Substrate requirements for let-7 function in the developing zebrafish embryo.
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.
Kloosterman
Wigard P
WP
The Hubrecht Laboratory, Centre for Biomedical Genetics, 3584 CT Utrecht, The Netherlands.
Wienholds
Erno
E
Ketting
René F
RF
Plasterk
Ronald H A
RH
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000818:Animals; D004252:DNA Mutational Analysis; D004625:Embryo, Nonmammalian / Q000033:anatomy & histology / Q000378:metabolism; D047108:Embryonic Development; D020868:Gene Silencing; D017930:Genes, Reporter; D035683:MicroRNAs / Q000008:administration & dosage / Q000235:genetics / Q000494:pharmacology*; D008845:Microinjections; D015027:Zebrafish / Q000033:anatomy & histology / Q000196:embryology* / Q000235:genetics
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D035683:MicroRNAs