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Novel and nodulation-regulated microRNAs in soybean roots.

Subramanian S, Fu Y, Sunkar R, Barbazuk WB, Zhu JK, Yu O - BMC Genomics (2008)

Bottom Line: Construction and analysis of a small RNA library led to the identification of 20 conserved and 35 novel miRNA families in soybean.The availability of complete and assembled genome sequence information will enable identification of many other miRNAs.The conserved miRNA loci and novel miRNAs identified in this study enable investigation of the role of miRNAs in rhizobial symbiosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Donald Danforth Plant Science Center, 975 N Warson Road, St Louis, MO 63132, USA. ssubramanian@danforthcenter.org

ABSTRACT

Background: Small RNAs regulate a number of developmental processes in plants and animals. However, the role of small RNAs in legume-rhizobial symbiosis is largely unexplored. Symbiosis between legumes (e.g. soybean) and rhizobia bacteria (e.g. Bradyrhizobium japonicum) results in root nodules where the majority of biological nitrogen fixation occurs. We sought to identify microRNAs (miRNAs) regulated during soybean-B. japonicum symbiosis.

Results: We sequenced approximately 350000 small RNAs from soybean roots inoculated with B. japonicum and identified conserved miRNAs based on similarity to miRNAs known in other plant species and new miRNAs based on potential hairpin-forming precursors within soybean EST and shotgun genomic sequences. These bioinformatics analyses identified 55 families of miRNAs of which 35 were novel. A subset of these miRNAs were validated by Northern analysis and miRNAs differentially responding to B. japonicum inoculation were identified. We also identified putative target genes of the identified miRNAs and verified in vivo cleavage of a subset of these targets by 5'-RACE analysis. Using conserved miRNAs as internal control, we estimated that our analysis identified approximately 50% of miRNAs in soybean roots.

Conclusion: Construction and analysis of a small RNA library led to the identification of 20 conserved and 35 novel miRNA families in soybean. The availability of complete and assembled genome sequence information will enable identification of many other miRNAs. The conserved miRNA loci and novel miRNAs identified in this study enable investigation of the role of miRNAs in rhizobial symbiosis.

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RNA secondary structure of the hairpin forming precursors of gma-MIR1507, MIR1509 and MIR482. The putative mature miRNA and miRNA* sequences identified in the library are shaded in pink and blue respectively. Nucleotide positions are numbered starting from the 5' end of the precursor sequence.
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Figure 3: RNA secondary structure of the hairpin forming precursors of gma-MIR1507, MIR1509 and MIR482. The putative mature miRNA and miRNA* sequences identified in the library are shaded in pink and blue respectively. Nucleotide positions are numbered starting from the 5' end of the precursor sequence.

Mentions: The 139 novel precursors were subjected to folding (see Methods) and these hairpin structures were manually examined if they fit the criteria (less than 8 unpaired nucleotides and no more than three consecutive unpaired nucleotides of which no more than two were asymmetrically bulged in the 25 bp stem encompassing the mature miRNA sequence) used by [23]. We selected 82 sequences that fit the criteria as candidate miRNA genes. The folded secondary structures of three of our candidates are shown in Figure 3 as examples. Finally, we clustered these sequences into 35 different families representing novel candidate miRNAs (Table 1). Of the 35 candidates, 3 had miRNA* sequences in our library (Table 1), and all three paired to their corresponding miRNAs with 2 nt 3' overhangs (Figure 3). This is strong evidence that the miRNA/miRNA* pair originated from a Dicer1-like (DCL1) processing indicating that these are genuine miRNAs [22,23]. Of the remaining 32 candidates, all of the 5 tested were validated by Northern expression analysis (Table 1, see below) suggesting that most of the novel miRNAs identified in the study could be genuine miRNAs.


Novel and nodulation-regulated microRNAs in soybean roots.

Subramanian S, Fu Y, Sunkar R, Barbazuk WB, Zhu JK, Yu O - BMC Genomics (2008)

RNA secondary structure of the hairpin forming precursors of gma-MIR1507, MIR1509 and MIR482. The putative mature miRNA and miRNA* sequences identified in the library are shaded in pink and blue respectively. Nucleotide positions are numbered starting from the 5' end of the precursor sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2335117&req=5

Figure 3: RNA secondary structure of the hairpin forming precursors of gma-MIR1507, MIR1509 and MIR482. The putative mature miRNA and miRNA* sequences identified in the library are shaded in pink and blue respectively. Nucleotide positions are numbered starting from the 5' end of the precursor sequence.
Mentions: The 139 novel precursors were subjected to folding (see Methods) and these hairpin structures were manually examined if they fit the criteria (less than 8 unpaired nucleotides and no more than three consecutive unpaired nucleotides of which no more than two were asymmetrically bulged in the 25 bp stem encompassing the mature miRNA sequence) used by [23]. We selected 82 sequences that fit the criteria as candidate miRNA genes. The folded secondary structures of three of our candidates are shown in Figure 3 as examples. Finally, we clustered these sequences into 35 different families representing novel candidate miRNAs (Table 1). Of the 35 candidates, 3 had miRNA* sequences in our library (Table 1), and all three paired to their corresponding miRNAs with 2 nt 3' overhangs (Figure 3). This is strong evidence that the miRNA/miRNA* pair originated from a Dicer1-like (DCL1) processing indicating that these are genuine miRNAs [22,23]. Of the remaining 32 candidates, all of the 5 tested were validated by Northern expression analysis (Table 1, see below) suggesting that most of the novel miRNAs identified in the study could be genuine miRNAs.

Bottom Line: Construction and analysis of a small RNA library led to the identification of 20 conserved and 35 novel miRNA families in soybean.The availability of complete and assembled genome sequence information will enable identification of many other miRNAs.The conserved miRNA loci and novel miRNAs identified in this study enable investigation of the role of miRNAs in rhizobial symbiosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Donald Danforth Plant Science Center, 975 N Warson Road, St Louis, MO 63132, USA. ssubramanian@danforthcenter.org

ABSTRACT

Background: Small RNAs regulate a number of developmental processes in plants and animals. However, the role of small RNAs in legume-rhizobial symbiosis is largely unexplored. Symbiosis between legumes (e.g. soybean) and rhizobia bacteria (e.g. Bradyrhizobium japonicum) results in root nodules where the majority of biological nitrogen fixation occurs. We sought to identify microRNAs (miRNAs) regulated during soybean-B. japonicum symbiosis.

Results: We sequenced approximately 350000 small RNAs from soybean roots inoculated with B. japonicum and identified conserved miRNAs based on similarity to miRNAs known in other plant species and new miRNAs based on potential hairpin-forming precursors within soybean EST and shotgun genomic sequences. These bioinformatics analyses identified 55 families of miRNAs of which 35 were novel. A subset of these miRNAs were validated by Northern analysis and miRNAs differentially responding to B. japonicum inoculation were identified. We also identified putative target genes of the identified miRNAs and verified in vivo cleavage of a subset of these targets by 5'-RACE analysis. Using conserved miRNAs as internal control, we estimated that our analysis identified approximately 50% of miRNAs in soybean roots.

Conclusion: Construction and analysis of a small RNA library led to the identification of 20 conserved and 35 novel miRNA families in soybean. The availability of complete and assembled genome sequence information will enable identification of many other miRNAs. The conserved miRNA loci and novel miRNAs identified in this study enable investigation of the role of miRNAs in rhizobial symbiosis.

Show MeSH