bicistronic mRNAs, we show that the siRNA-mediated depletion and cDNA-mediated overexpression of Staufen1 acutely regulates HIV-1 IRES activity. Furthermore, we show that Staufen1-vRNA interaction is required for the enhancement of HIV-1 IRES activity. Interestingly, we find that only Staufen1 harboring an intact dsRNA-binding domain 3 (dsRBD3) rescues HIV-1 IRES activity in Staufen1 CRISPR-Cas9 gene edited cells. Finally, we show that the expression of Staufen1-dsRBD3 alone enhances HIV-1 IRES activity. This study provides evidence of a novel role for Staufen1 as an ITAF promoting HIV-1 vRNA IRES activity.     

The hallmarks of myotonic dystrophy type 1 muscle dysfunction

Myotonic dystrophy type 1 (DM1) is the most prevalent form of muscular dystrophy in adults and yet there are currently no treatment options. Although this disease causes multisystemic symptoms, it is mainly characterised by myopathy or diseased muscles, which includes muscle weakness, atrophy, and myotonia, severely affecting the lives of patients worldwide. On a molecular level, DM1 is caused by an expansion of CTG repeats in the 3′ untranslated region (3′UTR) of the DM1 Protein Kinase (DMPK) gene which become pathogenic when transcribed into RNA forming ribonuclear foci comprised of auto complementary CUG hairpin structures that can bind proteins. This leads to the sequestration of the muscleblind-like (MBNL) family of proteins, depleting them, and the abnormal stabilisation of CUGBP Elav-like family member 1 (CELF1), enhancing it. Traditionally, DM1 research has focused on this RNA toxicity and how it alters MBNL and CELF1 functions as key splicing regulators. However, other proteins are affected by the toxic DMPK RNA and there is strong evidence that supports various signalling cascades playing an important role in DM1 pathogenesis. Specifically, the impairment of protein kinase B (AKT) signalling in DM1 increases autophagy, apoptosis, and ubiquitin–proteasome activity, which may also be affected in DM1 by AMP-activated protein kinase (AMPK) downregulation. AKT also regulates CELF1 directly, by affecting its subcellular localisation, and indirectly as it inhibits glycogen synthase kinase 3 beta (GSK3β), which stabilises the repressive form of CELF1 in DM1. Another kinase that contributes to CELF1 mis-regulation, in this case by hyperphosphorylation, is protein kinase C (PKC). Additionally, it has been demonstrated that fibroblast growth factor-inducible 14 (Fn14) is induced in DM1 and is associated with downstream signalling through the nuclear factor κB (NFκB) pathways, associating inflammation with this disease. Furthermore, MBNL1 and CELF1 play a role in cytoplasmic processes involved in DM1 myopathy, altering proteostasis and sarcomere structure. Finally, there are many other elements that could contribute to the muscular phenotype in DM1 such as alterations to satellite cells, non-coding RNA metabolism, calcium dysregulation, and repeat-associated non-ATG (RAN) translation. This review aims to organise the currently dispersed knowledge on the different pathways affected in DM1 and discusses the unexplored connections that could potentially help in providing new therapeutic targets in DM1 research.     

DAP5 promotes cap-independent translation of Bcl-2 and CDK1 to facilitate cell survival during mitosis

DAP5 is an eIF4G protein previously implicated in mediating cap-independent translation in response to cellular stresses. Here we report that DAP5 is crucial for continuous cell survival in nonstressed cells. The knockdown of endogenous DAP5 induced M phase-specific caspase-dependent apoptosis. Bcl-2 and CDK1 were identified by two independent screens as DAP5 translation targets. Notably, the activity of the Bcl-2 IRES was reduced in DAP5 knockdown cells and a selective shift of Bcl-2 mRNA toward light polysomal fractions was detected. Furthermore, a functional IRES was identified in the 5'UTR of CDK1. At the cellular level, attenuated translation of CDK1 by DAP5 knockdown decreased the phosphorylation of its M phase substrates. Ectopic expression of Bcl-2 or CDK1 proteins partially reduced the extent of caspase activation caused by DAP5 knockdown. Thus, DAP5 is necessary for maintaining cell survival during mitosis by promoting cap-independent translation of at least two prosurvival proteins.     

The unstable CCTG repeat responsible for myotonic dystrophy type 2 originates from an AluSx element insertion into an early primate genome

Myotonic dystrophy type 2 (DM2) is a subtype of the myotonic dystrophies, caused by expansion of a tetranucleotide CCTG repeat in intron 1 of the zinc finger protein 9 (ZNF9) gene. The expansions are extremely unstable and variable, ranging from 75-11,000 CCTG repeats. This unprecedented repeat size and somatic heterogeneity make molecular diagnosis of DM2 difficult, and yield variable clinical phenotypes. To better understand the mutational origin and instability of the ZNF9 CCTG repeat, we analyzed the repeat configuration and flanking regions in 26 primate species. The 3'-end of an AluSx element, flanked by target site duplications (5'-ACTRCCAR-3'or 5'-ACTRCCARTTA-3'), followed the CCTG repeat, suggesting that the repeat was originally derived from the Alu element insertion. In addition, our results revealed lineage-specific repetitive motifs: pyrimidine (CT)-rich repeat motifs in New World monkeys, dinucleotide (TG) repeat motifs in Old World monkeys and gibbons, and dinucleotide (TG) and tetranucleotide (TCTG and/or CCTG) repeat motifs in great apes and humans. Moreover, these di- and tetra-nucleotide repeat motifs arose from the poly (A) tail of the AluSx element, and evolved into unstable CCTG repeats during primate evolution. Alu elements are known to be the source of microsatellite repeats responsible for two other repeat expansion disorders: Friedreich ataxia and spinocerebellar ataxia type 10. Taken together, these findings raise questions as to the mechanism(s) by which Alu-mediated repeats developed into the large, extremely unstable expansions common to these three disorders.     

Tpa1p is part of an mRNP complex that influences translation termination, mRNA deadenylation, and mRNA turnover in Saccharomyces cerevisiae

In this report, we show that the Saccharomyces cerevisiae protein Tpa1p (for termination and polyadenylation) influences translation termination efficiency, mRNA poly(A) tail length, and mRNA stability. Tpa1p is encoded by the previously uncharacterized open reading frame YER049W. Yeast strains carrying a deletion of the TPA1 gene (tpa1Delta) exhibited increased readthrough of stop codons, and coimmunoprecipitation assays revealed that Tpa1p interacts with the translation termination factors eRF1 and eRF3. In addition, the tpa1Delta mutation led to a 1.5- to 2-fold increase in the half-lives of mRNAs degraded by the general 5'-->3' pathway or the 3'-->5' nonstop decay pathway. In contrast, this mutation did not have any affect on the nonsense-mediated mRNA decay pathway. Examination of mRNA poly(A) tail length revealed that poly(A) tails are longer than normal in a tpa1Delta strain. Consistent with a potential role in regulating poly(A) tail length, Tpa1p was also found to coimmunoprecipitate with the yeast poly(A) binding protein Pab1p. These results suggest that Tpa1p is a component of a messenger ribonucleoprotein complex bound to the 3' untranslated region of mRNAs that affects translation termination, deadenylation, and mRNA decay.     

The translation of an antiapoptotic protein HIAP2 is regulated by an upstream open reading frame

HIAP2 is a multifunctional protein that is critically involved in the regulation of cell survival and apoptosis. Here, we show that HIAP2 5' untranslated region functions as a strong inhibitor of translation. Sequence analysis of human, mouse and rat sequences revealed that there exists a short open reading frame (ORF) that is located just upstream of the HIAP2 coding sequence. The translation of this uORF severely inhibited translation of the downstream reporter gene in vivo but not in vitro. Point mutation that destroys the CUG initiating codon of uORF markedly enhanced translation of the reporter gene without affecting the mRNA levels. Our results identify a novel translational regulatory mechanism that controls the expression of HIAP2 and point to the importance of tight regulation of antiapoptotic gene expression.     

The translation initiation factor eIF2beta is an interactor of protein phosphatase-1

It is reasonably well understood how the initiation of translation is controlled by reversible phosphorylation of the eukaryotic translation initiation factors eIF2alpha, eIF2Bepsilon and eIF4E. Other initiation factors, including eIF2beta, are also established phosphoproteins but the physiological impact of their phosphorylation is not known. Using a sequence homology search we found that the central region of eIF2beta contains a putative PP1-(protein phosphatase-1) binding RVxF-motif. The predicted eIF2beta-PP1 interaction was confirmed by PP1 binding and co-immunoprecipitation assays on cell lysates as well as with the purified components. Site-directed mutagenesis showed that eIF2beta contains, in addition to an RVxF-motif, at least one other PP1-binding site in its C-terminal half. eIF2beta functioned as an inhibitor for the dephosphorylation of glycogen phosphorylase and Ser51 of eIF2alpha by PP1, but did not affect the dephosphorylation of Ser464 of eIF2Bepsilon by this phosphatase. Strikingly, eIF2beta emerged as an activator of its own dephosphorylation (Ser2, Ser67, Ser218) by associated PP1, since the substrate quality of eIF2beta was decreased by the mere mutation of its RVxF-motif. These results make eIF2beta an attractive candidate substrate for associated PP1 in vivo. The overexpression of wild-type eIF2beta or eIF2beta with a mutated RVxF-motif did not differentially affect the rate of translation, indicating that the binding of PP1 is not rate-limiting for translation under basal conditions.     

Confirmation of the type 2 myotonic dystrophy (CCTG)n expansion mutation in patients with proximal myotonic myopathy/proximal myotonic dystrophy of different European origins: a single shared haplotype indicates an ancestral founder effect

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, is a clinically and genetically heterogeneous neuromuscular disorder. DM is characterized by autosomal dominant inheritance, muscular dystrophy, myotonia, and multisystem involvement. Type 1 DM (DM1) is caused by a (CTG)(n) expansion in the 3' untranslated region of DMPK in 19q13.3. Multiple families, predominantly of German descent and with clinically variable presentation that included proximal myotonic myopathy (PROMM) and type 2 DM (DM2) but without the DM1 mutation, showed linkage to the 3q21 region and were recently shown to segregate a (CCTG)(n) expansion mutation in intron 1 of ZNF9. Here, we present linkage to 3q21 and mutational confirmation in 17 kindreds of European origin with PROMM and proximal myotonic dystrophy, from geographically distinct populations. All patients have the DM2 (CCTG)(n) expansion. To study the evolution of this mutation, we constructed a comprehensive physical map of the DM2 region around ZNF9. High-resolution haplotype analysis of disease chromosomes with five microsatellite and 22 single-nucleotide polymorphism markers around the DM2 mutation identified extensive linkage disequilibrium and a single shared haplotype of at least 132 kb among patients from the different populations. With the exception of the (CCTG)(n) expansion, the available markers indicate that the DM2 haplotype is identical to the most common haplotype in normal individuals. This situation is reminiscent of that seen in DM1. Taken together, these data suggest a single founding mutation in DM2 patients of European origin. We estimate the age of the founding haplotype and of the DM2 (CCTG) expansion mutation to be approximately 200-540 generations.     

The petunia restorer of fertility protein is part of a large mitochondrial complex that interacts with transcripts of the CMS-associated locus

A class of nuclear genes termed "restorers of fertility" (Rf) acts to suppress the expression of abnormal mitochondrial genes associated with cytoplasmic male sterility (CMS). In petunia, both the nuclear Rf gene and mitochondrial CMS-associated gene have previously been identified. The CMS-associated gene is an aberrant chimera in which portions of several mitochondrially encoded genes are fused to an unknown reading frame. The dominant Rf allele reduces the CMS-associated protein to nearly undetectable levels and alters the RNA population derived from the CMS locus, but its mechanism of action has not been determined. The petuniaRf gene is a member of the pentatricopeptide repeat gene family (PPR), an unusually large gene family in Arabidopsis (approximately 450 genes) compared with yeast (five genes) and mammalian genomes (six genes). The PPR gene family has been implicated in the control of organelle gene expression. To gain insight into the mode of action of PPR genes, we generated transgenic petunia plants expressing a functional tagged version of Rf. Analysis of the restorer protein revealed that it is part of a soluble mitochondrial inner-membrane-associated, RNase-sensitive high-molecular-weight protein complex. The complex is associated with mRNA derived from the CMS locus.     

The kinesin-related protein MCAK is a microtubule depolymerase that forms an ATP-hydrolyzing complex at microtubule ends

MCAK belongs to the Kin I subfamily of kinesin-related proteins, a unique group of motor proteins that are not motile but instead destabilize microtubules. We show that MCAK is an ATPase that catalytically depolymerizes microtubules by accelerating, 100-fold, the rate of dissociation of tubulin from microtubule ends. MCAK has one high-affinity binding site per protofilament end, which, when occupied, has both the depolymerase and ATPase activities. MCAK targets protofilament ends very rapidly (on-rate 54 micro M(-1).s(-1)), perhaps by diffusion along the microtubule lattice, and, once there, removes approximately 20 tubulin dimers at a rate of 1 s(-1). We propose that up to 14 MCAK dimers assemble at the end of a microtubule to form an ATP-hydrolyzing complex that processively depolymerizes the microtubule.     

Vaccinia virus G9 protein is an essential component of the poxvirus entry-fusion complex

The vaccinia virus G9R gene (VACWR087) encodes a protein of 340 amino acids with the following structural features that are conserved in all poxviruses: a site for N-terminal myristoylation, 14 cysteines, and a C-terminal transmembrane domain. Previous studies showed that G9 is one of eight proteins associated in a putative entry-fusion complex. Our attempt to isolate a mutant without the G9R gene was unsuccessful, suggesting that it is essential for virus replication. To further investigate its role, we constructed a recombinant vaccinia virus in which G9R is regulated by addition of an inducer. Induced G9 protein was associated with mature infectious virions and could be labeled with a membrane-impermeant biotinylation reagent, indicating surface exposure. Omission of inducer reduced the infectious-virus yield by about 1.5 logs; nevertheless, all stages of virus morphogenesis appeared normal and extracellular virions were present on the cell surface. Purified virions assembled without inducer had a specific infectivity of less than 5% of the normal level and a comparably small amount of G9, whereas their overall polypeptide composition, including other components of the entry-fusion complex, was similar to that of virions made in the presence of inducer or of wild-type virions. G9-deficient virions bound to cells, but penetration of cores into the cytoplasm and early viral RNA synthesis were barely detected, and cell-cell fusion was not triggered by low pH. Of the identified components of the multiprotein complex, G9 is the sixth that has been shown to be required for entry and membrane fusion.