Limits...
Regulation of small RNA accumulation in the maize shoot apex.

Nogueira FT, Chitwood DH, Madi S, Ohtsu K, Schnable PS, Scanlon MJ, Timmermans MC - PLoS Genet. (2009)

Bottom Line: Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity.Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well.Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.

ABSTRACT
MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) are essential to the establishment of adaxial-abaxial (dorsoventral) leaf polarity. Tas3-derived ta-siRNAs define the adaxial side of the leaf by restricting the expression domain of miRNA miR166, which in turn demarcates the abaxial side of leaves by restricting the expression of adaxial determinants. To investigate the regulatory mechanisms that allow for the precise spatiotemporal accumulation of these polarizing small RNAs, we used laser-microdissection coupled to RT-PCR to determine the expression profiles of their precursor transcripts within the maize shoot apex. Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity. We show that miR390, an upstream determinant in leaf polarity whose activity triggers tas3 ta-siRNA biogenesis, accumulates adaxially in leaves. The polar expression of miR390 is established and maintained independent of the ta-siRNA pathway. The comparison of small RNA localization data with the expression profiles of precursor transcripts suggests that miR166 and miR390 accumulation is also regulated at the level of biogenesis and/or stability. Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well. Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.

Show MeSH
MiR390 is an upstream component in the maize leaf polarity pathway.(A, B) In situ hybridization analyses with a miR390 complementary LNA probe shows this small RNA accumulates on the adaxial side (arrowheads) of incipient and developing leaf primorida of both wild-type (A) and lbl1-rgd1 (B) seedlings. Dashed lines mark the position of the incipient leaf. No hybridization signal is detected in the tip of the wild-type SAM. (C) In situ hybridization with an LNA-probe complementary to murine miR122a serves as a negative control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2602737&req=5

pgen-1000320-g003: MiR390 is an upstream component in the maize leaf polarity pathway.(A, B) In situ hybridization analyses with a miR390 complementary LNA probe shows this small RNA accumulates on the adaxial side (arrowheads) of incipient and developing leaf primorida of both wild-type (A) and lbl1-rgd1 (B) seedlings. Dashed lines mark the position of the incipient leaf. No hybridization signal is detected in the tip of the wild-type SAM. (C) In situ hybridization with an LNA-probe complementary to murine miR122a serves as a negative control.

Mentions: As the biogenesis of tas3 ta-siRNAs is uniquely triggered by miR390 activity [12], we determined the localization pattern of mature miR390 by in situ hybridization to ascertain whether it might be a restrictive component limiting tasiR-ARF biogenesis. Whereas an LNA probe against a murine miRNA yields no detectable hybridization signal, an LNA probe complementary to miR390 shows it accumulates adaxially within the incipient and developing leaves (Figure 3A and 3C). Additionally, miR390 is expressed in vascular bundles (Figure 3A). The adaxial accumulation of miR390 overlaps with that of tasiR-ARFs and the expression pattern of lbl1/SGS3 in these regions [3], and is opposite to the abaxial accumulation of miR166 in the incipient leaf (Figures 2C and 3A). Thus, miR390 is a restricting factor in the tas3 ta-siRNA pathway that limits the biogenesis of tasiR-ARFs to within developing leaves. That miR390 restricts the accumulation of tasiR-ARFs places it as one of the upstream factors in the maize leaf polarity network, as genetic and expression data position the tas3 ta-siRNA pathway upstream of miR166 as well as the hd-zipIII and yabby genes [3],[8].


Regulation of small RNA accumulation in the maize shoot apex.

Nogueira FT, Chitwood DH, Madi S, Ohtsu K, Schnable PS, Scanlon MJ, Timmermans MC - PLoS Genet. (2009)

MiR390 is an upstream component in the maize leaf polarity pathway.(A, B) In situ hybridization analyses with a miR390 complementary LNA probe shows this small RNA accumulates on the adaxial side (arrowheads) of incipient and developing leaf primorida of both wild-type (A) and lbl1-rgd1 (B) seedlings. Dashed lines mark the position of the incipient leaf. No hybridization signal is detected in the tip of the wild-type SAM. (C) In situ hybridization with an LNA-probe complementary to murine miR122a serves as a negative control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2602737&req=5

pgen-1000320-g003: MiR390 is an upstream component in the maize leaf polarity pathway.(A, B) In situ hybridization analyses with a miR390 complementary LNA probe shows this small RNA accumulates on the adaxial side (arrowheads) of incipient and developing leaf primorida of both wild-type (A) and lbl1-rgd1 (B) seedlings. Dashed lines mark the position of the incipient leaf. No hybridization signal is detected in the tip of the wild-type SAM. (C) In situ hybridization with an LNA-probe complementary to murine miR122a serves as a negative control.
Mentions: As the biogenesis of tas3 ta-siRNAs is uniquely triggered by miR390 activity [12], we determined the localization pattern of mature miR390 by in situ hybridization to ascertain whether it might be a restrictive component limiting tasiR-ARF biogenesis. Whereas an LNA probe against a murine miRNA yields no detectable hybridization signal, an LNA probe complementary to miR390 shows it accumulates adaxially within the incipient and developing leaves (Figure 3A and 3C). Additionally, miR390 is expressed in vascular bundles (Figure 3A). The adaxial accumulation of miR390 overlaps with that of tasiR-ARFs and the expression pattern of lbl1/SGS3 in these regions [3], and is opposite to the abaxial accumulation of miR166 in the incipient leaf (Figures 2C and 3A). Thus, miR390 is a restricting factor in the tas3 ta-siRNA pathway that limits the biogenesis of tasiR-ARFs to within developing leaves. That miR390 restricts the accumulation of tasiR-ARFs places it as one of the upstream factors in the maize leaf polarity network, as genetic and expression data position the tas3 ta-siRNA pathway upstream of miR166 as well as the hd-zipIII and yabby genes [3],[8].

Bottom Line: Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity.Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well.Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.

ABSTRACT
MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) are essential to the establishment of adaxial-abaxial (dorsoventral) leaf polarity. Tas3-derived ta-siRNAs define the adaxial side of the leaf by restricting the expression domain of miRNA miR166, which in turn demarcates the abaxial side of leaves by restricting the expression of adaxial determinants. To investigate the regulatory mechanisms that allow for the precise spatiotemporal accumulation of these polarizing small RNAs, we used laser-microdissection coupled to RT-PCR to determine the expression profiles of their precursor transcripts within the maize shoot apex. Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity. We show that miR390, an upstream determinant in leaf polarity whose activity triggers tas3 ta-siRNA biogenesis, accumulates adaxially in leaves. The polar expression of miR390 is established and maintained independent of the ta-siRNA pathway. The comparison of small RNA localization data with the expression profiles of precursor transcripts suggests that miR166 and miR390 accumulation is also regulated at the level of biogenesis and/or stability. Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well. Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.

Show MeSH