Coilin levels and modifications influence artificial reporter splicing.

Cajal bodies (CBs) and Gems are nuclear domains that contain factors responsible for spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis. The marker protein for CBs is coilin. In addition to snRNPs, coilin and other factors, canonical CBs contain the survivor of motor neuron protein (SMN). SMN can also localize to Gems. Considering the important role that coilin plays in the formation and composition of CBs, we tested the splicing efficiency of several cell lines that vary in regards to coilin level and modification using an artificial reporter substrate. We found that cells with both hypomethylated coilin and Gems are more efficient at reporter splicing compared to cells in which SMN localizes to CBs. In contrast, coilin reduction, which induces Gem formation, decreases cell proliferation and artificial reporter splicing. These findings demonstrate that coilin modifications or levels impact artificial reporter splicing, possibly by influencing snRNP biogenesis.

Gene-based approaches toward Friedreich ataxia therapeutics.

Friedreich ataxia is an autosomal recessive trinucleotide-repeat disease caused by expanded GAA repeats in the first intron of the FRDA gene. These GAA repeats are suspected to form unusual non-B DNA conformations that decrease transcription and subsequently reduce levels of the encoded protein, frataxin. GAA repeats also induce heterochromatin formation and silencing of the frataxin gene locus. Frataxin plays a crucial role in iron metabolism and detoxification and interacts with electron transport chain proteins. There is no effective therapy for Friedreich ataxia, but antioxidant therapy has shown promise and is currently in clinical trials. In this review we focus on the mechanisms by which expanded GAA repeats reduce transcription and discuss how these findings have lead to gene-based approaches that may be effective in treating Friedreich ataxia.

Role of tissue transglutaminase type 2 in calbindin-D28k interaction with ataxin-1.

Spinocerebellar ataxia-1 (SCA1) is caused by the expansion of a polyglutamine repeats within the disease protein, ataxin-1. The mutant ataxin-1 precipitates as large intranuclear aggregates in the affected neurons. These aggregates may protect neurons from mutant protein and/or trigger neuronal degeneration by encouraging recruitment of other essential proteins. Our previous studies have shown that calcium binding protein calbindin-D28k (CaB) associated with SCAl pathogenesis is recruited to ataxin-l aggregates in Purkinje cells of SCAl mice. Since our recent findings suggest that tissue transglutaminase 2 (TG2) may be involved in crosslinking and aggregation of ataxin-l, the present study was initiated to determine if TG2 has any role in CaB-ataxin-l interaction. The guinea pig TG2 covalently crosslinked purified rat brain CaB. Time dependent progressive increase in aggregation produced large multimers, which stayed on top of the gel. CaB interaction with ataxin-l was studied using HeLa cell lysates expressing GFP and GFP tagged ataxin-l with normal and expanded polyglutamine repeats (Q2, Q30 and Q82). The reaction products were analyzed by Western blots using anti-polyglutamine, CaB or GFP antibodies. CaB interacted with ataxin-1 independent of TG2 as the protein-protein crosslinker DSS stabilized CaB-ataxin-l complex. TG2 crosslinked CaB preferentially with Q82 ataxin-1. The crosslinking was inhibited with EGTA or TG2 inhibitor cystamine. The present data indicate that CaB may be a TG2 substrate. In addition, aggregates of mutant ataxin-l may recruit CaB via TG2 mediated covalent crosslinking, further supporting the argument that ataxin-l aggregates may be toxic to neurons.

Tissue transglutaminase crosslinks ataxin-1: possible role in SCA1 pathogenesis.

Transglutaminase type 2 (TG2) has recently been implicated in crosslinking of mutant huntingtin protein into aggregates. Here we show that TG2 also crosslinks spinocerebellar ataxia-1 (SCA1) gene product ataxin-1. HeLa cell lysates expressing GFP tagged ataxin-1 with 2, 30 or 82 glutamines showed covalent crosslinking of ataxin-1 when incubated with exogenously added TG2. This crosslinking was inhibited by TG2 inhibitor cystamine. SCA1 transgenic mice which overexpress the mutant ataxin-1 in cerebellar Purkinje cells showed elevated nuclear TG2 in the absence of ataxin-1 nuclear aggregates. The addition of purified TG2 to the nuclear extracts or addition of SCA1 nuclear TG2 to GFP-Q82 HeLa cell lysates resulted in the formation of insoluble aggregates. These data indicate that ataxin-1 is a substrate of TG2. Further, in SCA1 TG2 may translocate to the nucleus in response to nuclear accumulation of mutant ataxin-1 at early stages of the disease.

Coilin forms the bridge between Cajal bodies and SMN, the spinal muscular atrophy protein.

Spinal muscular atrophy (SMA) is a genetic disorder caused by mutations in the human survival of motor neuron 1 gene, SMN1. SMN protein is part of a large complex that is required for biogenesis of various small nuclear ribonucleoproteins (snRNPs). Here, we report that SMN interacts directly with the Cajal body signature protein, coilin, and that this interaction mediates recruitment of the SMN complex to Cajal bodies. Mutation or deletion of specific RG dipeptide residues within coilin inhibits the interaction both in vivo and in vitro. Interestingly, GST-pulldown experiments show that coilin also binds directly to SmB'. Competition studies show that coilin competes with SmB' for binding sites on SMN. Ectopic expression of SMN and coilin constructs in mouse embryonic fibroblasts lacking endogenous coilin confirms that recruitment of SMN and splicing snRNPs to Cajal bodies depends on the coilin C-terminal RG motif. A cardinal feature of SMA patient cells is a defect in the targeting of SMN to nuclear foci; our results uncover a role for coilin in this process.

Self-association of coilin reveals a common theme in nuclear body localization.

We have found that coilin, the marker protein for Cajal bodies (coiled bodies, CBs), is a self-interacting protein, and we have mapped the domain responsible for this activity to the amino-terminus. Together with a nuclear localization signal, the self-interaction domain is necessary and sufficient for localization to CBs. Overexpression of various wild-type and mutant coilin constructs in HeLa cells results in disruption of both CBs and survival motor neurons (SMN) gems. Additionally, we have identified a cryptic nucleolar localization signal (NoLS), within the coilin protein, which may be exposed in specific coilin phospho-isoforms. The implications of these findings are discussed in light of the fact that other proteins known to localize within nuclear bodies (e. g., PML, SMN and Sam68) can also self-associate. Thus protein self-interaction appears to be a general feature of nuclear body marker proteins.

Structure and characterization of the murine p80 coilin gene, Coil.

Cajal bodies (coiled bodies, CBs) are nuclear organelles of unknown function and are characterized by a wide variety of components including various basal transcription and cell cycle proteins, the nucleolar proteins fibrillarin and Nopp140, numerous small nuclear ribonucleoproteins, the survival motor neuron protein complex, and the marker protein, p80 coilin. To gain insight into the role of p80 coilin in CBs, we have cloned the murine gene Coil and have mapped it to the distal portion of chromosome band 11D. The approximately 2.6-kb transcript is detectable in all tissues analyzed, with the highest levels in brain and testis. Sequence analysis shows that, like its human counterpart, the mouse coilin gene is composed of seven exons and spans nearly 30 kb of genomic DNA. The predicted amino acid sequence reveals two conserved N- and C-terminal domains, and comparison with the Xenopus SPH-1 protein reveals that these three genes are indeed orthologous. These results should facilitate gene disruption experiments aimed at creating a genetic model system to study CBs.

Cell cycle-dependent localization of the CDK2-cyclin E complex in Cajal (coiled) bodies.

We have found that CDK2 and cyclin E, but not cyclin A, accumulates within Cajal bodies (CBs) in a cell cycle-dependent fashion. In the absence of cyclin E, CDK2 is not enriched in the CB compartment, suggesting that the translocation of CDK2 to CBs is dependent on cyclin E. CDK2 and cyclin E could be recruited to CBs as a functional complex or CBs may serve as 'docking stations' for CDK2-cyclin E activation by CAKs during the G(1)/S transition. Notably, CDK7-cyclin H-Mat1 complexes are known to accumulate in CBs. Treatment of cells with inhibitors of either CDKs (olomoucine, 200 microM) or RNA polymerase I (actinomycin D, 0.05 microgram/ml), results in a striking reorganization of CDK2 and p80 coilin to the nucleolar periphery. Furthermore, we demonstrate that p80 coilin can be phosphorylated by purified CDK2-cyclin E complexes in vitro. Thus coilin and other CB proteins appear to be downstream targets of CDK2-cyclin E complex-mediated signaling pathways regulating cell cycle progression and controlling aspects of CB function. Possible roles for CDK2 and cyclin E in the well-documented association of CBs, histone gene clusters and RNA 3' end processing factors are discussed.

Twin carriers of X-linked agammaglobulinemia (XLA) due to germline mutation in the Btk gene.

We report on an X-linked agammaglobulinemia (XLA) family in which mothers of two affected cousins were monozygotic twins. We analyzed the Btk gene of several members in three generations of the family by SSCP analysis, DNA sequencing, and RFLP analysis following polymerase chain reaction-amplification of the individual exons. We identified a missense point mutation, G1817C (R562P), in exon 17 of the Btk gene in the affected cousins. The same mutation was also present in both mothers (twin sisters) of the cousins identifying them as carriers. However, the mutation was absent in all other relatives including the grandmother of the cousins (mother of the twin sisters). This strongly suggests that the mutation in the Btk gene had originated in one of the germ lines or in the zygote. This may be the first demonstration of a germ line (or zygotic) mutation in XLA.

Regulation of ornithine utilization in Pseudomonas aeruginosa (PAO1) is mediated by a transcriptional regulator, OruR.

We have used transpositional mutagenesis of a proline auxotroph (PAO951) to isolate an ornithine utilization (oru) mutant of Pseudomonas aeruginosa (PAO951-4) that was unable to use ornithine efficiently as the sole carbon and nitrogen source. DNA sequence analysis of the inactivated locus confirmed that the transposon had inserted into a locus whose product demonstrated significant primary sequence homology to members of the AraC family of transcriptional activators. DNA mobility shift assays affirmed this potential regulatory function and indicated that the inactivated gene encodes a transcriptional regulator, which has been designated OruR. In trying to define the ornithine utilization phenotype further, a similar inactivation was engineered in the wild-type strain, PAO1. The resulting isolate (PAO1R4) was totally unable to use ornithine as the sole carbon source. Despite the intensified phenotype, this isolate failed to demonstrate significant changes in any of the catabolic or anabolic enzymes that are known to be subject to regulation by the presence of either ornithine or arginine. It did, however, show modified levels of an enzyme, ornithine acetyltransferase (OAcT), that was previously thought to have merely an anaplerotic activity. Definition of this oruR locus and its effects upon OAcT activity provide evidence that control of ornithine levels in P. aeruginosa may have a significant impact upon how the cell is able to monitor and regulate the use of arginine and glutamate as sources of either carbon or nitrogen.