ABSTRACT
Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3'-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.
Subject(s)
Gene Expression Regulation , Morpholinos/pharmacology , Oligonucleotides, Antisense/pharmacology , Poly A/metabolism , Protein Biosynthesis/drug effects , RNA, Messenger, Stored/metabolism , 3' Untranslated Regions , Animals , Asterina/genetics , Asterina/metabolism , Cyclin B/genetics , Cyclin B/metabolism , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/genetics , Down-Regulation , Gene Knockdown Techniques , Injections , Morpholinos/administration & dosage , Oligonucleotides, Antisense/administration & dosage , Oocytes/drug effects , Oocytes/metabolism , Polyadenylation/drug effects , RNA, Messenger, Stored/chemistry , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/antagonists & inhibitors , Zebrafish Proteins/geneticsABSTRACT
Recent studies have showed that transcription elongation factors regulate early development. Foggy/Spt5 is a subunit of DRB sensitivity-inducing factor, which negatively and positively regulates transcription elongation. Here, we report that the positive function of Foggy/Spt5 is required for gata1 expression during zebrafish embryonic hematopoiesis. Antisense morpholino oligonucleotide (MO)-mediated knockdown of foggy/spt5 has led to a reduction in the expression of gata1 and the gata1 target genes alas2 and hbae3 and inhibited proper hemoglobin production. By contrast, expression of hematopoietic stem cell and endothelial markers, including scl, lmo2, gata2, fli-1, and flk-1, and expression of biklf, whose product directs gata1 expression via its direct binding to the gata1 promoter, were unaltered, suggesting that gata1 is a functionally important target gene of Foggy/Spt5. The MO-mediated gata1 repression was relieved by forced expression of wild-type foggy/spt5, but not by a mutant lacking the positive function. Therefore, this study provides evidence that Foggy/Spt5 plays an important role in gata1 gene expression and erythropoiesis through its transcriptional activation domain.
Subject(s)
Erythropoiesis/genetics , GATA1 Transcription Factor/genetics , Gene Expression Regulation, Developmental , Nuclear Proteins/physiology , Transcription Factors/physiology , Zebrafish Proteins/genetics , Zebrafish Proteins/physiology , Zebrafish/embryology , Amino Acid Sequence , Animals , DNA-Binding Proteins , Hematopoietic Stem Cells/metabolism , Molecular Sequence Data , Nuclear Proteins/genetics , RNA-Binding Proteins , Transcription Factors/genetics , Zebrafish/geneticsABSTRACT
The transcription elongation factor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) regulates RNA polymerase II (RNAPII) processivity by promoting, in concert with negative elongation factor (NELF), promoter-proximal pausing of RNAPII. DSIF is also reportedly involved in transcriptional activation. However, the role of DSIF in transcriptional activation by DNA-binding activators is unclear. Here we show that DSIF acts cooperatively with a DNA-binding activator, Gal4-VP16, to promote transcriptional activation. In the absence of DSIF, Gal4-VP16-activated transcription resulted in frequent pausing of RNAPII during elongation in vitro. The presence of DSIF reduced pausing, thereby supporting Gal4-VP16-mediated activation. We found that DSIF exerts its positive effects within a short time-frame from initiation to elongation, and that NELF does not affect the positive regulatory function of DSIF. Knockdown of the gene encoding the large subunit of DSIF, human Spt5 (hSpt5), in HeLa cells reduced Gal4-VP16-mediated activation of a reporter gene, but had no effect on expression in the absence of activator. Together, these results provide evidence that higher-level transcription has a stronger requirement for DSIF, and that DSIF contributes to efficient transcriptional activation by preventing RNAPII pausing during transcription elongation.
