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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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Cleavage sites of selected miRNA targets as identified by 5'-RACE analysis. For each miRNA, the target sequence is shown at the top and the miRNA sequence at the bottom. Perfectly complementary bases are shown connected by solid lines and G:U wobbles by dotted lines. The red arrows indicate the site of cleavage. Targets are labeled with TC or EST IDs assigned in The Gene Index [52].
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Figure 6: Cleavage sites of selected miRNA targets as identified by 5'-RACE analysis. For each miRNA, the target sequence is shown at the top and the miRNA sequence at the bottom. Perfectly complementary bases are shown connected by solid lines and G:U wobbles by dotted lines. The red arrows indicate the site of cleavage. Targets are labeled with TC or EST IDs assigned in The Gene Index [52].

Mentions: For a subset of miRNAs, we experimentally verified the cleavage of selected targets by 5'-RACE analysis. We isolated polyA RNA from mock-inoculated and B. japonicum-inoculated (3 h) soybean roots and subjected the RNA to a 5'-RACE reaction (see Methods). We examined the presence of miRNA-directed cleavage products by PCR using primers specific to the target of interest. We were able to verify the cleavage of target miRNA for four of the identified miRNA families (Figure 6). The targets of miR166 and miR393 are well-conserved in other species. Consistently, we also observed cleavage of transcripts encoding a homeodomain protein [TGI:TC221756; 4 clones] and a TIR1-like protein [TGI:TC225843; 3 clones] by these miRNAs respectively. The identified targets of miR168 [TGI:BG882680; 1 clone] and miR396 [TGI:TC206710; 2 clones] in soybean seem to be non-conserved. These targets encode a putative protein kinase and a cysteine protease respectively. In all these cases, the site of cleavage corresponded to the position between the 10th and 11th nt of the miRNA.


Novel and nodulation-regulated microRNAs in soybean roots.

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

Cleavage sites of selected miRNA targets as identified by 5'-RACE analysis. For each miRNA, the target sequence is shown at the top and the miRNA sequence at the bottom. Perfectly complementary bases are shown connected by solid lines and G:U wobbles by dotted lines. The red arrows indicate the site of cleavage. Targets are labeled with TC or EST IDs assigned in The Gene Index [52].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Cleavage sites of selected miRNA targets as identified by 5'-RACE analysis. For each miRNA, the target sequence is shown at the top and the miRNA sequence at the bottom. Perfectly complementary bases are shown connected by solid lines and G:U wobbles by dotted lines. The red arrows indicate the site of cleavage. Targets are labeled with TC or EST IDs assigned in The Gene Index [52].
Mentions: For a subset of miRNAs, we experimentally verified the cleavage of selected targets by 5'-RACE analysis. We isolated polyA RNA from mock-inoculated and B. japonicum-inoculated (3 h) soybean roots and subjected the RNA to a 5'-RACE reaction (see Methods). We examined the presence of miRNA-directed cleavage products by PCR using primers specific to the target of interest. We were able to verify the cleavage of target miRNA for four of the identified miRNA families (Figure 6). The targets of miR166 and miR393 are well-conserved in other species. Consistently, we also observed cleavage of transcripts encoding a homeodomain protein [TGI:TC221756; 4 clones] and a TIR1-like protein [TGI:TC225843; 3 clones] by these miRNAs respectively. The identified targets of miR168 [TGI:BG882680; 1 clone] and miR396 [TGI:TC206710; 2 clones] in soybean seem to be non-conserved. These targets encode a putative protein kinase and a cysteine protease respectively. In all these cases, the site of cleavage corresponded to the position between the 10th and 11th nt of the miRNA.

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