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2.
Nat Protoc ; 6(12): 2035-49, 2011 Dec 01.
Article in English | MEDLINE | ID: mdl-22134127

ABSTRACT

MicroRNAs (miRNAs) regulate gene expression by pairing with complementary sequences in the 3' untranslated regions (UTRs) of transcripts. Although the molecular mechanism underlying miRNA biogenesis and activity is becoming better understood, determining the physiological role of the interaction of an miRNA with its target remains a challenge. A number of methods have been developed to inhibit individual miRNAs, but it can be difficult to determine which specific targets are responsible for any observed phenotypes. To address this problem, we use target protector (TP) morpholinos that interfere with a single miRNA-mRNA pair by binding specifically to the miRNA target sequence in the 3' UTR. In this protocol, we detail the steps for identifying miRNA targets, validating their regulation and using TPs to interrogate their function in zebrafish. Depending on the biological context, this set of experiments can be completed in 6-8 weeks.


Subject(s)
MicroRNAs/physiology , Morpholinos/chemistry , RNA, Messenger/physiology , Zebrafish/genetics , 3' Untranslated Regions , Animals , Binding, Competitive , Embryo, Nonmammalian , Gene Expression Regulation , Genetic Techniques , Luciferases/analysis , MicroRNAs/chemistry , RNA, Messenger/chemistry , Zebrafish/embryology
3.
Nat Genet ; 43(3): 204-11, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21258340

ABSTRACT

microRNAs (miRNAs) function as genetic rheostats to control gene output. Based on their role as modulators, it has been postulated that miRNAs canalize development and provide genetic robustness. Here, we uncover a previously unidentified regulatory layer of chemokine signaling by miRNAs that confers genetic robustness on primordial germ cell (PGC) migration. In zebrafish, PGCs are guided to the gonad by the ligand Sdf1a, which is regulated by the sequestration receptor Cxcr7b. We find that miR-430 regulates sdf1a and cxcr7 mRNAs. Using target protectors, we demonstrate that miR-430-mediated regulation of endogenous sdf1a (also known as cxcl12a) and cxcr7b (i) facilitates dynamic expression of sdf1a by clearing its mRNA from previous expression domains, (ii) modulates the levels of the decoy receptor Cxcr7b to avoid excessive depletion of Sdf1a and (iii) buffers against variation in gene dosage of chemokine signaling components to ensure accurate PGC migration. Our results indicate that losing miRNA-mediated regulation can expose otherwise buffered genetic lesions leading to developmental defects.


Subject(s)
Cell Movement/genetics , Chemokine CXCL12/genetics , Germ Cells/physiology , Animals , Chemokine CXCL12/metabolism , Gene Expression Regulation , MicroRNAs/physiology , Models, Biological , Receptors, CXCR/genetics , Receptors, CXCR/metabolism , Signal Transduction/genetics , Zebrafish , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
4.
Genes Dev ; 23(5): 619-32, 2009 Mar 01.
Article in English | MEDLINE | ID: mdl-19240126

ABSTRACT

microRNAs (miRNAs) represent approximately 4% of the genes in vertebrates, where they regulate deadenylation, translation, and decay of the target messenger RNAs (mRNAs). The integrated role of miRNAs to regulate gene expression and cell function remains largely unknown. Therefore, to identify the targets coordinately regulated by muscle miRNAs in vivo, we performed gene expression arrays on muscle cells sorted from wild type, dicer mutants, and single miRNA knockdown embryos. Our analysis reveals that two particular miRNAs, miR-1 and miR-133, influence gene expression patterns in the zebrafish embryo where they account for >54% of the miRNA-mediated regulation in the muscle. We also found that muscle miRNA targets (1) tend to be expressed at low levels in wild-type muscle but are more highly expressed in dicer mutant muscle, and (2) are enriched for actin-related and actin-binding proteins. Loss of dicer function or down-regulation of miR-1 and miR-133 alters muscle gene expression and disrupts actin organization during sarcomere assembly. These results suggest that miR-1 and miR-133 actively shape gene expression patterns in muscle tissue, where they regulate sarcomeric actin organization.


Subject(s)
Actins/metabolism , Gene Expression Regulation, Developmental , MicroRNAs/metabolism , Muscle, Skeletal/metabolism , Sarcomeres/metabolism , Zebrafish/embryology , Animals , Gene Knockdown Techniques , Mutation , RNA, Messenger/metabolism , Reproducibility of Results , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
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