PUBLICATION
Substrate requirements for let-7 function in the developing zebrafish embryo
- Authors
- Kloosterman, W.P., Wienholds, E., Ketting, R.F., and Plasterk, R.H.
- ID
- ZDB-PUB-041213-5
- Date
- 2004
- Source
- Nucleic acids research 32(21): 6284-6291 (Journal)
- Registered Authors
- Kloosterman, Wigard, Plasterk, Ronald H.A., Wienholds, Erno
- Keywords
- none
- MeSH Terms
-
- 3' Untranslated Regions
- 5' Untranslated Regions
- Animals
- DNA Mutational Analysis
- Embryo, Nonmammalian/anatomy & histology
- Embryo, Nonmammalian/metabolism
- Embryonic Development
- Gene Silencing*
- Genes, Reporter
- MicroRNAs/administration & dosage
- MicroRNAs/genetics
- MicroRNAs/pharmacology*
- Microinjections
- Zebrafish/anatomy & histology
- Zebrafish/embryology*
- Zebrafish/genetics
- PubMed
- 15585662 Full text @ Nucleic Acids Res.
Citation
Kloosterman, W.P., Wienholds, E., Ketting, R.F., and Plasterk, R.H. (2004) Substrate requirements for let-7 function in the developing zebrafish embryo. Nucleic acids research. 32(21):6284-6291.
Abstract
MicroRNAs (miRNAs) are involved in the regulation of gene expression at the post-transcriptional level by base pairing to the 3'-UTR (untranslated region) of mRNAs. The let-7 miRNA was first discovered in Caenorhabditis elegans and is evolutionarily conserved. We used zebrafish embryos as a vertebrate in vivo system to study substrate requirements for function of let-7. Injection of a double-stranded let-7 miRNA into the zygotes of zebrafish and frogs causes specific phenotypic defects. Only the antisense strand of the let-7 duplex has biological activity. In addition, co-injected mRNA of gfp fused to the 3'-UTR of a zebrafish lin-41 ortholog (a presumed target of let-7) is silenced by let-7. Point mutant studies revealed that the two let-7 target sites in the lin-41 3'-UTR are both essential and sufficient for silencing. let-7 and mir221 together, but not either of them alone, can silence a construct with one of the let-7 target sites replaced by a target site for mir221, showing that two different miRNAs can provide the required cooperative effect. let-7 target sites can be moved around: they are also functional when positioned in the coding sequence or even in the 5'-UTR of gfp. We took advantage of reporter and phenotypic assays to analyze the activity of all possible point mutant derivatives of let-7 and found that only the 5' region is critical for function of let-7.
Errata / Notes
See note
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping