A family of microRNAs present in plants and animals

Abstract

Although many miRNAs are deeply conserved within each kingdom, none are known to be conserved between plants and animals. We identified Arabidopsis thaliana miR854 and miR855, two microRNAs (miRNAs) with multiple binding sites in the 3' untranslated region (3'UTR) of OLIGOURIDYLATE binding PROTEIN1b (At UBP1b), forming miRNA:mRNA interactions similar to those that cause translational repression/mRNA cleavage in animals. At UBP1b encodes a member of a heterogeneous nuclear RNA binding protein (hnRNP) family. The 3'UTR of At UBP1b is sufficient to repress reporter protein expression in tissues expressing miR854 or miR855 (rosette leaves and flowers, respectively) but not where both miRNAs are absent (cauline leaves). Intergenic regions containing sequences closely resembling miR854 are predicted to fold into stable miRNA precursors in animals, and members of the miR854 family are expressed in Caenorhabditis elegans, Mus musculus, and Homo sapiens, all with imperfect binding sites in the 3'UTR of genes encoding the T cell Intracellular Antigen-Related protein, an hnRNP of the UBP1 family. Potential binding sites for miR854 are absent from UBP1-like genes in fungi lacking the miRNA biogenetic machinery. Our results indicate that plants and animals share miRNAs of the miR854 family, suggesting a common origin of these miRNAs as regulators of basal transcriptional mechanisms.

Figure 1.

Strategy for the Prediction of Candidate miRNAs and Their Target 3′UTRs in Arabidopsis. A Perl script was designed to compare an intergenic regions database built from the Arabidopsis genome against a database containing nonredundant 21- and 22-nucleotide sequences derived from a 3′UTR database. Larger genomic regions containing sequences that respected the established criteria were analyzed for subsequent prediction of potential RNA secondary structure using MFOLD (Zuker, 2003). Experimental validation for all candidate miRNAs was performed by RNA gel blot analysis (see main text for details). nt, nucleotides.

Figure 2.

Expression Patterns of Candidate miRNAs. RNA gel blot analysis was conducted by hybridizing low molecular weight RNA from rosette leaves, stems, cauline leaves, and developing inflorescences of adult Arabidopsis plants with radiolabeled probes complementary to each candidate miRNA. rRNA bands were visualized by ethidium bromide staining of polyacrylamide gels and served as loading controls. A labeled RNA oligonucleotide was used as a size marker.

Figure 3.

Predicted Precursors for miR854, miR855, and Os miR854 and Their Potential Binding Sites in the 3′UTR of At UBP1b. (A) Foldback secondary structures of the predicted precursor for miR855 as determined by MFOLD; the intergenic region corresponds to coordinates 4,681,515 to 4,681,536 in chromosome II. (B) Foldback secondary structures of the predicted precursor for miR854 as determined by MFOLD and localization of miR854 predicted precursor in the ATHILA6A_I retroelement; the intergenic region corresponds to coordinates 11,855,326 to 11,855,546 in chromosome V. (C) Foldback secondary structures of the predicted precursor for Os miR854 as determined by MFOLD; the intergenic region corresponds to coordinates 7,974,551 to 7,974,531 in chromosome IV. For Arabidopsis, coordinates follow TAIR release 6.0 annotation.

Figure 4.

miR854 Is Produced by the miRNA Biogenetic Pathway. RNA gel blot analysis was conducted by hybridizing low molecular weight flower RNA from dcl1-9, hyl1-1, hen1-1, and rdr2-1 mutant individuals and wild-type plants with radiolabeled probes complementary to miR854. Whereas dcl1-9, hyl1-1, and hen1-1 mutants are affected in the miRNA biogenesis pathway, the rdr2-1 mutant is affected in siRNA production. The membrane was subsequently reprobed with an ath-miR170 complementary end-labeled oligonucleotide as a positive control. rRNA bands were visualized by ethidium bromide staining of polyacrylamide gels and served as loading controls. A labeled RNA oligonucleotide was used as a size marker.

Figure 5.

Patterns of GUS Expression in Pro_35S:GUS:3′UTR-UBP1b Transgenic Plants. (A) to (E) Pro_35S:GUS:3′UTR-UBP1b transgenic Arabidopsis plants. (A) Germinating seedling at growth stage 0.5. Bar = 2.5 mm. (B) Seedling at growth stage 0.7. Bar = 2.5 mm. (C) Seedling at growth stage 1.0. Bar = 2.5 mm. (D) Seedling at growth stage 1.05. Bar = 2.5 mm. (E) Plant at growth stage 3.20. Bar = 2.5 mm. (F) Individual leaves from developing rosettes at growth stage 1.10 and inforescences at growth stage 6.1 of Pro_35S:GUS transgenic plants. Bar = 2.5 mm (G) Individual leaves from developing rosettes at growth stage 1.10 and inforescences at growth stage 6.1 of Pro_35S:GUS:3′UTR-UBP1b transgenic plants. Bar = 2.5 mm Growth stages are as previously described in Boyes et al. (2001). C, cotyledon; H, hypocotyl; Hy, hydathodes; L, rosette leaves; R, root; S, stem.

Figure 6.

Posttranscriptional Gene Silencing Conferred by the 3′UTR of At UBP1b. (A) GUS activity in rosette leaves from Pro_35S:GUS:3′UTR-UBP1b (+) and Pro_3S:GUS (−) transgenic and wild-type Arabidopsis plants (w) at growth stages 1.10 and 6.1. (B) Immunoblot analysis of GUS protein in rosette leaves from Pro_35S:GUS:3′UTR-UBP1b (+), Pro_35S:GUS (−) transgenic, and wild-type plants at 1.10 growth stage. The 68.4-kD GUS protein is indicated by an asterisk. (C) Coomassie blue staining shown as a protein loading control. (D) RNA gel blot analysis of GUS mRNA in rosette leaves from Pro_35S:GUS:3′UTR-UBP1b (+), Pro_35S:GUS (−), and wild-type plants at 1.10 growth stage. The arrowhead indicates the 1802-nucleotide GUS transcript for (−); transcript for GUS:3′UTR-UBP1b (+) is 2048 nucleotides. (E) rRNA was visualized by ethidium bromide staining of polyacrylamide gels and is shown as a loading control. (F) GUS activiy in T2 seedlings and developing flowers from Pro_35S:GUS:3′UTR-UBP1b (+) and Pro_35S:GUS (−) transgenic and wild-type plants at 1.0 growth stage. Bars = 2.5 mm.

Figure 7.

Predicted Target Sites of miR854 and miR855 in the 3′UTR of At UBP1b. The distance in nucleotides is indicated between target sites.

Figure 8.

Conservation of miR854 Family in Animal Species. (A) Alignment of miR854 family members in plants and animals; underlined letters indicate unique nucleotide substitutions compared with Arabidopsis. (B) RNA gel blot analysis of animal miRNAs of the miR854 family using RNA from HepG2 human cells (lane 1), adult C. elegans individuals (lane 2), mice stem cells (lane 3), and mice kidney tissue (lane 4). rRNA is shown as a loading control. (C) Predicted precursors for animal miRNAs of the miR854 family: Pt miR854 (P. troglodytes), Hs miR854 (H. sapiens), Mm miR854 (M. musculus) and Ce miR854 (C. elegans) as predicted by MFOLD (Zuker, 2003). (D) Predicted target sites in the 3′UTR of TIAR family members for miR854, Hs miR854, Ce miR854, Mm miR854, and Pt miR854; gray letters indicate single nucleotide substitutions compared with miR854.