DDX6 regulates sequestered nuclear CUG-expanded DMPK-mRNA in dystrophia myotonica type 1

Myotonic dystrophy type 1 (DM1) is caused by CUG triplet expansions in the 3′ UTR of dystrophia myotonica protein kinase (DMPK) messenger ribonucleic acid (mRNA). The etiology of this multi-systemic disease involves pre-mRNA splicing defects elicited by the ability of the CUG-expanded mRNA to ‘sponge’ splicing factors of the muscleblind family. Although nuclear aggregation of CUG-containing mRNPs in distinct foci is a hallmark of DM1, the mechanisms of their homeostasis have not been completely elucidated. Here we show that a DEAD-box helicase, DDX6, interacts with CUG triplet-repeat mRNA in primary fibroblasts from DM1 patients and with CUG–RNA in vitro. DDX6 overexpression relieves DM1 mis-splicing, and causes a significant reduction in nuclear DMPK-mRNA foci. Conversely, knockdown of endogenous DDX6 leads to a significant increase in DMPK-mRNA foci count and to increased sequestration of MBNL1 in the nucleus. While the level of CUG-expanded mRNA is unaffected by increased DDX6 expression, the mRNA re-localizes to the cytoplasm and its interaction partner MBNL1 becomes dispersed and also partially re-localized to the cytoplasm. Finally, we show that DDX6 unwinds CUG-repeat duplexes in vitro in an adenosinetriphosphate-dependent manner, suggesting that DDX6 can remodel and release nuclear DMPK messenger ribonucleoprotein foci, leading to normalization of pathogenic alternative splicing events.     

Removal of cellular water prevents the reformation of the interphase nucleus

The mature snRNP (small nuclear ribonucleoprotein) particles are localized quantitatively in the interphase nucleus. Like many nuclear antigens, they distribute throughout the cytoplasm after the nuclear envelope breaks down during mitosis and then return to the newly formed daughter nuclei in early G1. Their abundance and stability and the availability of monoclonal antibodies that recognize them, make the snRNP particles a useful model system for studying the reformation of the nucleus at the completion of mitosis. A wide variety of metabolic inhibitors and alterations in normal culture conditions were investigated for their ability to interfere with the return of the snRNP particles to daughter nuclei after mitosis. None of the well-characterized cytoskeletal inhibitors, biosynthetic inhibitors, calcium antagonists, nor ionophores were effective in interfering with this return. However, the removal of cellular water by exposure of cells to hypertonic medium during mitosis blocked the reformation of the nucleus and trapped the snRNP particles in the cytoplasm. In medium of twice the normal tonicity, the function of the mitotic spindle and the cleavage furrow are inhibited, however, the cells reattach to the substratum as if returning to interphase. The chromatin stays condensed and does not form a normal interphase nucleus and the snRNP particles stay dispersed throughout the cytoplasm. This condition is reversible and after return to normal medium the nucleus reforms and the snRNP particles collect in the new nuclei. After gentle extraction of metaphase cells, about 30% of the snRNP particles are soluble, however, the remainder are associated with an insoluble remnant. These data are consistent with the notion that the snRNP particles accumulate in the nucleus due to both preferential solubility and specific binding sites in the interphase nucleus.     

Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of Muscleblind-like-1 isoforms

Muscleblind-like-1 (MBNL1) is a splicing regulatory factor controlling the fetal-to-adult alternative splicing transitions during vertebrate muscle development. Its capture by nuclear CUG expansions is one major cause for type 1 myotonic dystrophy (DM1). Alternative splicing produces MBNL1 isoforms that differ by the presence or absence of the exonic regions 3, 5, and 7. To understand better their respective roles and the consequences of the deregulation of their expression in DM1, here we studied the respective roles of MBNL1 alternative and constitutive exons. By combining genetics, molecular and cellular approaches, we found that (i) the exon 5 and 6 regions are both needed to control the nuclear localization of MBNL1; (ii) the exon 3 region strongly enhances the affinity of MBNL1 for its pre-mRNA target sites; (iii) the exon 3 and 6 regions are both required for the splicing regulatory activity, and this function is not enhanced by an exclusive nuclear localization of MBNL1; and finally (iv) the exon 7 region enhances MBNL1-MBNL1 dimerization properties. Consequently, the abnormally high inclusion of the exon 5 and 7 regions in DM1 is expected to enhance the potential of MBNL1 of being sequestered with nuclear CUG expansions, which provides new insight into DM1 pathophysiology.     

Cytoplasmic CUG RNA Foci Are Insufficient to Elicit Key DM1 Features

The genetic basis of myotonic dystrophy type I (DM1) is the expansion of a CTG tract located in the 3′ untranslated region of DMPK. Expression of mutant RNAs encoding expanded CUG repeats plays a central role in the development of cardiac disease in DM1. Expanded CUG tracts form both nuclear and cytoplasmic aggregates, yet the relative significance of such aggregates in eliciting DM1 pathology is unclear. To test the pathophysiology of CUG repeat encoding RNAs, we developed and analyzed mice with cardiac-specific expression of a beta-galactosidase cassette in which a (CTG)₄₀₀ repeat tract was positioned 3′ of the termination codon and 5′ of the bovine growth hormone polyadenylation signal. In these animals CUG aggregates form exclusively in the cytoplasm of cardiac cells. A key pathological consequence of expanded CUG repeat RNA expression in DM1 is aberrant RNA splicing. Abnormal splicing results from the functional inactivation of MBNL1, which is hypothesized to occur due to MBNL1 sequestration in CUG foci or from elevated levels of CUG-BP1. We therefore tested the ability of cytoplasmic CUG foci to elicit these changes. Aggregation of CUG RNAs within the cytoplasm results both in Mbnl1 sequestration and in approximately a two fold increase in both nuclear and cytoplasmic Cug-bp1 levels. Significantly, despite these changes RNA splice defects were not observed and functional analysis revealed only subtle cardiac dysfunction, characterized by conduction defects that primarily manifest under anesthesia. Using a human myoblast culture system we show that this transgene, when expressed at similar levels to a second transgene, which encodes expanded CTG tracts and facilitates both nuclear focus formation and aberrant splicing, does not elicit aberrant splicing. Thus the lack of toxicity of cytoplasmic CUG foci does not appear to be a consequence of low expression levels. Our results therefore demonstrate that the cellular location of CUG RNA aggregates is an important variable that influences toxicity and support the hypothesis that small molecules that increase the rate of transport of the mutant DMPK RNA from the nucleus into the cytoplasm may significantly improve DM1 pathology.     

Cell cycle-dependent assembly and disassembly of cytoplasmic microtubules in the plasmodium of the myxomycete Physarum polycephalum.

The giant syncytium of Physarum plasmodia possesses a complex cytoplasmic microtubule network except during the occurrence of the intranuclear mitosis. In early prophase stages, intranuclear spindles assemble concomitantly as the cytoplasmic microtubule network disassembles. No cytoplasmic microtubules are present in metaphase. They begin to reassemble in telophase. The complex cytoplasmic microtubule network reappears in early reconstruction stages. The assembly of cytoplasmic microtubules occurs on cytoplasmic foci, both in telophase stage and during rewarming after cold microtubule disassembly. These foci, independent of the nuclei, correspond to the foci observed in the cytoplasm during interphase, both by immunofluorescence and electron microscopy. As cytoplasmic and intranuclear microtubule-organizing centers are spatially distinct, plasmodial syncytia offer the possibility to study the effects of cell regulatory pathways on two types of microtubule-organizing centers that differ in their nucleating activity during the cell cycle.     

Tau exon 2 responsive elements deregulated in myotonic dystrophy type I are proximal to exon 2 and synergistically regulated by MBNL1 and MBNL2

The splicing of the microtubule-associated protein Tau is regulated during development and is found to be deregulated in a growing number of pathological conditions such as myotonic dystrophy type I (DM1), in which a reduced number of isoforms is expressed in the adult brain. DM1 is caused by a dynamic and unstable CTG repeat expansion in the DMPK gene, resulting in an RNA bearing long CUG repeats (n > 50) that accumulates in nuclear foci and sequesters CUG-binding splicing factors of the muscleblind-like (MBNL) family, involved in the splicing of Tau pre-mRNA among others. However, the precise mechanism leading to Tau mis-splicing and the role of MBNL splicing factors in this process are poorly understood. We therefore used new Tau minigenes that we developed for this purpose to determine how MBNL1 and MBNL2 interact to regulate Tau exon 2 splicing. We demonstrate that an intronic region 250 nucleotides downstream of Tau exon 2 contains cis-regulatory splicing enhancers that are sensitive to MBNL and that bind directly to MBNL1. Both MBNL1 and MBNL2 act as enhancers of Tau exon 2 inclusion. Intriguingly, the interaction of MBNL1 and MBNL2 is required to fully reverse the mis-splicing of Tau exon 2 induced by the trans-dominant effect of long CUG repeats, similar to the DM1 condition. In conclusion, both MBNL1 and MBNL2 are involved in the regulation of Tau exon 2 splicing and the mis-splicing of Tau in DM1 is due to the combined inactivation of both.     

A major 62-kD intranuclear matrix polypeptide is a component of metaphase chromosomes

We have isolated and partially characterized a major intranuclear matrix polypeptide from rat liver. This polypeptide, which is reversibly stabilized into the intranuclear matrix under conditions which promote intermolecular disulfide bond formation, has a Mr of 62,000 and pI of 6.8-7.2 as determined by two-dimensional IEF/SDS-PAGE. A chicken polyclonal antiserum was raised against the polypeptide purified from two-dimensional polyacrylamide gels. Affinity-purified anti-62-kD IgG was prepared and used to immunolocalize this polypeptide in rat liver tissue hepatocytes. In interphase hepatocytes the 62-kD antigen is localized in small, discrete patches within the nucleus consistent with the distribution of chromatin. The staining is most prominent at the nuclear periphery and somewhat less dense in the nuclear interior. Nucleoli and cytoplasm are devoid of staining. During mitosis the 62-kD antigen localizes to the condensed chromosomes with no apparent staining of cytoplasmic areas. The chromosomal staining during mitosis is uniform with no suggestion of the patching seen in interphase nuclei. Fractionation and immunoblotting studies using rat hepatoma tissue culture cells blocked in metaphase with colcemid confirm the chromosomal localization of this 62-kD intranuclear protein during mitosis. The 62-kD polypeptide fractionates completely with metaphase chromosome scaffolds generated by sequential treatment of isolated chromosomes with DNAse I and 1.6 M NaCl, suggesting that this major 62-kD intranuclear protein may be involved in maintaining metaphase chromosomal architecture.     

contribution to the study of nuclear total proteins and DNA during the mitotic cycle in fibroblasts cultivated in vitro and in Ehrlich ascites cells

Summary The dry weight and total protein content of nuclei has been measured by interferometry in living or fixed cells cultivated in vitro (freshly prepared chick, mouse or rat embryo fibroblasts) and in fixed Ehrlich ascites tumor cells of the mouse growing in vivo. The DNA content was estimated by cytophotometry after Feulgen reaction in the same nuclei. The dry weight of nucleoli in fibroblasts and the dry weight and DNA content of chromosomes in dividing fibroblasts and Ehrlich tumor cells have also been measured.During the interphase in fibroblasts, the dry weight of the living nucleus and the nuclear total protein content as measured in fixed cells doubles during the preparation for mitosis, as the DNA content does. In chick and mammal fibroblasts and within the limits of accuracy of our measurements, the synthesis curves for nuclear proteins and DNA do not seem to be necessarily identical.In our fibroblasts, the nucleolar total dry weight per nucleus doubles during the interphase (nucleolar preparation for mitosis); it increases in proportion to the nuclear total protein content, even in polyploid nuclei.During the mitosis, the chromosomes contain all the DNA of the nucleus but some nuclear proteins (non chromosomal proteins) seem to move into the cytoplasm during the mitosis and return into the nucleus at the post-telophase.According to our observations, Ehrlich ascites mouse tumor cells are near-tetraploid as far as the number of chromosomes, nuclear total protein content and DNA content are concerned. During the preparation for mitosis, these amounts double but no necessary close time relation seems to link these premitotic syntheses. Prom this point of view, our results show no clear-cut differences between these tumor cells and the fibroblasts. Except the polyploidy, the behaviour of nuclear proteins and DNA during mitosis in the tumor cells is the same as that observed in our fibroblasts.The effects of various antimitotic agents on rat fibroblasts cultivated in vitro have also been studied with our cytochemical methods. Our measurements of nuclear protein, DNA and nucleolar material content have been made in cells in which mitosis was prevented by alkylating agents, beryllium sulphate, RNase or neutral DNase. The effects of colchicine on these cellular parameters have also been studied.     

MPF localization is controlled by nuclear export.

In eukaryotes, mitosis is initiated by M phase promoting factor (MPF), composed of B-type cyclins and their partner protein kinase, CDK1. In animal cells, MPF is cytoplasmic in interphase and is translocated into the nucleus after mitosis has begun, after which it associates with the mitotic apparatus until the cyclins are degraded in anaphase. We have used a fusion protein between human cyclin B1 and green fluorescent protein (GFP) to study this dynamic behaviour in real time, in living cells. We found that when we injected cyclin B1-GFP, or cyclin B1-GFP bound to CDK1 (i.e. MPF), into interphase nuclei it is rapidly exported into the cytoplasm. Cyclin B1 nuclear export is blocked by leptomycin B, an inhibitor of the recently identified export factor, exportin 1 (CRM1). The nuclear export of MPF is mediated by a nuclear export sequence in cyclin B1, and an export-defective cyclin B1 accumulates in interphase nuclei. Therefore, during interphase MPF constantly shuttles between the nucleus and the cytoplasm, but the bulk of MPF is retained in the cytoplasm by rapid nuclear export. We found that a cyclin mutant with a defective nuclear export signal does not enhance the premature mitosis caused by interfering with the regulatory phosphorylation of CDK1, but is more sensitive to inhibition by the Wee1 kinase.     

Mitosis in the dermatophyteMicrosporum canis as revealed by freeze substitution electron microscopy

Summary Mitosis in the dermatophyteMicrosporum canis was studied by freeze substitution and electron microscopy, and analyzed by three dimensional reconstruction from serial sections of the mitotic nuclei. The interphase nucleus has associated nucleus-associated organelle (NAO) on a portion of the outer surface of the nuclear envelope, subjacent to which there was dense intranuclear material. The NAO divided and separated on the envelope, and a spindle was formed. The spindle was composed mostly of microtubules extended between opposite NAOs. Pairing of kinetochores was observed in the spindle from an early stage of development, when chromosomes were not so condensed, and remained unchanged while chromosome condensation proceeded until metaphase. Before the completion of nuclear division, daughter nuclei were connected by a narrow spindle channel, and then the nucleolus, whose structure underwent minimal change during mitosis, was eliminated into the cytoplasm.     

Mechanistic determinants of MBNL activity

Muscleblind-like (MBNL) proteins are critical RNA processing factors in development. MBNL activity is disrupted in the neuromuscular disease myotonic dystrophy type 1 (DM1), due to the instability of a non-coding microsatellite in the DMPK gene and the expression of CUG expansion (CUG^exp) RNAs. Pathogenic interactions between MBNL and CUG^exp RNA lead to the formation of nuclear complexes termed foci and prevent MBNL function in pre-mRNA processing. The existence of multiple MBNL genes, as well as multiple protein isoforms, raises the question of whether different MBNL proteins possess unique or redundant functions. To address this question, we coexpressed three MBNL paralogs in cells at equivalent levels and characterized both specific and redundant roles of these proteins in alternative splicing and RNA foci dynamics. When coexpressed in the same cells, MBNL1, MBNL2 and MBNL3 bind the same RNA motifs with different affinities. While MBNL1 demonstrated the highest splicing activity, MBNL3 showed the lowest. When forming RNA foci, MBNL1 is the most mobile paralog, while MBNL3 is rather static and the most densely packed on CUG^exp RNA. Therefore, our results demonstrate that MBNL paralogs and gene-specific isoforms possess inherent functional differences, an outcome that could be enlisted to improve therapeutic strategies for DM1.     

Monoclonal antibody against microtubule associated protein-1 produces immunofluorescent spots in the nucleus and centrosome of cultured mammalian cells

A monoclonal antibody was raised against the highest molecular weight protein associated with microtubules (MAP-1). Its specific binding to MAP-1 was determined by immunoblotting of the gel electrophoretogram of microtubule proteins prepared from porcine brain. The antibody reacted only with MAP-1, not with MAP-2, tau or tubulin. Indirect immunofluorescent staining by this antibody showed bright intranuclear spots, the centrosome and the faint meshwork of the cytoplasm in several types of cultured mammalian cells; HeLa, PtK2, human skin fibroblasts, mouse melanoma cells, Chinese hamster ovary cells. The nuclear spots in the interphase cells, were replaced by diffuse enhanced fluorescence throughout the cell except for chromosomes during mitosis. They reappeared in late telophase, first in the cytoplasm, late in the nucleus. The punctate pattern of nuclear immunofluorescence was not affected by microtubule-depolymerizing agents. The result that it persisted on residual cell structures after extraction with a high salt concentration buffer containing Triton X-100 followed by digestion with DNase I and RNase A suggests that the antigen is associated with the nuclear skeleton.     

Manipulation of the amount of RNA in post-mitotic nuclei

Because all (or almost all) nuclear RNAs are liberated to the cytoplasm during mitosis and then return to the post-mitotic nuclei, we expected that if cytoplasm were amputated from mitotic cells the post-division nuclei would possess less than normal amounts of RNA. Experiments performed with amebae (A. proteus) show that this is in fact what happens. Furthermore, since the enucleate fragment cut from a mitotic cell possesses an “excess” of returnable nuclear RNAs, a normal interphase nucleus implanted into such mitotic cytoplasm might be expected to acquire above-normal amounts of RNA. Experiments reported here show that this expectation also is realized. Thus, the regulation of the normal nuclear concentration of these RNAs involves mechanisms other than a limited number of intranuclear “binding” sites and most likely is restricted by the rate of synthesis of these RNAs.The demonstration that nuclei can be depleted or enriched for RNAs, many of which are unique to nuclei, makes it possible to determine the consequences for cell metabolism of altered amounts of nuclear RNA. Hopefully, such studies will reveal the function(s) of these RNAs.     

Expanded CUG repeats Dysregulate RNA splicing by altering the stoichiometry of the muscleblind 1 complex

To understand the role of the splice regulator muscleblind 1 (MBNL1) in the development of RNA splice defects in myotonic dystrophy I (DM1), we purified RNA-independent MBNL1 complexes from normal human myoblasts and examined the behavior of these complexes in DM1 myoblasts. Antibodies recognizing MBNL1 variants (MBNL1(CUG)), which can sequester in the toxic CUG RNA foci that develop in DM1 nuclei, were used to purify MBNL1(CUG) complexes from normal myoblasts. In normal myoblasts, MBNL1(CUG) bind 10 proteins involved in remodeling ribonucleoprotein complexes including hnRNP H, H2, H3, F, A2/B1, K, L, DDX5, DDX17, and DHX9. Of these proteins, only MBNL1(CUG) colocalizes extensively with DM1 CUG foci (>80% of foci) with its partners being present in <10% of foci. Importantly, the stoichiometry of MBNL1(CUG) complexes is altered in DM1 myoblasts, demonstrating an increase in the steady state levels of nine of its partner proteins. These changes are recapitulated by the expression of expanded CUG repeat RNA in Cos7 cells. Altered stoichiometry of MBNL1(CUG) complexes results from aberrant protein synthesis or stability and is unlinked to PKCα function. Modeling these changes in normal myoblasts demonstrates that increased levels of hnRNP H, H2, H3, F, and DDX5 independently dysregulate splicing in overlapping RNA subsets. Thus expression of expanded CUG repeats alters the stoichiometry of MBNL1(CUG) complexes to allow both the reinforcement and expansion of RNA processing defects.     

MITOSIS IN THE AQUATIC FUNGUS RHIZOPHYDIUM SPHEROTHECA (CHYTRIDIALES)

The structure of centric, intranuclear mitosis and of organelles associated with nuclei are described in developing zoosporangia of the chytrid Rhizophydium spherotheca. Frequently dictyosomes partially encompass the sides of diplosomes (paired centrioles). A single, incomplete layer of endoplasmic reticulum with tubular connections to the nuclear envelope is found around dividing nuclei. The nuclear envelope remains intact during mitosis except for polar fenestrae which appear during spindle incursion. During prophase, when diplosomes first define the nuclear poles, secondary centrioles occur adjacent and at right angles to the sides of primary centrioles. By late metaphase the centrioles in a diplosome are positioned at a 40° angle to each other and are joined by an electron-dense band; by telophase the centrioles lie almost parallel to each other. Astral microtubules radiate into the cytoplasm from centrioles during interphase, but by metaphase few cytoplasmic microtubules are found. Cytoplasmic microtubules increase during late anaphase and telophase as spindle microtubules gradually disappear. The mitotic spindle, which contains chromosomal and interzonal microtubules, converges at the base of the primary centriole. Throughout mitosis the semipersistent nucleolus is adjacent to the nuclear envelope and remains in the interzonal region of the nucleus as chromosomes separate and the nucleus elongates. During telophase the nuclear envelope constricts around the chromosomal mass, and the daughter nuclei separate from each end of the interzonal region of the nucleus. The envelope of the interzonal region is relatively intact and encircles the nucleolus, but later the membranes of the interzonal region scatter and the nucleolus disperses. The structure of the mitotic apparatus is similar to that of the chytrid Phlyctochytrium irregulare.