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Genome-wide analysis of leafbladeless1-regulated and phased small RNAs underscores the importance of the TAS3 ta-siRNA pathway to maize development.

Dotto MC, Petsch KA, Aukerman MJ, Beatty M, Hammell M, Timmermans MC - PLoS Genet. (2014)

Bottom Line: Interestingly, in contrast to data from other plant species, we found no evidence for the existence of phased siRNAs generated via the one-hit model.The phenotypes of Arabidopsis and Medicago ta-siRNA mutants, while strikingly different, likewise result from misexpression of the tasiR-ARF target ARF3.Instead, we propose that divergence in the gene networks downstream of the ARF3 transcription factors or the spatiotemporal pattern during leaf development in which these proteins act constitute key factors underlying the distinct contributions of the ta-siRNA pathway to development in maize, Arabidopsis, and possibly other plant species as well.

View Article: PubMed Central - PubMed

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

ABSTRACT
Maize leafbladeless1 (lbl1) encodes a key component in the trans-acting short-interfering RNA (ta-siRNA) biogenesis pathway. Correlated with a great diversity in ta-siRNAs and the targets they regulate, the phenotypes conditioned by mutants perturbing this small RNA pathway vary extensively across species. Mutations in lbl1 result in severe developmental defects, giving rise to plants with radial, abaxialized leaves. To investigate the basis for this phenotype, we compared the small RNA content between wild-type and lbl1 seedling apices. We show that LBL1 affects the accumulation of small RNAs in all major classes, and reveal unexpected crosstalk between ta-siRNA biogenesis and other small RNA pathways regulating transposons. Interestingly, in contrast to data from other plant species, we found no evidence for the existence of phased siRNAs generated via the one-hit model. Our analysis identified nine TAS loci, all belonging to the conserved TAS3 family. Information from RNA deep sequencing and PARE analyses identified the tasiR-ARFs as the major functional ta-siRNAs in the maize vegetative apex where they regulate expression of AUXIN RESPONSE FACTOR3 (ARF3) homologs. Plants expressing a tasiR-ARF insensitive arf3a transgene recapitulate the phenotype of lbl1, providing direct evidence that deregulation of ARF3 transcription factors underlies the developmental defects of maize ta-siRNA biogenesis mutants. The phenotypes of Arabidopsis and Medicago ta-siRNA mutants, while strikingly different, likewise result from misexpression of the tasiR-ARF target ARF3. Our data indicate that diversity in TAS pathways and their targets cannot fully account for the phenotypic differences conditioned by ta-siRNA biogenesis mutants across plant species. Instead, we propose that divergence in the gene networks downstream of the ARF3 transcription factors or the spatiotemporal pattern during leaf development in which these proteins act constitute key factors underlying the distinct contributions of the ta-siRNA pathway to development in maize, Arabidopsis, and possibly other plant species as well.

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Organization of TAS3 loci generating phased 21-nt ta-siRNAs.(A, B) Normalized read counts (RPM) for ta-siRNAs in phase with the 3′ miR390 cleavage site (top graphs) and for out of phase small RNAs (bottom graphs) are shown for tas3c (A) and tas3e (B). Red bars, miR390 binding sites; vertical dashed lines, the 21-nt register. Annotations of the TAS3 precursors are shown beneath, with alignments of miR390 to the tas3c and tas3e precursors and the number of PARE signatures in the small window (WS) over the large window (WL) at the 3′ miR390 target sites indicated. Black brackets; ta-siRNAs detected in the vegetative apex libraries; dashed lines, predicted ta-siRNAs not detected in our libraries; red brackets, miR390 binding sites; green bars, tasiR-ARFs. (C) Size distribution profiles for small RNAs derived from tas3c and tas3e showing most are 21-nt long. Values shown are the mean normalized read counts (RPM) and SD from three independent biological replicates. (D) Alignment of the eleven tasiR-ARFs processed from the maize TAS3 loci. Asterisks indicate 100% identity.
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pgen-1004826-g002: Organization of TAS3 loci generating phased 21-nt ta-siRNAs.(A, B) Normalized read counts (RPM) for ta-siRNAs in phase with the 3′ miR390 cleavage site (top graphs) and for out of phase small RNAs (bottom graphs) are shown for tas3c (A) and tas3e (B). Red bars, miR390 binding sites; vertical dashed lines, the 21-nt register. Annotations of the TAS3 precursors are shown beneath, with alignments of miR390 to the tas3c and tas3e precursors and the number of PARE signatures in the small window (WS) over the large window (WL) at the 3′ miR390 target sites indicated. Black brackets; ta-siRNAs detected in the vegetative apex libraries; dashed lines, predicted ta-siRNAs not detected in our libraries; red brackets, miR390 binding sites; green bars, tasiR-ARFs. (C) Size distribution profiles for small RNAs derived from tas3c and tas3e showing most are 21-nt long. Values shown are the mean normalized read counts (RPM) and SD from three independent biological replicates. (D) Alignment of the eleven tasiR-ARFs processed from the maize TAS3 loci. Asterisks indicate 100% identity.

Mentions: A closer analysis of the three novel phased siRNA loci indicates that these represent new members of the TAS3 family. Transcripts from these loci contain two miR390 binding sites and have the potential to generate small RNAs homologous to tasiR-ARFs (Fig. 2A-D; S4A-G Figure). As mentioned above, two additional low copy regions in the genome (GRMZM2G155490 and GRMZM2G588623) generate 21-nt LBL1-dependent small RNAs from predicted non-coding transcripts (Fig. 1C; S1 Dataset). While both loci did not pass the P-score filter in the phased siRNA analysis (S2 Dataset), a closer analysis indicates that both contain two miR390 binding sites (S4D, F Figure). Both loci generate relatively few small RNAs in vegetative apices with many being out of phase (S4 D, F Figure), presenting a likely explanation for the observed low P-score. Supporting this, a similar P-score analysis of 21-nt small RNAs in Arabidopsis failed to detect the confirmed TAS4 ta-siRNA locus due to the low abundance of its reads [21]. Data from PARE (parallel analysis of RNA ends; [35]) libraries generated from B73 apices, which allow the detection of small RNA-directed mRNA cleavage products, confirms that the 3′ miR390 target site in transcripts generated from all nine loci is cleaved (Fig. 2A-B). As such, the new loci were named tas3e to tas3i (Table 1).


Genome-wide analysis of leafbladeless1-regulated and phased small RNAs underscores the importance of the TAS3 ta-siRNA pathway to maize development.

Dotto MC, Petsch KA, Aukerman MJ, Beatty M, Hammell M, Timmermans MC - PLoS Genet. (2014)

Organization of TAS3 loci generating phased 21-nt ta-siRNAs.(A, B) Normalized read counts (RPM) for ta-siRNAs in phase with the 3′ miR390 cleavage site (top graphs) and for out of phase small RNAs (bottom graphs) are shown for tas3c (A) and tas3e (B). Red bars, miR390 binding sites; vertical dashed lines, the 21-nt register. Annotations of the TAS3 precursors are shown beneath, with alignments of miR390 to the tas3c and tas3e precursors and the number of PARE signatures in the small window (WS) over the large window (WL) at the 3′ miR390 target sites indicated. Black brackets; ta-siRNAs detected in the vegetative apex libraries; dashed lines, predicted ta-siRNAs not detected in our libraries; red brackets, miR390 binding sites; green bars, tasiR-ARFs. (C) Size distribution profiles for small RNAs derived from tas3c and tas3e showing most are 21-nt long. Values shown are the mean normalized read counts (RPM) and SD from three independent biological replicates. (D) Alignment of the eleven tasiR-ARFs processed from the maize TAS3 loci. Asterisks indicate 100% identity.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263373&req=5

pgen-1004826-g002: Organization of TAS3 loci generating phased 21-nt ta-siRNAs.(A, B) Normalized read counts (RPM) for ta-siRNAs in phase with the 3′ miR390 cleavage site (top graphs) and for out of phase small RNAs (bottom graphs) are shown for tas3c (A) and tas3e (B). Red bars, miR390 binding sites; vertical dashed lines, the 21-nt register. Annotations of the TAS3 precursors are shown beneath, with alignments of miR390 to the tas3c and tas3e precursors and the number of PARE signatures in the small window (WS) over the large window (WL) at the 3′ miR390 target sites indicated. Black brackets; ta-siRNAs detected in the vegetative apex libraries; dashed lines, predicted ta-siRNAs not detected in our libraries; red brackets, miR390 binding sites; green bars, tasiR-ARFs. (C) Size distribution profiles for small RNAs derived from tas3c and tas3e showing most are 21-nt long. Values shown are the mean normalized read counts (RPM) and SD from three independent biological replicates. (D) Alignment of the eleven tasiR-ARFs processed from the maize TAS3 loci. Asterisks indicate 100% identity.
Mentions: A closer analysis of the three novel phased siRNA loci indicates that these represent new members of the TAS3 family. Transcripts from these loci contain two miR390 binding sites and have the potential to generate small RNAs homologous to tasiR-ARFs (Fig. 2A-D; S4A-G Figure). As mentioned above, two additional low copy regions in the genome (GRMZM2G155490 and GRMZM2G588623) generate 21-nt LBL1-dependent small RNAs from predicted non-coding transcripts (Fig. 1C; S1 Dataset). While both loci did not pass the P-score filter in the phased siRNA analysis (S2 Dataset), a closer analysis indicates that both contain two miR390 binding sites (S4D, F Figure). Both loci generate relatively few small RNAs in vegetative apices with many being out of phase (S4 D, F Figure), presenting a likely explanation for the observed low P-score. Supporting this, a similar P-score analysis of 21-nt small RNAs in Arabidopsis failed to detect the confirmed TAS4 ta-siRNA locus due to the low abundance of its reads [21]. Data from PARE (parallel analysis of RNA ends; [35]) libraries generated from B73 apices, which allow the detection of small RNA-directed mRNA cleavage products, confirms that the 3′ miR390 target site in transcripts generated from all nine loci is cleaved (Fig. 2A-B). As such, the new loci were named tas3e to tas3i (Table 1).

Bottom Line: Interestingly, in contrast to data from other plant species, we found no evidence for the existence of phased siRNAs generated via the one-hit model.The phenotypes of Arabidopsis and Medicago ta-siRNA mutants, while strikingly different, likewise result from misexpression of the tasiR-ARF target ARF3.Instead, we propose that divergence in the gene networks downstream of the ARF3 transcription factors or the spatiotemporal pattern during leaf development in which these proteins act constitute key factors underlying the distinct contributions of the ta-siRNA pathway to development in maize, Arabidopsis, and possibly other plant species as well.

View Article: PubMed Central - PubMed

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

ABSTRACT
Maize leafbladeless1 (lbl1) encodes a key component in the trans-acting short-interfering RNA (ta-siRNA) biogenesis pathway. Correlated with a great diversity in ta-siRNAs and the targets they regulate, the phenotypes conditioned by mutants perturbing this small RNA pathway vary extensively across species. Mutations in lbl1 result in severe developmental defects, giving rise to plants with radial, abaxialized leaves. To investigate the basis for this phenotype, we compared the small RNA content between wild-type and lbl1 seedling apices. We show that LBL1 affects the accumulation of small RNAs in all major classes, and reveal unexpected crosstalk between ta-siRNA biogenesis and other small RNA pathways regulating transposons. Interestingly, in contrast to data from other plant species, we found no evidence for the existence of phased siRNAs generated via the one-hit model. Our analysis identified nine TAS loci, all belonging to the conserved TAS3 family. Information from RNA deep sequencing and PARE analyses identified the tasiR-ARFs as the major functional ta-siRNAs in the maize vegetative apex where they regulate expression of AUXIN RESPONSE FACTOR3 (ARF3) homologs. Plants expressing a tasiR-ARF insensitive arf3a transgene recapitulate the phenotype of lbl1, providing direct evidence that deregulation of ARF3 transcription factors underlies the developmental defects of maize ta-siRNA biogenesis mutants. The phenotypes of Arabidopsis and Medicago ta-siRNA mutants, while strikingly different, likewise result from misexpression of the tasiR-ARF target ARF3. Our data indicate that diversity in TAS pathways and their targets cannot fully account for the phenotypic differences conditioned by ta-siRNA biogenesis mutants across plant species. Instead, we propose that divergence in the gene networks downstream of the ARF3 transcription factors or the spatiotemporal pattern during leaf development in which these proteins act constitute key factors underlying the distinct contributions of the ta-siRNA pathway to development in maize, Arabidopsis, and possibly other plant species as well.

Show MeSH
Related in: MedlinePlus