PUF60-activated exons uncover altered 3' splice-site selection by germline missense mutations in a single RRM.

PUF60 is a splicing factor that binds uridine (U)-rich tracts and facilitates association of the U2 small nuclear ribonucleoprotein with primary transcripts. PUF60 deficiency (PD) causes a developmental delay coupled with intellectual disability and spinal, cardiac, ocular and renal defects, but PD pathogenesis is not understood. Using RNA-Seq, we identify human PUF60-regulated exons and show that PUF60 preferentially acts as their activator. PUF60-activated internal exons are enriched for Us upstream of their 3' splice sites (3'ss), are preceded by longer AG dinucleotide exclusion zones and more distant branch sites, with a higher probability of unpaired interactions across a typical branch site location as compared to control exons. In contrast, PUF60-repressed exons show U-depletion with lower estimates of RNA single-strandedness. We also describe PUF60-regulated, alternatively spliced isoforms encoding other U-bound splicing factors, including PUF60 partners, suggesting that they are co-regulated in the cell, and identify PUF60-regulated exons derived from transposed elements. PD-associated amino-acid substitutions, even within a single RNA recognition motif (RRM), altered selection of competing 3'ss and branch points of a PUF60-dependent exon and the 3'ss choice was also influenced by alternative splicing of PUF60. Finally, we propose that differential distribution of RNA processing steps detected in cells lacking PUF60 and the PUF60-paralog RBM39 is due to the RBM39 RS domain interactions. Together, these results provide new insights into regulation of exon usage by the 3'ss organization and reveal that germline mutation heterogeneity in RRMs can enhance phenotypic variability at the level of splice-site and branch-site selection.

A t(4;19) pediatric undifferentiated sarcoma with a novel variant of the CIC-DUX4 fusion transcript.

We report cytogenetic and molecular genetic analysis of a pediatric tumor positive for the CIC-DUX4 fusion. The tumor belongs to a rare, diagnostically challenging subgroup of undifferentiated small round cell sarcomas. A balanced t(4;19)(q35;q13.1-2) was identified by G-banding, as a sole cytogenetic finding. The translocation was also identified by the M-FISH technique. After RT-PCR, the tumor sample was positive for the CIC-DUX4 fusion. The PCR product contains a novel, so far unreported variant of the CIC-DUX4 fusion transcript, with a fusion of the exon 20 from the CIC gene and the exon 1 from the DUX4 gene.

SART3-Dependent Accumulation of Incomplete Spliceosomal snRNPs in Cajal Bodies.

Cajal bodies (CBs) are evolutionarily conserved nuclear structures involved in the metabolism of spliceosomal small nuclear ribonucleoprotein particles (snRNPs). CBs are not present in all cell types, and the trigger for their formation is not yet known. Here, we depleted cells of factors required for the final steps of snRNP assembly and assayed for the presence of stalled intermediates in CBs. We show that depletion induces formation of CBs in cells that normally lack these nuclear compartments, suggesting that CB nucleation is triggered by an imbalance in snRNP assembly. Accumulation of stalled intermediates in CBs depends on the di-snRNP assembly factor SART3. SART3 is required for both the induction of CB formation as well as the tethering of incomplete snRNPs to coilin, the CB scaffolding protein. We propose a model wherein SART3 monitors tri-snRNP assembly and sequesters incomplete particles in CBs, thereby allowing cells to maintain a homeostatic balance of mature snRNPs in the nucleoplasm.

The splicing factor U1-70K interacts with the SMN complex and is required for nuclear gem integrity.

The nuclear SMN complex localizes to specific structures called nuclear gems. The loss of gems is a cellular marker for several neurodegenerative diseases. Here, we identify that the U1-snRNP-specific protein U1-70K localizes to nuclear gems, and we show that U1-70K is necessary for gem integrity. Furthermore, we show that the interaction between U1-70K and the SMN complex is RNA independent, and we map the SMN complex binding site to the unstructured N-terminal tail of U1-70K. Consistent with these results, the expression of the U1-70K N-terminal tail rescues gem formation. These findings show that U1-70K is an SMN-complex-associating protein, and they suggest a new function for U1-70K in the formation of nuclear gems.

Enzyme-linked electrochemical DNA ligation assay using magnetic beads.

DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5'-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

The C-terminal domain of Brd2 is important for chromatin interaction and regulation of transcription and alternative splicing.

Brd2 is a member of the bromodomain extra terminal (BET) protein family, which consists of four chromatin-interacting proteins that regulate gene expression. Each BET protein contains two N-terminal bromodomains, which recognize acetylated histones, and the C-terminal protein-protein interaction domain. Using a genome-wide screen, we identify 1450 genes whose transcription is regulated by Brd2. In addition, almost 290 genes change their alternative splicing pattern upon Brd2 depletion. Brd2 is specifically localized at promoters of target genes, and our data show that Brd2 interaction with chromatin cannot be explained solely by histone acetylation. Using coimmunoprecipitation and live-cell imaging, we show that the C-terminal part is crucial for Brd2 association with chromatin. Live-cell microscopy also allows us to map the average binding time of Brd2 to chromatin and quantify the contributions of individual Brd2 domains to the interaction with chromatin. Finally, we show that bromodomains and the C-terminal domain are equally important for transcription and splicing regulation, which correlates with the role of these domains in Brd2 binding to chromatin.

Changes in MYCN expression in human neuroblastoma cell lines following cisplatin treatment may not be related to MYCN copy numbers.

Neuroblastoma is a tumor accounting for approximately 10% of all childhood malignancies and 50% of all childhood cancer-related deaths. MYCN gene copy number variation represents the most important prognostic factor in neuroblastoma. Prognostic significance of MYCN gene expression is more complicated and may depend on other factors such as MYCN gene copy number status. In the present study, we assessed MYCN gene expression using real-time RT-PCR following cisplatin treatment in three human neuroblastoma cell lines (UKF-NB-3, UKF-NB-4 and SK-N-AS) and their cisplatin-resistant counterparts. We also examined MYCN gene status and copy number (gain and amplification) variations using interphase and metaphase fluorescent in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA). Only cisplatin-sensitive UKF-NB-4 cells exhibited decreased MYCN expression following treatment with cisplatin. Other sensitive neuroblastoma cells did not exhibit a change in MYCN expression. In contrast, cisplatin-resistant UKF-NB-4 and SK-N-AS cells exhibited increased MYCN expression irrespective of the number of MYCN copies or concentration of cisplatin in the medium. In MYCN-amplified neuroblastoma cells we did not observe any significant change in the number of MYCN copies after cisplatin treatment, whereas MYCN-non-amplified SK-N-AS cells revealed during cisplatin treatment an increased number of MYCN gene copies caused by 2p gain in the majority of cells by FISH. We postulated that cisplatin treatment does not result directly in altered transcription of MYCN. A functional change in MYCN mRNA levels and increased MYCN expression in cisplatin-resistant neuroblastoma cells do not have a clear relationship to MYCN copy numbers. These findings may further contribute to the understanding of cisplatin chemotherapy in connection with MYCN expression, and the possible copy number variations in MYCN neuroblastoma cells may be of importance since targeting of MYCN is being tested as neuroblastoma therapy.

Cytogenetic and array comparative genomic hybridization analysis of a series of hepatoblastomas.

Hepatoblastoma is the most common primary hepatic tumor in children, and only a limited number of detailed karyotypic analyses have been reported to date. In the present study, cytogenetic abnormalities were identified in nine cases of hepatoblastoma from a single institution. Among characteristic chromosomal changes detected were simple numerical aberrations, structural alterations of chromosomes 1, 2, and 8, and the recurrent unbalanced rearrangements der(4)t(1;4)(q25.2;q35.1) and der(6)t(1;6)(q21;q26). Array comparative genomic hybridization was applied in four of the cases. The combined cytogenetic, molecular cytogenetic, and histopathologic analyses are presented here, together with clinical data. The results substantially confirm previous findings of aberrations involving chromosomal loci on 1q, 2 or 2q, 4q, 6q, 8 or 8q, and 20 as significant in the development and clinical course of this disease.

A novel variant of SYT-SSX1 fusion gene in a case of spindle cell synovial sarcoma.

Synovial sarcoma (SS) is a rare soft-tissue tumor that affects children and young adults. It is characterized by chromosomal translocation t(X;18)(p11.2;q11.2), which results in the fusion of the gene SYT on chromosome 18 with SSX genes on chromosome X. Heterogeneity within SS fusion junctions is rare. We report a case of a 9-year-old boy with a high-grade spindle cell sarcoma. Reverse transcriptase-polymerase chain reaction revealed a characteristic translocation of SSs. However, this sarcoma showed a longer-than-expected PCR product after gel electrophoresis. Direct sequencing of the product disclosed a novel SYT/SSX1 fusion transcript. Detection of fusion transcripts is useful for diagnostics of SS. In each case, when considering this diagnosis on the morphologic grounds an attempt to analyze the translocation using PCR should be made, including the recognition of its uncommon variants.

Teratoma in an adolescent with malignant transformation into embryonal rhabdomyosarcoma: case report.

BACKGROUND: The somatic type tumors are occasionally found in nonseminomatous germ cell tumors in men. These malignancies are presumed to arise from malignant transformation (MT) of teratoma or by differentiation of totipotential germ cell. OBSERVATION: A case of MT of germ cell tumor in 17-year-old male into embryonal rhabdomyosarcoma is described. The histopathologic diagnosis was that of embryonal rhabdomyosarcoma in which no germ cell elements were found. The germ cell origin of transformed histology is supported by cytogenetic analysis (isochromosome 12p), and elevated alpha(1)-fetoprotein. Despite intensive therapy the patient died. CONCLUSIONS: MT of teratoma is rare entity with poor prognosis.

Primary mediastinal (thymic) large B-cell lymphoma with a der(14)t(8;14)(q24;q32) and a translocation of MYC to the derivative chromosome 14 with a deleted IgH locus.

We report a case of primary mediastinal (thymic) large B-cell lymphoma (PMBL) with an initial karyotype containing numerical chromosomal aberrations: +X, +9, +12, +21, and a novel translocation t(2;11)(q?31; q23 approximately 24) with a duplication of the derivative chromosome 11. Subsequent multicolor fluorescence in situ hybridization (M-FISH) analysis revealed a der(14)t(8;14)(q24;q32). Further analysis using fluorescence in situ hybridization (FISH) with locus-specific probes revealed loss of the entire IgH locus from the der(14)t(8;14) and relocation of MYC to this derivative chromosome 14. Our data show definitively the existence of the t(8;14) in PMBL, previously only suspected. This finding supplies additional evidence that a translocation-mediated MYC activation may be an important event in the pathogenesis of this unique lymphoma.

Malignant peripheral primitive neuroectodermal tumor of the kidney.

Ewing family of tumors is a group of highly aggressive neoplasias that occur most commonly in the first two decades of life. These tumors are most frequently localized in bones, less frequently in soft tissues. They usually appear as undifferentiated small round-cell tumors. With current treatment regiments, 5-year disease-free survival rates exceed 60% in patients with a localized disease. Patients with metastatic disease at the time of their first presentation have a poor prognosis. We describe a rare case of visceral primitive neuroectodermal tumor with the involvement of the kidney in a 9-year-old girl. The tumor was studied with immunohistochemistry, cytogenetics, and molecular biology methods. Strong expression of protein MIC(2) by immunochemistry (antibody HBA 71) with subsequent demonstration of a translocation consistent with t(11;22)(q24;q12) using cytogenetic and reverse transcriptase polymerase chain reaction (RT-PCR) confirmed the histopathological diagnosis of peripheral primitive neuroectodermal tumor. We detected minimal residual disease in bone marrow using RT-PCR.