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Network of Coregulated Spliceosome Components Revealed by Zebrafish Mutant in Recycling Factor P110

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Network of Coregulated Spliceosome Components Revealed by Zebrafish Mutant in Recycling Factor P110 Network of coregulated spliceosome components revealed by zebrafish mutant in recycling factor p110 Nikolaus S. Trede*, Jan Medenbach†, Andrey Damianov†, Lee-Hsueh Hung†, Gerhard J. Weber‡, Barry H. Paw§, Yi Zhou‡, Candace Hersey‡, Agustin Zapata¶, Matthew Keefe‡, Bruce A. Barut‡, Andrew B. Stuartʈ, Tammisty Katz*, Chris T. Amemiyaʈ, Leonard I. Zon‡**, and Albrecht Bindereif†,†† †Institute of Biochemistry, Justus-Liebig-University, D-35392 Giessen, Germany; *Department of Pediatrics, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112; ‡Howard Hughes Medical Institute, Division of Hematology/Oncology, Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115; §Division of Hematology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; ¶Department of Cell Biology, Faculty of Biology, Complutense University, 28040 Madrid, Spain; and ʈBenaroya Research Institute at Virginia Mason, Department of Biology, University of Washington, Seattle, WA 98101 Communicated by Christine Guthrie, University of California, San Francisco, CA, March 2, 2007 (received for review December 20, 2006) The spliceosome cycle consists of assembly, catalysis, and recycling genetic screen for mutants of T cell and thymus development. phases. Recycling of postspliceosomal U4 and U6 small nuclear Surprisingly, the embryonically lethal mutation was mapped in ribonucleoproteins (snRNPs) requires p110/SART3, a general splic- the p110 gene. Biochemical characterization of egy mutant ing factor. In this article, we report that the zebrafish earl grey embryos demonstrated the role of p110 in U4/U6 snRNP (egy) mutation maps in the p110 gene and results in a phenotype recycling in vivo. Through microarray expression profiling of the characterized by thymus hypoplasia, other organ-specific defects, egy mutant, we discovered an extensive network of coregulated and death by 7 to 8 days postfertilization. U4/U6 snRNPs were components of the spliceosome cycle, which would provide a disrupted in egy mutant embryos, demonstrating the importance mechanism compensating for the recycling defect. In sum, these of p110 for U4/U6 snRNP recycling in vivo. Surprisingly, expression data illustrate the usefulness of zebrafish as a vertebrate model profiling of the egy mutant revealed an extensive network of system to investigate the role of splicing factors in organ devel- coordinately up-regulated components of the spliceosome cycle, opment and human disease. providing a mechanism compensating for the recycling defect. Together, our data demonstrate that a mutation in a general Results and Discussion splicing factor can lead to distinct defects in organ development egy Phenotype and Locus. Here we report that the zebrafish egy and cause disease. mutation maps in the p110 gene. egy was identified in a genetic screen for mutants of T cell and thymus development by using small nuclear RNA ͉ small nuclear ribonucleoprotein ͉ splicing ͉ N-ethyl-N-nitrosourea as a mutagen. Phenotypically, the egy genetic screen ͉ thymus mutant is characterized by microcephaly, microphthalmia (Fig. 1A), and death by 7 to 8 days postfertilization (dpf). Identifica- essenger RNA splicing requires the ordered assembly of tion of the egy phenotype was based on the absence of T cells in Mthe spliceosome from Ͼ100 protein components and five the bilateral thymic organ [by using rag1 whole-mount in situ small nuclear RNAs (snRNAs): U1, U2, U4, U5, and U6 hybridization (WISH); Fig. 1B]. Disruption in a number of (reviewed in refs. 1–3). After splicing catalysis and mRNA pathways can lead to this phenotype (reviewed in refs. 10 and release, the spliceosome disassembles, and its components un- 11). Disturbance of other hematopoietic lineages was excluded dergo a recycling phase, which still is poorly understood. In with the demonstration of normal erythropoiesis in egy mutants humans, recycling of postspliceosomal U4 and U6 small nuclear (Fig. 1C). Similarly, neural crest cells and their migration to the ribonucleoproteins (snRNPs) to functional U4/U6 snRNPs re- pharyngeal area (Fig. 1D), as well as patterning of the endoderm quires in vitro p110/SART3, a general splicing factor referred to (Fig. 1E), were intact. However, pharyngeal arch formation was as p110 in the present article (4, 5). In addition, p110 functions defective (Fig. 1F), resulting in a thymus devoid of lymphocytes in recycling of the U4atac/U6atac snRNP (6). Characteristically, (Fig. 1G). Characterization of the effect of the egy mutation on p110 associates only transiently with the U6 and U4/U6 snRNPs other organs revealed that, surprisingly, although insulin expres- but is absent from the U4/U6.U5 tri-snRNP and spliceosomes. sion indicated normal development of the endocrine pancreas in The domain structure of the human p110 protein is composed of at least seven tetratricopeptide repeats (TPR) in the N- terminal half, followed by two RNA recognition motifs (RRMs) Author contributions: N.S.T., J.M., and A.D. contributed equally to this work; N.S.T., J.M., in the C-terminal half, as well as a stretch of 10 highly conserved A.D., L.-H.H., C.T.A., L.I.Z., and A.B. designed research; N.S.T., J.M., A.D., L.-H.H., G.J.W., amino acids at the C terminus (C10 domain). The N-terminal B.H.P., Y.Z., C.H., A.Z., M.K., B.A.B., A.B.S., T.K., and C.T.A. performed research; N.S.T., J.M., A.D., L.-H.H., G.J.W., B.H.P., A.Z., A.B.S., C.T.A., L.I.Z., and A.B. analyzed data; and N.S.T., TPR domain functions in interaction with the U4/U6 snRNP- L.I.Z., and A.B. wrote the paper. specific 90K protein, the RRMs are important for U6 snRNA The authors declare no conflict of interest. binding, and the conserved C10 domain is critical for interacting Abbreviations: snRNP, small nuclear ribonucleoprotein; dpf, days postfertilization; snRNA, with the U6-specific LSm proteins (5, 7, 8). Thus, multiple small nuclear RNA; TPR, tetratricopeptide repeats; RRM, RNA recognition motif; WISH, contacts mediate the interaction between p110 and the U4 and whole-mount in situ hybridization; m3G cap, trimethyl cap structure; NCBI, National Center U6 components. for Biotechnology Information. This p110 domain organization is conserved in many other **To whom correspondence may be addressed at: Howard Hughes Medical Institute, eukaryotes, including Caenorhabditis elegans, Arabidopsis thali- Department of Hematology/Oncology, Children’s Hospital, Harvard Medical School, Karp Family Research Laboratories, 300 Longwood Avenue, Boston, MA 02115. E-mail: ana, Schizosaccharomyces pombe, and Drosophila melanogaster [email protected]. (5). The Saccharomyces cerevisiae Prp24 protein, although func- ††To whom correspondence may be addressed at: Institute of Biochemistry, Justus-Liebig- tionally related to human p110, is an exception in that it lacks the University, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany. E-mail: albrecht.bindereif@ entire N-terminal half with the TPR domain (9). chemie.bio.uni-giessen.de. Here we use the zebrafish system to study the system-wide role This article contains supporting information online at www.pnas.org/cgi/content/full/ and in vivo function of p110. We describe the phenotype of a 0701919104/DC1. zebrafish mutant, called earl grey (egy), that originated from a © 2007 by The National Academy of Sciences of the USA 6608–6613 ͉ PNAS ͉ April 17, 2007 ͉ vol. 104 ͉ no. 16 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0701919104 Downloaded by guest on September 30, 2021 ABCD E F GH IJK Fig. 1. Phenotype of egy mutant zebrafish: organ-selective defects. (Upper) Wild-type animals. (Lower) Mutant animals. (A) Wild-type and egy mutant larvae exhibiting microcephaly and microphthalmia at 5 dpf. (B) Absence of T cells shown by WISH with a rag1-specific probe. Ventral view shows rag1 expression in 5-dpf wild-type animal, with the arrow indicating the left thymic region. No rag1 signal was detected in egy mutants. (C) Normal primitive hematopoiesis in mutants is shown by WISH with an ikaros probe at 19 h postfertilization. No difference was detected between wild-type and mutant embryos in the hematopoietic intermediate cell mass. (D) Hoxa3 expression at 2 dpf is equivalent between wild-type and mutant animals, indicating normal neural crest cell development. Red arrows point to rhombomeres 5 and 6, the black arrowhead shows hindbrain expression, and the black arrow indicates pharyngeal area. (E) Nkx2.3 expression by WISH is normal in egy mutants, indicating normal patterning of endoderm. (F) Alcian blue staining of wild-type and mutant larvae at 7 dpf indicates a defect in pharyngeal arch formation. Gill arches are marked and numbered from anterior to posterior. (G) Thin section of thymus (arrow) from wild-type and mutant larvae at 7 dpf shows only a small rim of epithelium devoid of T cells in egy mutants. The arrow points to the thymus, and the arrowhead points to the otic vesicle epithelium. (H) No difference in insulin expression was detected between wild-type and mutant larvae at 5 dpf. The arrow points to the pancreatic endocrine ␤-islet cells. For better visualization of the pancreas, larvae are shown with anterior to the right. (I) Trypsin, a marker of pancreatic exocrine glands, was strongly expressed in wild-type but not in egy mutant animals at 5 dpf. (J) MyoD expression suggests that formation of somites and skeletal muscle were not affected in egy mutants at 2 dpf. (K) Heart looping to right and expression of cardiac myosin light chain-2 (cmlc-2) was equivalent in wild-type and egy mutant animals at 2 dpf. egy mutants (Fig. 1H), markers for exocrine pancreas (repre- which phenocopied the absence of T cells and the arch defect in sented by trypsin in Fig. 1I) were strongly reduced or absent. egy mutants. The analysis of p110 expression in 19-h postfertil- Besides normal endocrine pancreatic development, somites were ization and 2-dpf embryos showed no spatially restricted expres- normally formed (Fig.
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