Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets - PubMed (original) (raw)

Comparative Study

Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets

Xiu-Jie Wang et al. Genome Biol. 2004.

Abstract

Background: A class of eukaryotic non-coding RNAs termed microRNAs (miRNAs) interact with target mRNAs by sequence complementarity to regulate their expression. The low abundance of some miRNAs and their time- and tissue-specific expression patterns make experimental miRNA identification difficult. We present here a computational method for genome-wide prediction of Arabidopsis thaliana microRNAs and their target mRNAs. This method uses characteristic features of known plant miRNAs as criteria to search for miRNAs conserved between Arabidopsis and Oryza sativa. Extensive sequence complementarity between miRNAs and their target mRNAs is used to predict miRNA-regulated Arabidopsis transcripts.

Results: Our prediction covered 63% of known Arabidopsis miRNAs and identified 83 new miRNAs. Evidence for the expression of 25 predicted miRNAs came from northern blots, their presence in the Arabidopsis Small RNA Project database, and massively parallel signature sequencing (MPSS) data. Putative targets functionally conserved between Arabidopsis and O. sativa were identified for most newly identified miRNAs. Independent microarray data showed that the expression levels of some mRNA targets anti-correlated with the accumulation pattern of their corresponding regulatory miRNAs. The cleavage of three target mRNAs by miRNA binding was validated in 5' RACE experiments.

Conclusions: We identified new plant miRNAs conserved between Arabidopsis and O. sativa and report a wide range of transcripts as potential miRNA targets. Because MPSS data are generated from polyadenylated RNA molecules, our results suggest that at least some miRNA precursors are polyadenylated at certain stages. The broad range of putative miRNA targets indicates that miRNAs participate in the regulation of a variety of biological processes.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Flowchart of the Arabidopsis miRNA prediction procedure. The number of predicted miRNA candidates and potential miRNA precursors (hairpins) is shown in blue bars. The number of known Arabidopsis miRNAs included in each prediction step is shown in parentheses. Known Arabidopsis miRNAs rejected by each prediction step are shown in red boxes.

Figure 2

Figure 2

Northern blot analysis of predicted miRNAs. Total RNA (20 μg) from 2-day-old seedlings (Se), 4-week-old adult plants (Pl), root-regenerated calluses (Ca), and mixed-stage flowers (Fl) was resolved in a 15% polyacrylamide/8 M urea gel for northern blot analysis. (a) Hybridization signal from confirmed miRNAs. (b) Antisense and sense oligonucleotides (indicated by AS and S, respectively) were used to confirm the polarity of miR417. (c) Hybridization signal for miR158 and 5S rRNA as indicated. The number next to each panel represents the position of RNA markers in nucleotides. In all cases the number in parentheses indicates the time of film exposure in days.

Figure 3

Figure 3

Putative secondary structures of selected miRNA precursors. (a-c) Secondary structures of predicted precursors of Arabidopsis miR393a, miR416 and miR396b, respectively. (d) pri-mir structure of proposed O. sativa homolog of Arabidopsis miR396b shown in (c). Sequences of mature miRNAs are marked with a red box.

Figure 4

Figure 4

Comparison of predicted miRNAs with sequences in the Arabidopsis ASRP database. Sequences from the ASRP database are named as 'sRNA' followed by clone numbers. Sequences of predicted miRNAs and sequences from ASRP database are shown in red; miRNA sequences extended according to cloned RNA sequences are in black. The final miRNA sequences reported in Additional data file 1 are marked with asterisks.

Figure 5

Figure 5

Clusters of predicted miRNAs with known Arabidopsis miRNAs. Identical nucleotides in predicted (underlined names) and known Arabidopsis miRNAs are highlighted in red; differences are highlighted in black; adjacent genomic sequences are shown in black in parentheses. NB indicates miRNAs whose expression was detected as positive by northern blot hybridization; ASRP indicates sequences present in the ASRP database.

Figure 6

Figure 6

Validation of a miRNA-cleaved mRNA target. (a) Northern blot analysis of miR393a showing its expression pattern. Samples are identical to those in Figure 2b. (b) The 5' RACE product for the predicted target gene At3g26810 amplified by PCR is shown in the ethidium bromide-stained agarose gel. (c) The 5' end of the cleaved product determined by sequencing is indicated by an arrow in the miRNA:mRNA base-pairing diagram, along with the number of clones analyzed. The gene structure of At3g26810 and location of the miRNA-binding site are shown at the bottom.

Similar articles

Cited by

References

    1. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297. doi: 10.1016/S0092-8674(04)00045-5. - DOI - PubMed
    1. Aravin AA, Lagos-Quintana M, Yalcin A, Zavolan M, Marks D, Snyder B, Gaasterland T, Meyer J, Tuschl T. The small RNA profile during Drosophila melanogaster development. Dev Cell. 2003;5:337–350. doi: 10.1016/S1534-5807(03)00228-4. - DOI - PubMed
    1. Dostie J, Mourelatos Z, Yang M, Sharma A, Dreyfuss G. Numerous microRNPs in neuronal cells containing novel microRNAs. RNA. 2003;9:180–186. doi: 10.1261/rna.2141503. - DOI - PMC - PubMed
    1. Grad Y, Aach J, Hayes GD, Reinhart BJ, Church GM, Ruvkun G, Kim J. Computational and experimental identification of C. elegans microRNAs. Mol Cell. 2003;11:1253–1263. doi: 10.1016/S1097-2765(03)00153-9. - DOI - PubMed
    1. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science. 2001;294:853–858. doi: 10.1126/science.1064921. - DOI - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources