ABSTRACT
Proprotein convertase subtilisin/kexin (PCSK) enzymes convert proproteins into bioactive end products. Although other PCSK enzymes are known to be essential for biological processes ranging from cholesterol metabolism to host defense, the in vivo importance of the evolutionarily ancient PCSK7 has remained enigmatic. Here, we quantified the expressions of all pcsk genes during the 1st week of fish development and in several tissues. pcsk7 expression was ubiquitous and evident already during the early development. To compare mammalian and zebrafish PCSK7, we prepared homology models, which demonstrated remarkable structural conservation. When the PCSK7 function in developing larvae was inhibited, we found that PCSK7-deficient fish have defects in various organs, including the brain, eye, and otic vesicle, and these result in mortality within 7 days postfertilization. A genome-wide analysis of PCSK7-dependent gene expression showed that, in addition to developmental processes, several immune system-related pathways are also regulated by PCSK7. Specifically, the PCSK7 contributed to the mRNA expression and proteolytic cleavage of the cytokine TGFß1a. Consequently, tgfß1a morphant fish displayed phenotypical similarities with pcsk7 morphants, underscoring the importance of this cytokine in the zebrafish development. Targeting PCSK activity has emerged as a strategy for treating human diseases. Our results suggest that inhibiting PCSK7 might interfere with normal vertebrate development.
Subject(s)
Subtilisins/metabolism , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta/metabolism , Zebrafish Proteins/metabolism , Amino Acid Sequence , Animals , Brain/growth & development , Brain/metabolism , Catalytic Domain , Conserved Sequence , Gene Expression Regulation, Developmental , Humans , Larva/growth & development , Larva/metabolism , Molecular Sequence Data , Organ Specificity , Proteolysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology , Subtilisins/chemistry , Subtilisins/genetics , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta1/genetics , Zebrafish/growth & development , Zebrafish/metabolism , Zebrafish Proteins/chemistry , Zebrafish Proteins/geneticsABSTRACT
PURPOSE: Small nucleolar RNAs (snoRNAs) direct sequence-specific modifications to ribosomal RNA. We hypothesized that the expression of snoRNAs may be altered in leukemic cells. METHODS: The expression of snoRNAs was analyzed in various leukemic cell lines by massive parallel sequencing (SOLiD). Quantitative real-time PCR (RT-qPCR) was used to validate the expression profiles. RESULTS: Our results show characteristic differences in the expression patterns of snoRNAs between cell lines representing the main subgroups of leukemia, AML, pre-B-ALL and T-ALL, respectively. In RT-qPCR analyses, several snoRNAs were found to be differentially expressed in T-ALL as compared to pre-B-ALL cell lines. CONCLUSIONS: snoRNAs are differentially expressed in various leukemic cell lines and could, therefore, be potentially useful in the classification of leukemia subgroups.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Neoplastic , RNA, Neoplasm/genetics , RNA, Small Nucleolar/genetics , Cell Line, Tumor , Cluster Analysis , Gene Library , HL-60 Cells , HeLa Cells , Humans , Jurkat Cells , Leukemia/classification , Leukemia/genetics , Leukemia/pathology , Oligonucleotide Array Sequence Analysis/methods , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
The Sin3A-associated proteins SAP30 and SAP30L share 70% sequence identity and are part of the multiprotein Sin3A corepressor complex. They participate in gene repression events by linking members of the complex and stabilizing interactions among the protein members as well as between proteins and DNA. While most organisms have both SAP30 and SAP30L, the zebrafish is exceptional because it only has SAP30L. Here we demonstrate that SAP30L is expressed ubiquitously in embryonic and adult zebrafish tissues. Knockdown of SAP30L using morpholino-mediated technology resulted in a morphant phenotype manifesting as cardiac insufficiency and defective hemoglobinization of red blood cells. A microarray analysis of gene expression in SAP30L morphant embryos revealed changes in the expression of genes involved in regulation of transcription, TGF-beta signaling, Wnt-family transcription factors, and nuclear genes encoding mitochondrial proteins. The expression of the heart-specific nkx2.5 gene was markedly down-regulated in SAP30L morphants, and the cardiac phenotype could be partially rescued by nkx2.5 mRNA. In addition, changes were detected in the expression of genes known to be important in hemoglobin synthesis and erythropoiesis. Our results demonstrate that SAP30L regulates several transcriptional pathways in zebrafish embryos and is involved in the development of cardiac and hematopoietic systems.
