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Identification of novel microRNAs in Hevea brasiliensis and computational prediction of their targets.

Gébelin V, Argout X, Engchuan W, Pitollat B, Duan C, Montoro P, Leclercq J - BMC Plant Biol. (2012)

Bottom Line: The results showed the most abundant size for miRNAs to be 24 nucleotides, except for seven families.Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs when the complete genome is not yet available.Our study provided additional information for evolutionary studies and revealed potentially specific regulation of the control of redox status in Hevea.

View Article: PubMed Central - HTML - PubMed

Affiliation: CIRAD, UMR AGAP, F-34398 Montpellier, France.

ABSTRACT

Background: Plants respond to external stimuli through fine regulation of gene expression partially ensured by small RNAs. Of these, microRNAs (miRNAs) play a crucial role. They negatively regulate gene expression by targeting the cleavage or translational inhibition of target messenger RNAs (mRNAs). In Hevea brasiliensis, environmental and harvesting stresses are known to affect natural rubber production. This study set out to identify abiotic stress-related miRNAs in Hevea using next-generation sequencing and bioinformatic analysis.

Results: Deep sequencing of small RNAs was carried out on plantlets subjected to severe abiotic stress using the Solexa technique. By combining the LeARN pipeline, data from the Plant microRNA database (PMRD) and Hevea EST sequences, we identified 48 conserved miRNA families already characterized in other plant species, and 10 putatively novel miRNA families. The results showed the most abundant size for miRNAs to be 24 nucleotides, except for seven families. Several MIR genes produced both 20-22 nucleotides and 23-27 nucleotides. The two miRNA class sizes were detected for both conserved and putative novel miRNA families, suggesting their functional duality. The EST databases were scanned with conserved and novel miRNA sequences. MiRNA targets were computationally predicted and analysed. The predicted targets involved in "responses to stimuli" and to "antioxidant" and "transcription activities" are presented.

Conclusions: Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs when the complete genome is not yet available. Our study provided additional information for evolutionary studies and revealed potentially specific regulation of the control of redox status in Hevea.

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Gel electrophoresis of stem-loop RT-PCR. A) Stem-loop RT-PCR from seven precursors of conserved miRNA family. B) Stem-loop RT-PCR from nine precursors of putatively new miRNAs.
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Figure 6: Gel electrophoresis of stem-loop RT-PCR. A) Stem-loop RT-PCR from seven precursors of conserved miRNA family. B) Stem-loop RT-PCR from nine precursors of putatively new miRNAs.

Mentions: Conserved and putative novel miRNA precursors were sought in the Hevea clone PB260 transcript sequence databases obtained from various organs using the LeARN pipeline [51]. Eight conserved miRNA families that mapped against RNA sequences displayed a stem-loop structure (Table 4 and Additional Table 1). A single miRNA precursor transcript was found for the HbmiR156, HbmiR159, HbmiR396, HbmiR476 and HbmiR2910 families. Two miRNA precursor transcripts were identified for the HbmiR166, HbmiR319 and HbmiR408 families. In addition, the LeARN pipeline predicted 10 putative novel miRNAs with their unique precursors (Table 5 and Additional Table 2). Mapping the sequences of the small RNA dataset on the precursor sequences led to the identification of miRNA*for all eight conserved families (HbmiR156, HbmiR159, HbmiR166, HbmiR319, HbmiR396, HbmiR408, HbmiR476 and HbmiR2910) and for the five putatively new families HbmiRn3, HbmiRn4, HbmiRn8, HbmiRn9 and HbmiRn10. Stem-loop reverse transcription polymerase chain reactions (RT-PCR) were successfully performed on seven precursors for the conserved families and on nine for the putatively new families (Figure 6).


Identification of novel microRNAs in Hevea brasiliensis and computational prediction of their targets.

Gébelin V, Argout X, Engchuan W, Pitollat B, Duan C, Montoro P, Leclercq J - BMC Plant Biol. (2012)

Gel electrophoresis of stem-loop RT-PCR. A) Stem-loop RT-PCR from seven precursors of conserved miRNA family. B) Stem-loop RT-PCR from nine precursors of putatively new miRNAs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368772&req=5

Figure 6: Gel electrophoresis of stem-loop RT-PCR. A) Stem-loop RT-PCR from seven precursors of conserved miRNA family. B) Stem-loop RT-PCR from nine precursors of putatively new miRNAs.
Mentions: Conserved and putative novel miRNA precursors were sought in the Hevea clone PB260 transcript sequence databases obtained from various organs using the LeARN pipeline [51]. Eight conserved miRNA families that mapped against RNA sequences displayed a stem-loop structure (Table 4 and Additional Table 1). A single miRNA precursor transcript was found for the HbmiR156, HbmiR159, HbmiR396, HbmiR476 and HbmiR2910 families. Two miRNA precursor transcripts were identified for the HbmiR166, HbmiR319 and HbmiR408 families. In addition, the LeARN pipeline predicted 10 putative novel miRNAs with their unique precursors (Table 5 and Additional Table 2). Mapping the sequences of the small RNA dataset on the precursor sequences led to the identification of miRNA*for all eight conserved families (HbmiR156, HbmiR159, HbmiR166, HbmiR319, HbmiR396, HbmiR408, HbmiR476 and HbmiR2910) and for the five putatively new families HbmiRn3, HbmiRn4, HbmiRn8, HbmiRn9 and HbmiRn10. Stem-loop reverse transcription polymerase chain reactions (RT-PCR) were successfully performed on seven precursors for the conserved families and on nine for the putatively new families (Figure 6).

Bottom Line: The results showed the most abundant size for miRNAs to be 24 nucleotides, except for seven families.Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs when the complete genome is not yet available.Our study provided additional information for evolutionary studies and revealed potentially specific regulation of the control of redox status in Hevea.

View Article: PubMed Central - HTML - PubMed

Affiliation: CIRAD, UMR AGAP, F-34398 Montpellier, France.

ABSTRACT

Background: Plants respond to external stimuli through fine regulation of gene expression partially ensured by small RNAs. Of these, microRNAs (miRNAs) play a crucial role. They negatively regulate gene expression by targeting the cleavage or translational inhibition of target messenger RNAs (mRNAs). In Hevea brasiliensis, environmental and harvesting stresses are known to affect natural rubber production. This study set out to identify abiotic stress-related miRNAs in Hevea using next-generation sequencing and bioinformatic analysis.

Results: Deep sequencing of small RNAs was carried out on plantlets subjected to severe abiotic stress using the Solexa technique. By combining the LeARN pipeline, data from the Plant microRNA database (PMRD) and Hevea EST sequences, we identified 48 conserved miRNA families already characterized in other plant species, and 10 putatively novel miRNA families. The results showed the most abundant size for miRNAs to be 24 nucleotides, except for seven families. Several MIR genes produced both 20-22 nucleotides and 23-27 nucleotides. The two miRNA class sizes were detected for both conserved and putative novel miRNA families, suggesting their functional duality. The EST databases were scanned with conserved and novel miRNA sequences. MiRNA targets were computationally predicted and analysed. The predicted targets involved in "responses to stimuli" and to "antioxidant" and "transcription activities" are presented.

Conclusions: Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs when the complete genome is not yet available. Our study provided additional information for evolutionary studies and revealed potentially specific regulation of the control of redox status in Hevea.

Show MeSH