title: Identification of microRNAs and microRNA targets in Xenopus gastrulae: The role of miR-26 in the regulation of Smad1.
abstract: MicroRNAs (miRNAs) are known to play diverse roles in the regulation of vertebrate development. To investigate miRNA-target mRNA relationships in embryonic development, we have carried out small-RNA sequencing to identify miRNAs expressed in the early gastrula of Xenopus laevis. We identify a total of 180 miRNAs, and we have identified the locations of the miRNA precursor sequences in the X. laevis genome. Of these miRNAs, 141 represent miRs previously identified in Xenopus tropicalis. Alignment to human miRNAs led to the identification of 39 miRNAs that have not previously been described for Xenopus. We have also used a biochemical approach to isolate mRNAs that are associated with the RNA-Induced Silencing Complex (RISC) in early gastrulae and thus candidate targets of miRNA-dependent regulation. Interrogation of this RISC-associated mRNA pool by RT-PCR indicates that a number of genes essential for early patterning and specification may be under regulation by miRNAs. Smad1 transcripts are associated with the RISC; target prediction algorithms identify a single miRNA-binding site for miR-26, which is common to the 3'UTRs of Smad1a and Smad1b. Disruption of the interaction between miR-26 and the Smad1 3'UTR via a Target Protector Morpholino Oligonucleotide (TPMO) leads to a 2-fold increase in Smad1 protein accumulation, moderate increases in the expression of BMP4/Smad1 target genes, and a reduction in organizer gene expression, as well as a partially ventralized phenotype in approximately 25% of embryos. Overexpression of miR-26 resulted in moderately decreased expression of Smad1-dependent genes and an expansion of the region expressing the Organizer gene not1. Our findings indicate that interactions between miR-26 and the Smad1 3'UTR modulate Smad1 function in the establishment of axial patterning; they also establish a foundation for the functional analysis of miRNAs and their regulatory interactions during gastrulation.
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, United States.
authors.forename: Chen
authors.initials: C
authors.lastname: Liu
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, United States.
authors.forename: Chih-Hong
authors.initials: CH
authors.lastname: Lou
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, United States.
authors.forename: Vrutant
authors.initials: V
authors.lastname: Shah
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, United States.
authors.forename: Ruth
authors.initials: R
authors.lastname: Ritter
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, United States.
authors.forename: Julia
authors.initials: J
authors.lastname: Talley
authors.affiliation: Department of Stem Cell Engineering, Texas Heart Institute at St. Luke's Episcopal Hospital, Houston, TX 77225, United States.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Soibam
authors.affiliation: Department of Stem Cell Engineering, Texas Heart Institute at St. Luke's Episcopal Hospital, Houston, TX 77225, United States.
authors.forename: Ashley
authors.initials: A
authors.lastname: Benham
authors.affiliation: Department of Physics, University of Houston, Houston, TX 77204, United States.
authors.forename: Huifeng
authors.initials: H
authors.lastname: Zhu
authors.affiliation: Department of Mathematics and Statistics, University of Houston-Downtown, Houston, TX 77002, United States.
authors.forename: Eloy
authors.initials: E
authors.lastname: Perez
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, United States.
authors.forename: Yi-Er
authors.initials: YE
authors.lastname: Shieh
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, United States.
authors.forename: Preethi H
authors.initials: PH
authors.lastname: Gunaratne
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, United States.
authors.forename: Amy K
authors.initials: AK
authors.lastname: Sater
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004625:Embryo, Nonmammalian / Q000378:metabolism; D047108:Embryonic Development / Q000235:genetics; D005775:Gastrula / Q000196:embryology / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010641:Phenotype; D011485:Protein Binding / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D051898:Smad1 Protein / Q000235:genetics* / Q000378:metabolism; D014981:Xenopus / Q000196:embryology* / Q000235:genetics*; D029867:Xenopus Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D060565:Argonaute Proteins; C000607854:MIRN26 microRNA, Xenopus; D035683:MicroRNAs; D012333:RNA, Messenger; C000607853:SMAD1 protein, Xenopus; D051898:Smad1 Protein; D029867:Xenopus Proteins
keywords: Argonaute; Gastrula; Smad1; Xenopus; microRNA