Femoral Structure and Biomechanical Characteristics in Sanfilippo Syndrome Type-B Mice.

Sanfilippo syndrome Type-B, also known as mucopolysaccharidosis IIIB (MPS IIIB), accounts for approximately one-third of all Sanfilippo syndrome patients and is characterized by a similar natural history as Type-A. Patients suffer from developmental regression, bone malformation, organomegaly, GI distress, and profound neurological deficits. Despite human trials of enzyme replacement therapy (ERT) (SBC-103, AX250) in MPS IIIB, there is currently no FDA approved treatment and a few palliative options. The major concerns of ERT and gene therapy for the treatment of bone malformation are the inadequate biodistribution of the missing enzyme, N-acetyl-α-glucosaminidase (NAGLU), and that the skeleton is a poorly hit target tissue in ERT and gene therapy. Each of the four known human types of MPS III (A, B, C, and D) is usually regarded as having mild bone manifestations, yet it remains poorly characterized. This study aimed to determine bone mineral content (BMC), volumetric bone mineral density (vBMD), and biomechanical properties in femurs MPS IIIB C57BL/6 mice compared to phenotypic control C57BL/6 mice. Significant differences were observed in MPS IIIB mice within various cortical and cancellous bone parameters for both males and females (p < 0.05). Here, we establish some osteogenic manifestations of MPS IIIB within the mouse model by radiographic and biomechanical tests, which are also differentially affected by age and sex. This suggests that some skeletal features of the MPS IIIB mouse model may be used as biomarkers of peripheral disease correction for preclinical treatment of MPS IIIB.

The Scd6/Lsm14 protein xRAPB has properties different from RAP55 in selecting mRNA for early translation or intracellular distribution in Xenopus oocytes.

Oocytes accumulate mRNAs in the form of maternal ribonucleoprotein (RNP) particles, the protein components of which determine the location and stability of individual mRNAs prior to translation. Scd6/Lsm14 proteins, typified by RAP55, function in a wide range of eukaryotes in repressing translation and relocating mRNPs to processing bodies and stress granules. In Xenopus laevis, the RAP55 orthologue xRAPA fulfils these functions. Here we describe the properties of a variant of xRAPA, xRAPB, which is a member of the Lsm14B group. xRAPB differs from xRAPA in various respects: it is expressed at high concentration earlier in oogenesis; it interacts specifically with the DDX6 helicase Xp54; it is detected in polysomes and stalled translation initiation complexes; its over-expression leads to selective binding to translatable mRNA species without evidence of translation repression or mRNA degradation. Since both Xp54 and xRAPA are repressors of translation, activation appears to be effected through targeting of xRAPB/Xp54.

Using oocyte nuclei for studies on chromatin structure and gene expression.

The giant nucleus of amphibian oocytes is generally referred to as the germinal vesicle (GV). Its size allows relatively easy manual isolation from the rest of the oocyte and also presents a large target in situ for microinjection of macromolecules including plasmid DNA, RNA species, antibodies and other proteins and even whole organelles, including somatic cell nuclei. Thus the use of GVs is excellent for two major types of study: the function of endogenous nuclear processes such as gene transcription, RNA processing and intra-nuclear dynamics; and the use of the nuclear components to effect processes such as chromatin assembly, expression of foreign genes and nucleocytoplasmic transport of injected biomolecules. This article outlines some basic techniques appropriate for GV studies, particularly the preparation of oocytes for microinjection and the isolation of germinal vesicles into an oil phase. As an aid to the targeting of the GV within the nucleus, descriptions are given of the use of oocytes from albino animals.

Dynamic regulation of histone modifications in Xenopus oocytes through histone exchange.

Histone H3 lysine 9 (H3K9) methylation has broad roles in transcriptional repression, gene silencing, maintenance of heterochromatin, and epigenetic inheritance of heterochromatin. Using Xenopus laevis oocytes, we have previously shown that targeting G9a, an H3K9 histone methyltransferase, to chromatin increases H3K9 methylation and consequently represses transcription. Here we report that treatment with trichostatin A induces histone acetylation and is sufficient to activate transcription repressed by G9a, and this activation is accompanied by a reduction in dimethyl H3K9 (H3K9me2). We tested the possibility that the reduction in H3K9me2 was due to the replacement of methylated H3 with unmethylated H3.3. Surprisingly, we found that both free H3 and H3.3 are continually exchanged with chromatin-associated histones. This dynamic exchange of chromatin-associated H3 with free H3/H3.3 was not affected by alterations in transcriptional activity, elongation, acetylation, H3K9 methylation, or DNA replication. In support of this continual histone exchange model, we show that maintenance of H3K9 methylation at a specific site requires the continual presence of an H3K9 histone methyltransferase. Upon dissociation of the methyltransferase, H3K9 methylation decreases. Taken together, our data suggest that chromatin-associated and non-chromatin-associated histones are continually exchanged in the Xenopus oocyte, creating a highly dynamic chromatin environment.

Xp54 and related (DDX6-like) RNA helicases: roles in messenger RNP assembly, translation regulation and RNA degradation.

The DEAD-box RNA helicase Xp54 is an integral component of the messenger ribonucleoprotein (mRNP) particles of Xenopus oocytes. In oocytes, several abundant proteins bind pre-mRNA transcripts to modulate nuclear export, RNA stability and translational fate. Of these, Xp54, the mRNA-masking protein FRGY2 and its activating protein kinase CK2alpha, bind to nascent transcripts on chromosome loops, whereas an Xp54-associated factor, RapA/B, binds to the mRNP complex in the cytoplasm. Over-expression, mutation and knockdown experiments indicate that Xp54 functions to change the conformation of mRNP complexes, displacing one subset of proteins to accommodate another. The sequence of Xp54 is highly conserved in a wide spectrum of organisms. Like Xp54, Drosophila Me31B and Caenorhabditis CGH-1 are required for proper meiotic development, apparently by regulating the translational activation of stored mRNPs and also for sorting certain mRNPs into germplasm-containing structures. Studies on yeast Dhh1 and mammalian rck/p54 have revealed a key role for these helicases in mRNA degradation and in earlier remodelling of mRNP for entry into translation, storage or decay pathways. The versatility of Xp54 and related helicases in modulating the metabolism of mRNAs at all stages of their lifetimes marks them out as key regulators of post-transcriptional gene expression.

RAP55: insights into an evolutionarily conserved protein family.

The RAP55 protein family is evolutionarily conserved in eukaryotes. Two highly conserved paralogues, RAP55A and RAP55B, exist in vertebrates; their functional properties and expression patterns remain to be compared. RAP55 proteins share multiple domains: the LSm14 domain, a serine/threonine rich region, an FDF (phenylalanine-aspartate-phenylalanine) motif, an FFD-TFG box and RGG (arginine-glycine-glycine) repeats. Together these domains are responsible for RAP55 proteins participating in translational repression, incorporation into mRNP particles, protein-protein interactions, P-body formation and stress granule localisation. All RAP55A proteins localise to P-body-like complexes either in the germline or in somatic cells. Xenopus laevis RAP55B has been shown to be part of translationally repressed mRNP complexes in early oocytes. Together these findings suggest that this protein family has evolved a common and fundamental role in the control of mRNA translation. Furthermore human RAP55A is an autoantigen detected in the serum of patients with primary biliary cirrhosis (PBC). The link between RAP55A, P-bodies and PBC remains to be elucidated.

Signal recognition particle assembly in relation to the function of amplified nucleoli of Xenopus oocytes.

The signal recognition particle (SRP) is a ribonucleoprotein machine that controls the translation and intracellular sorting of membrane and secreted proteins. The SRP contains a core RNA subunit with which six proteins are assembled. Recent work in both yeast and mammalian cells has identified the nucleolus as a possible initial site of SRP assembly. In the present study, SRP RNA and protein components were identified in the extrachromosomal, amplified nucleoli of Xenopus laevis oocytes. Fluorescent SRP RNA microinjected into the oocyte nucleus became specifically localized in the nucleoli, and endogenous SRP RNA was also detected in oocyte nucleoli by RNA in situ hybridization. An initial step in the assembly of SRP involves the binding of the SRP19 protein to SRP RNA. When green fluorescent protein (GFP)-tagged SRP19 protein was injected into the oocyte cytoplasm it was imported into the nucleus and became concentrated in the amplified nucleoli. After visiting the amplified nucleoli, GFP-tagged SRP19 protein was detected in the cytoplasm in a ribonucleoprotein complex, having a sedimentation coefficient characteristic of the SRP. These results suggest that the amplified nucleoli of Xenopus oocytes produce maternal stores not only of ribosomes, the classical product of nucleoli, but also of SRP, presumably as a global developmental strategy for stockpiling translational machinery for early embryogenesis.

RNA helicase p54 (DDX6) is a shuttling protein involved in nuclear assembly of stored mRNP particles.

Previously, we showed that an integral component of stored mRNP particles in Xenopus oocytes, Xp54, is a DEAD-box RNA helicase with ATP-dependent RNA-unwinding activity. Xp54 belongs to small family of helicases (DDX6) that associate with mRNA molecules encoding proteins required for progress through meiosis. Here we describe the nucleocytoplasmic translocation of recombinant Xp54 in microinjected oocytes and in transfected culture cells. We demonstrate that Xp54 is present in oocyte nuclei, its occurrence in both soluble and particle-bound forms and its ability to shuttle between nucleus and cytoplasm. Translocation of Xp54 from the nucleus to the cytoplasm appears to be dependent on the presence of a leucine-rich nuclear export signal (NES) and is blocked by leptomycin B, a specific inhibitor of the CRM1 receptor pathway. However, the C-terminal region of Xp54 can act to retain the protein in the cytoplasm of full-grown oocytes and culture cells. Cytoplasmic retention of Xp54 is overcome by activation of transcription. That Xp54 interacts directly with nascent transcripts is shown by immunostaining of the RNP matrix of lampbrush chromosome loops and co-immunoprecipitation with de novo-synthesized RNA. However, we are unable to show that nuclear export of this RNA is affected by either treatment with leptomycin B or mutation of the NES. We propose that newly synthesized Xp54 is regulated in its nucleocytoplasmic distribution: in transcriptionally quiescent oocytes it is largely restricted to the cytoplasm and, if imported into the nucleus, it is rapidly exported again by the CRM1 pathway. In transcriptionally active oocytes, it binds to a major set of nascent transcripts, accompanies mRNA sequences to the cytoplasm by an alternative export pathway and remains associated with masked mRNA until the time of translation activation at meiotic maturation and early embryonic cell division.

Expression in Xenopus oocytes shows that WT1 binds transcripts in vivo, with a central role for zinc finger one.

The Wilms' tumour suppressor gene WT1 encodes a protein involved in urogenital development and disease. The salient feature of WT1 is the presence of four 'Krüppel'-type C(2)-H(2) zinc fingers in the C-terminus. Uniquely to WT1, an evolutionarily conserved alternative splicing event inserts three amino acids (KTS) between the third and fourth zinc fingers, which disrupts DNA binding. The ratio of +KTS:-KTS isoforms is crucial for normal development. Previous work has shown that WT1 (+KTS) interacts with splice factors and that WT1 zinc fingers, particularly zinc finger one, bind to RNA in vitro. In this study we investigate the role of zinc finger one and the +KTS splice in vivo by expressing tagged proteins in mammalian cells and Xenopus oocytes. We find that both full-length +/-KTS isoforms and deletion constructs that include zinc finger one co-sediment with ribonucleoprotein particles (RNP) on density gradients. In Xenopus oocytes both isoforms located to the lateral loops of lampbrush chromosomes. Strikingly, only the +KTS isoform was detected in B-snurposomes, but not when co-expressed with -KTS. However, co-expression of the C-terminus (amino acids 233-449, +KTS) resulted in snurposome staining, which is consistent with an in vivo interaction between isoforms via the N-terminus. Expressed WT1 was also detected in the RNA-rich granular component of nucleoli and co-immunoprecipitated with oocyte transcripts. Full-length WT1 was most stably bound to transcripts, followed by the C-terminus; the least stably bound was CTDeltaF1 (C-terminus minus zinc finger one). Expression of the transcription factor early growth response 1 (EGR1), whose three zinc fingers correspond to WT1 zinc fingers 2-4, caused general chromosomal loop retraction and transcriptional shut-down. However, a construct in which WT1 zinc finger one was added to EGR1 mimicked the properties of WT1 (-KTS). We suggest that in evolution, WT1 has acquired the ability to interact with transcripts and splice factors because of the modification of zinc finger one and the +KTS alternative splice.

Nuclear import and activity of histone deacetylase in Xenopus oocytes is regulated by phosphorylation.

Most of the histone deacetylase (HDAC) activity detected in oocytes and early embryos of Xenopus can be accounted for by the presence of a protein complex that contains the maternal HDACm protein. This complex appears to fulfil the conditions required of a 'deposition' histone deacetylase, its primary function being to deacetylate the core histones incorporated into newly-synthesized chromatin during the rapid cell cycles leading up to blastula. A major event in the assembly and accumulation of the HDAC complex is the translocation of the HDACm protein into the germinal vesicle during oogenesis. Here we examine the features of HDACm that are responsible for its nuclear uptake and enzyme activity, identifying the charged C-terminal domain as a target for modification by phosphorylation. Whereas, one phosphorylation site lying within the putative nuclear localization signal, T445, is required for efficient nuclear import of a GST-carboxy-tail fusion, two others, S421 and S423, appear to effect release from the import receptors. Although overexpression of recombinant HDACm in oocytes leads to premature condensation of endogenous chromatin, this effect is abrogated in vivo by mutation of S421A and S423A. Thus, both translocation and activity of HDACm appear to be regulated by specific phosphorylation events. These results have implications for techniques involving the transfer of somatic nuclei into enucleated oocytes.