MiR-RACE, a new efficient approach to determine the precise sequences of computationally identified trifoliate orange (Poncirus trifoliata) microRNAs

Abstract

Background: Among the hundreds of genes encoding miRNAs in plants reported, much more were predicted by numerous computational methods. However, unlike protein-coding genes defined by start and stop codons, the ends of miRNA molecules do not have characteristics that can be used to define the mature miRNAs exactly, which made computational miRNA prediction methods often cannot predict the accurate location of the mature miRNA in a precursor with nucleotide-level precision. To our knowledge, there haven't been reports about comprehensive strategies determining the precise sequences, especially two termini, of these miRNAs.

Methods: In this study, we report an efficient method to determine the precise sequences of computationally predicted microRNAs (miRNAs) that combines miRNA-enriched library preparation, two specific 5' and 3' miRNA RACE (miR-RACE) PCR reactions, and sequence-directed cloning, in which the most challenging step is the two specific gene specific primers designed for the two RACE reactions. miRNA-mediated mRNA cleavage by RLM-5' RACE and sequencing were carried out to validate the miRNAs detected. Real-time PCR was used to analyze the expression of each miRNA.

Results: The efficiency of this newly developed method was validated using nine trifoliate orange (Poncirus trifoliata) miRNAs predicted computationally. The miRNAs computationally identified were validated by miR-RACE and sequencing. Quantitative analysis showed that they have variable expression. Eight target genes have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in Poncirus trifoliate.

Conclusion: The efficient and powerful approach developed herein can be successfully used to validate the sequences of miRNAs, especially the termini, which depict the complete miRNA sequence in the computationally predicted precursor.

Figure 1

a. Determination of the precise miRNA sequence by 5′ and 3′ miR-RACE. (a) miRNA cDNA library construction. (b) Analysis of miRNA 5′RACE and 3′RACE. Sequences for the 5′RNA adaptor, dT(30)RT primer, MirRacer 3′ Primer, MirRacer 5′ Primer, GSP1, and GSP2 are listed in Table 2.

Figure 2. Predicted fold-back structures of the identified ptr-miRNAs.

The mature miRNA sequences are shaded. The miRNA precursors may be slightly longer than the sequences shown in this figure. Predicted fold-back structures of the identified ptr-miRNAs. The mature miRNA sequences are shaded. The miRNA precursors may be slightly longer than the sequences shown in this figure.

Figure 3. The 3′ RACE and 5′ RACE products of ptr-miRNAs amplified by PCR are shown in an ethidium bromide-stained agarose gel.

The sizes of the molecular weight markers of the bottom and the second from bottom bands are 50 bp and 100bp, respectively. Lanes 1–9 are 3′RACE products of ptr-miR156, ptr-miR164, ptr-miR167, ptr-miR171, ptr-miR319, ptr-miR482a, ptr-miR482b, ptr-miR435, and ptr-miR1446, respectively, and lanes 10–18 are the 5′RACE products of them.

Figure 4. Relative expression levels of trifoliate orange miRNAs in different the trifoliate orange tisues of root, stem, leaf, flower, and fruit.

Each reaction was repeated three times and the template amount was corrected by 5.8 s rRNAs.

Figure 5. Mapping of the mRNA cleavage sites by RNA ligase-mediated 5′ RACE.

Each top strand (black) depicts a miRNA complementary site, and each bottom strand depicts the miRNA (red). Watson-Crick pairing (vertical dashes) and G∶U wobble pairing (circles) are indicated. The arrows indicate the 5′ termini of mRNA fragments isolated from citrus, as identified by cloned 5′RACE products, with the frequency of clones shown. Only the cloned sequences that matched the correct gene and had 5′ ends within a 100 nt window centered on the miRNA validation are included (Table 1). The miRNA sequence shown corre (1 out of 4 PCR clones) is indicated in lower case and corresponds to the most common miRNA supported by the miRNA PCR. RNA ligase-mediated 5′RACE was used to map the cleavage sites. The partial mRNA sequences from the target genes were aligned with the miRNAs. The numbers indicate the fraction of cloned PCR products terminating at different positions. Pt-SPL9 (accession FJ502237), Pt-SPL13 (accession FJ502238), Pt-NAC1 (accession FJ619349), Pt-ARF8 (UC46-16450), Pt-SCL6 (accession GQ505957), Pt-TCP4 (GQ505958), Pt-GRAS (accession FC901464). Pt-SPL9 (accession FJ502237) was similar to AT2G42200 (NM_129782) SPL9 (squamosa promoter-binding protein 9); Pt-SPL13 (accession FJ502238) was similar to AT5G50670 (NM_124445) SPL (squamosa promoter-binding protein); Pt-NAC1 (accession FJ619349) was similar to AT5G61430 ( NM_125536) ANAC100 (ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 100); Pt-ARF8 (UC46-16450) was similar to AT5G37020 (NM_001085203) ARF8 (AUXIN RESPONSE FACTOR 8); Pt-SCL6 (accession GQ505957) was similar to AT4G00150 (NM_116232) SCL6 (scarecrow-like transcription factor 6); Pt-TCP4 (GQ505958) was similar to AT3G15030 (NM_180258) TCP4 (TCP family transcription factor 4); UC46-36616 was similar to AT1G12220 (NM_101094) RPS5 (RESISTANT TO P. SYRINGAE 5); Pt-GRAS (accession GU072592) was similar to IPR005202 (XM_002318667) GRAS71 (GRAS family transcription factor).