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The miRNAome of globe artichoke: conserved and novel micro RNAs and target analysis.

De Paola D, Cattonaro F, Pignone D, Sonnante G - BMC Genomics (2012)

Bottom Line: In addition to conserved miRNAs, found in a wide range of plant species a number of novel species-specific miRNAs, displaying lower levels of expression can be found.New target genes were identified.We suggest that the generation of secondary short-interfering RNAs from miR393 target can be a general rule in the plant kingdom.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Plant Genetics (IGV), National Research Council (CNR), Via Amendola 165/A, 70126 Bari - Italy.

ABSTRACT

Background: Plant microRNAs (miRNAs) are involved in post-transcriptional regulatory mechanisms of several processes, including the response to biotic and abiotic stress, often contributing to the adaptive response of the plant to adverse conditions. In addition to conserved miRNAs, found in a wide range of plant species a number of novel species-specific miRNAs, displaying lower levels of expression can be found. Due to low abundance, non conserved miRNAs are difficult to identify and isolate using conventional approaches. Conversely, deep-sequencing of small RNA (sRNA) libraries can detect even poorly expressed miRNAs.No miRNAs from globe artichoke have been described to date. We analyzed the miRNAome from artichoke by deep sequencing four sRNA libraries obtained from NaCl stressed and control leaves and roots.

Results: Conserved and novel miRNAs were discovered using accepted criteria. The expression level of selected miRNAs was monitored by quantitative real-time PCR. Targets were predicted and validated for their cleavage site. A total of 122 artichoke miRNAs were identified, 98 (25 families) of which were conserved with other plant species, and 24 were novel. Some miRNAs were differentially expressed according to tissue or condition, magnitude of variation after salt stress being more pronounced in roots. Target function was predicted by comparison to Arabidopsis proteins; the 43 targets (23 for novel miRNAs) identified included transcription factors and other genes, most of which involved in the response to various stresses. An unusual cleaved transcript was detected for miR393 target, transport inhibitor response 1.

Conclusions: The miRNAome from artichoke, including novel miRNAs, was unveiled, providing useful information on the expression in different organs and conditions. New target genes were identified. We suggest that the generation of secondary short-interfering RNAs from miR393 target can be a general rule in the plant kingdom.

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Target validation. (a) Separation of 5' RACE products of target genes for cca-miR-160 (left gel), and cca-miR-393, 397, 398 (right gel, from left to right). Asterisks indicate cleaved fragments. (b) Partial sequence of TIR1 homolog from globe artichoke (acc. No. JN382008). 21 nucleotide register phasing of sense (red) siRNAs were observed following 5' RACE analysis. Canonical and observed miR393 cleavage sites are indicated by green arrows.
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Figure 3: Target validation. (a) Separation of 5' RACE products of target genes for cca-miR-160 (left gel), and cca-miR-393, 397, 398 (right gel, from left to right). Asterisks indicate cleaved fragments. (b) Partial sequence of TIR1 homolog from globe artichoke (acc. No. JN382008). 21 nucleotide register phasing of sense (red) siRNAs were observed following 5' RACE analysis. Canonical and observed miR393 cleavage sites are indicated by green arrows.

Mentions: For a subset of artichoke miRNAs, the cleavage of putative targets was evaluated by 5' RACE assay. For cca-miR160 target, an auxin responsive factor, a fragment of the size corresponding to the cleaved sequence was observed on agarose gel (Figure 3). Sequence of the 5' RACE cleaved product confirmed a precise slice at the miRNA binding site, between position 10 and 11. Analysis for putative targets of cca-miR397 and cca-miR398 revealed no evidence of 3' cleaved product, and only fragments corresponding to unprocessed target sequence were detected for both targets (Figure 3). Uncleaved transcripts were confirmed by sequencing.


The miRNAome of globe artichoke: conserved and novel micro RNAs and target analysis.

De Paola D, Cattonaro F, Pignone D, Sonnante G - BMC Genomics (2012)

Target validation. (a) Separation of 5' RACE products of target genes for cca-miR-160 (left gel), and cca-miR-393, 397, 398 (right gel, from left to right). Asterisks indicate cleaved fragments. (b) Partial sequence of TIR1 homolog from globe artichoke (acc. No. JN382008). 21 nucleotide register phasing of sense (red) siRNAs were observed following 5' RACE analysis. Canonical and observed miR393 cleavage sites are indicated by green arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285030&req=5

Figure 3: Target validation. (a) Separation of 5' RACE products of target genes for cca-miR-160 (left gel), and cca-miR-393, 397, 398 (right gel, from left to right). Asterisks indicate cleaved fragments. (b) Partial sequence of TIR1 homolog from globe artichoke (acc. No. JN382008). 21 nucleotide register phasing of sense (red) siRNAs were observed following 5' RACE analysis. Canonical and observed miR393 cleavage sites are indicated by green arrows.
Mentions: For a subset of artichoke miRNAs, the cleavage of putative targets was evaluated by 5' RACE assay. For cca-miR160 target, an auxin responsive factor, a fragment of the size corresponding to the cleaved sequence was observed on agarose gel (Figure 3). Sequence of the 5' RACE cleaved product confirmed a precise slice at the miRNA binding site, between position 10 and 11. Analysis for putative targets of cca-miR397 and cca-miR398 revealed no evidence of 3' cleaved product, and only fragments corresponding to unprocessed target sequence were detected for both targets (Figure 3). Uncleaved transcripts were confirmed by sequencing.

Bottom Line: In addition to conserved miRNAs, found in a wide range of plant species a number of novel species-specific miRNAs, displaying lower levels of expression can be found.New target genes were identified.We suggest that the generation of secondary short-interfering RNAs from miR393 target can be a general rule in the plant kingdom.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Plant Genetics (IGV), National Research Council (CNR), Via Amendola 165/A, 70126 Bari - Italy.

ABSTRACT

Background: Plant microRNAs (miRNAs) are involved in post-transcriptional regulatory mechanisms of several processes, including the response to biotic and abiotic stress, often contributing to the adaptive response of the plant to adverse conditions. In addition to conserved miRNAs, found in a wide range of plant species a number of novel species-specific miRNAs, displaying lower levels of expression can be found. Due to low abundance, non conserved miRNAs are difficult to identify and isolate using conventional approaches. Conversely, deep-sequencing of small RNA (sRNA) libraries can detect even poorly expressed miRNAs.No miRNAs from globe artichoke have been described to date. We analyzed the miRNAome from artichoke by deep sequencing four sRNA libraries obtained from NaCl stressed and control leaves and roots.

Results: Conserved and novel miRNAs were discovered using accepted criteria. The expression level of selected miRNAs was monitored by quantitative real-time PCR. Targets were predicted and validated for their cleavage site. A total of 122 artichoke miRNAs were identified, 98 (25 families) of which were conserved with other plant species, and 24 were novel. Some miRNAs were differentially expressed according to tissue or condition, magnitude of variation after salt stress being more pronounced in roots. Target function was predicted by comparison to Arabidopsis proteins; the 43 targets (23 for novel miRNAs) identified included transcription factors and other genes, most of which involved in the response to various stresses. An unusual cleaved transcript was detected for miR393 target, transport inhibitor response 1.

Conclusions: The miRNAome from artichoke, including novel miRNAs, was unveiled, providing useful information on the expression in different organs and conditions. New target genes were identified. We suggest that the generation of secondary short-interfering RNAs from miR393 target can be a general rule in the plant kingdom.

Show MeSH
Related in: MedlinePlus