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Evidence for antisense transcription associated with microRNA target mRNAs in Arabidopsis.

Luo QJ, Samanta MP, Köksal F, Janda J, Galbraith DW, Richardson CR, Ou-Yang F, Rock CD - PLoS Genet. (2009)

Bottom Line: Antisense smRNAs were also found associated with MIRNA genes.Results showed that antisense transcripts associated with miRNA targets were mainly elevated in hen1-1 and sgs3-14 to a lesser extent, and somewhat reduced in dcl11-7, hyl11-2, or rdr6-15 mutants.Our overall analysis reveals a more widespread role for miRNA-associated transitivity with implications for functions of antisense transcription in gene regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Texas Tech University, Lubbock, Texas, USA.

ABSTRACT
Antisense transcription is a pervasive phenomenon, but its source and functional significance is largely unknown. We took an expression-based approach to explore microRNA (miRNA)-related antisense transcription by computational analyses of published whole-genome tiling microarray transcriptome and deep sequencing small RNA (smRNA) data. Statistical support for greater abundance of antisense transcription signatures and smRNAs was observed for miRNA targets than for paralogous genes with no miRNA cleavage site. Antisense smRNAs were also found associated with MIRNA genes. This suggests that miRNA-associated "transitivity" (production of small interfering RNAs through antisense transcription) is more common than previously reported. High-resolution (3 nt) custom tiling microarray transcriptome analysis was performed with probes 400 bp 5' upstream and 3' downstream of the miRNA cleavage sites (direction relative to the mRNA) for 22 select miRNA target genes. We hybridized RNAs labeled from the smRNA pathway mutants, including hen1-1, dcl1-7, hyl1-2, rdr6-15, and sgs3-14. Results showed that antisense transcripts associated with miRNA targets were mainly elevated in hen1-1 and sgs3-14 to a lesser extent, and somewhat reduced in dcl11-7, hyl11-2, or rdr6-15 mutants. This was corroborated by semi-quantitative reverse transcription PCR; however, a direct correlation of antisense transcript abundance in MIR164 gene knockouts was not observed. Our overall analysis reveals a more widespread role for miRNA-associated transitivity with implications for functions of antisense transcription in gene regulation. HEN1 and SGS3 may be links for miRNA target entry into different RNA processing pathways.

Show MeSH
Average topology of sense and antisense transcript signals spanning miRNA target sites.(A) Validated miRNA targets (n = 78); (B) Predicted miRNA targets (n = 188); (C) miRNA genes (n = 159); (D) paralogous non-targets (n = 120). Data was collected from two published whole genome tiling microarray experiments with five samples from Arabidopsis flowers, leaves, roots, and two suspension cultures [11],[13]. For validated and predicted targets, each data point on the plot is the average of the normalized total signal from five tissue samples spanning 800 n.t. upstream and downstream of the validated or predicted miRNA cleavage sites. For MIRNA genes or paralogous non-targets, data for the same length of region spanning miRNA* sites or pseudo-binding sites was plotted. Signals on the sense strand are indicated by gray line and open arrow, while antisense signals are displayed by black line and black arrow. In panel A, antisense signals within the 200 n.t. range upstream (black arrow) and sense signals within the 200 n.t. range downstream (open arrow) of the miRNA cleavage site (coordinate 0 on x-axis) for validated targets have significantly higher signal intensity than elsewhere on the plot and than those in the same region of paralogs (95% confidence interval, see Tables S3 and S4). In panel B, antisense signals within the 200 n.t. range upstream of the predicted miRNA cleavage site (black arrow) is also statistically higher than those in the same region of paralogs.
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pgen-1000457-g001: Average topology of sense and antisense transcript signals spanning miRNA target sites.(A) Validated miRNA targets (n = 78); (B) Predicted miRNA targets (n = 188); (C) miRNA genes (n = 159); (D) paralogous non-targets (n = 120). Data was collected from two published whole genome tiling microarray experiments with five samples from Arabidopsis flowers, leaves, roots, and two suspension cultures [11],[13]. For validated and predicted targets, each data point on the plot is the average of the normalized total signal from five tissue samples spanning 800 n.t. upstream and downstream of the validated or predicted miRNA cleavage sites. For MIRNA genes or paralogous non-targets, data for the same length of region spanning miRNA* sites or pseudo-binding sites was plotted. Signals on the sense strand are indicated by gray line and open arrow, while antisense signals are displayed by black line and black arrow. In panel A, antisense signals within the 200 n.t. range upstream (black arrow) and sense signals within the 200 n.t. range downstream (open arrow) of the miRNA cleavage site (coordinate 0 on x-axis) for validated targets have significantly higher signal intensity than elsewhere on the plot and than those in the same region of paralogs (95% confidence interval, see Tables S3 and S4). In panel B, antisense signals within the 200 n.t. range upstream of the predicted miRNA cleavage site (black arrow) is also statistically higher than those in the same region of paralogs.

Mentions: The high percentage of MPSS normalized antisense signatures for the validated miRNA targets prompted us to perform a systematic survey of antisense transcription for miRNA targets and MIRNA genes. We collectively plotted the sense and antisense transcript abundance as a function of miRNA cleavage sites for validated targets (n = 78), predicted targets (n = 188), non-target paralogs (n = 120), and the miRNA* sites of MIRNA genes (as potential cleavage sites by miRNAs [29], n = 159) (See Text S1 and Table S3). This analysis excluded PPR genes, ARGONAUTE1 (AGO1), DICER-LIKE1 (DCL1) (which harbors MIR838 within intron 14), and the ARF2/3/4 targets of ta-siRNAs derived from miR390 cleavage of TAS3 (AT3G17185), because these are reported evidence for miRNA target-associated transitivity [16],[20],[23],[28]. Figure 1 presents the sense and antisense strand expression as a function of the miRNA target sites. We identified a pair of expression peaks associated with validated miRNA targets flanking the miRNA cleavage site on the sense and antisense strands, which was not seen in paralogs relative to their cryptic pseudo miRNA-binding sites (Figure 1A and D). For the validated targets, an expression peak was observed immediately downstream of the miRNA cleavage site on the sense strand (Figure 1A open arrow, referred to as “downstream sense signal” hereafter). This could be a manifestation of higher stability of the 3′ RISC cleavage fragment for miRNA target mRNAs. This interpretation is consistent with previous reports describing the accumulation of 3′ endonucleolytic cleavage products of miRNA targets by Northern blot [6], reverse genetic analysis [30], and deep sequencing of non-capped polyA+ “degradome” libraries [31],[32],[33]. Associated with this downstream sense signal was an additional peak of transcription signal located in a 200 n.t. region upstream of the miRNA target sites on the antisense strand (Figure 1A black arrow, referred to as “upstream antisense signal” hereafter). Figure S1 provides additional examples of this phenomenon for high downstream-sense coupled to corresponding upstream-antisense transcript signals around the miRNA binding site for twelve different miRNAs, in which target genes also produce smRNAs. For the predicted miRNA targets, an expression pattern similar to that of validated targets was observed spanning the predicted cleavage sites (Figure 1B, open arrow for downstream sense signal and black arrow for upstream antisense signal). Statistical analysis indicated that the downstream sense and upstream antisense signals were significantly higher than the average signal elsewhere on either sense or antisense strand for validated miRNA targets and predicted targets (P<0.01, one-sided Student's t-test, equal variance model; Table S3). The pairs of downstream sense and upstream antisense signals for the validated targets were significantly higher compared to the same region for paralogs (Table S4, 95% confidence interval calculated). In line with the recent report of miR172-mediated cleavage of the pri-miR172b transcripts [29], we observed some sense expression signals immediately downstream of the miRNA* sites of MIRNA genes along with some antisense expression signals immediately upstream of the miRNA* sites (Figure 1C). This implies that MIRNA genes may share the same process of antisense transcription with the validated miRNA targets, possibly by miRNA interaction with miRNA primary transcripts. These observations suggested a causal relationship between miRNA target site regulation and antisense transcripts of miRNA targets and MIRNA genes that warranted further study.


Evidence for antisense transcription associated with microRNA target mRNAs in Arabidopsis.

Luo QJ, Samanta MP, Köksal F, Janda J, Galbraith DW, Richardson CR, Ou-Yang F, Rock CD - PLoS Genet. (2009)

Average topology of sense and antisense transcript signals spanning miRNA target sites.(A) Validated miRNA targets (n = 78); (B) Predicted miRNA targets (n = 188); (C) miRNA genes (n = 159); (D) paralogous non-targets (n = 120). Data was collected from two published whole genome tiling microarray experiments with five samples from Arabidopsis flowers, leaves, roots, and two suspension cultures [11],[13]. For validated and predicted targets, each data point on the plot is the average of the normalized total signal from five tissue samples spanning 800 n.t. upstream and downstream of the validated or predicted miRNA cleavage sites. For MIRNA genes or paralogous non-targets, data for the same length of region spanning miRNA* sites or pseudo-binding sites was plotted. Signals on the sense strand are indicated by gray line and open arrow, while antisense signals are displayed by black line and black arrow. In panel A, antisense signals within the 200 n.t. range upstream (black arrow) and sense signals within the 200 n.t. range downstream (open arrow) of the miRNA cleavage site (coordinate 0 on x-axis) for validated targets have significantly higher signal intensity than elsewhere on the plot and than those in the same region of paralogs (95% confidence interval, see Tables S3 and S4). In panel B, antisense signals within the 200 n.t. range upstream of the predicted miRNA cleavage site (black arrow) is also statistically higher than those in the same region of paralogs.
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Related In: Results  -  Collection

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pgen-1000457-g001: Average topology of sense and antisense transcript signals spanning miRNA target sites.(A) Validated miRNA targets (n = 78); (B) Predicted miRNA targets (n = 188); (C) miRNA genes (n = 159); (D) paralogous non-targets (n = 120). Data was collected from two published whole genome tiling microarray experiments with five samples from Arabidopsis flowers, leaves, roots, and two suspension cultures [11],[13]. For validated and predicted targets, each data point on the plot is the average of the normalized total signal from five tissue samples spanning 800 n.t. upstream and downstream of the validated or predicted miRNA cleavage sites. For MIRNA genes or paralogous non-targets, data for the same length of region spanning miRNA* sites or pseudo-binding sites was plotted. Signals on the sense strand are indicated by gray line and open arrow, while antisense signals are displayed by black line and black arrow. In panel A, antisense signals within the 200 n.t. range upstream (black arrow) and sense signals within the 200 n.t. range downstream (open arrow) of the miRNA cleavage site (coordinate 0 on x-axis) for validated targets have significantly higher signal intensity than elsewhere on the plot and than those in the same region of paralogs (95% confidence interval, see Tables S3 and S4). In panel B, antisense signals within the 200 n.t. range upstream of the predicted miRNA cleavage site (black arrow) is also statistically higher than those in the same region of paralogs.
Mentions: The high percentage of MPSS normalized antisense signatures for the validated miRNA targets prompted us to perform a systematic survey of antisense transcription for miRNA targets and MIRNA genes. We collectively plotted the sense and antisense transcript abundance as a function of miRNA cleavage sites for validated targets (n = 78), predicted targets (n = 188), non-target paralogs (n = 120), and the miRNA* sites of MIRNA genes (as potential cleavage sites by miRNAs [29], n = 159) (See Text S1 and Table S3). This analysis excluded PPR genes, ARGONAUTE1 (AGO1), DICER-LIKE1 (DCL1) (which harbors MIR838 within intron 14), and the ARF2/3/4 targets of ta-siRNAs derived from miR390 cleavage of TAS3 (AT3G17185), because these are reported evidence for miRNA target-associated transitivity [16],[20],[23],[28]. Figure 1 presents the sense and antisense strand expression as a function of the miRNA target sites. We identified a pair of expression peaks associated with validated miRNA targets flanking the miRNA cleavage site on the sense and antisense strands, which was not seen in paralogs relative to their cryptic pseudo miRNA-binding sites (Figure 1A and D). For the validated targets, an expression peak was observed immediately downstream of the miRNA cleavage site on the sense strand (Figure 1A open arrow, referred to as “downstream sense signal” hereafter). This could be a manifestation of higher stability of the 3′ RISC cleavage fragment for miRNA target mRNAs. This interpretation is consistent with previous reports describing the accumulation of 3′ endonucleolytic cleavage products of miRNA targets by Northern blot [6], reverse genetic analysis [30], and deep sequencing of non-capped polyA+ “degradome” libraries [31],[32],[33]. Associated with this downstream sense signal was an additional peak of transcription signal located in a 200 n.t. region upstream of the miRNA target sites on the antisense strand (Figure 1A black arrow, referred to as “upstream antisense signal” hereafter). Figure S1 provides additional examples of this phenomenon for high downstream-sense coupled to corresponding upstream-antisense transcript signals around the miRNA binding site for twelve different miRNAs, in which target genes also produce smRNAs. For the predicted miRNA targets, an expression pattern similar to that of validated targets was observed spanning the predicted cleavage sites (Figure 1B, open arrow for downstream sense signal and black arrow for upstream antisense signal). Statistical analysis indicated that the downstream sense and upstream antisense signals were significantly higher than the average signal elsewhere on either sense or antisense strand for validated miRNA targets and predicted targets (P<0.01, one-sided Student's t-test, equal variance model; Table S3). The pairs of downstream sense and upstream antisense signals for the validated targets were significantly higher compared to the same region for paralogs (Table S4, 95% confidence interval calculated). In line with the recent report of miR172-mediated cleavage of the pri-miR172b transcripts [29], we observed some sense expression signals immediately downstream of the miRNA* sites of MIRNA genes along with some antisense expression signals immediately upstream of the miRNA* sites (Figure 1C). This implies that MIRNA genes may share the same process of antisense transcription with the validated miRNA targets, possibly by miRNA interaction with miRNA primary transcripts. These observations suggested a causal relationship between miRNA target site regulation and antisense transcripts of miRNA targets and MIRNA genes that warranted further study.

Bottom Line: Antisense smRNAs were also found associated with MIRNA genes.Results showed that antisense transcripts associated with miRNA targets were mainly elevated in hen1-1 and sgs3-14 to a lesser extent, and somewhat reduced in dcl11-7, hyl11-2, or rdr6-15 mutants.Our overall analysis reveals a more widespread role for miRNA-associated transitivity with implications for functions of antisense transcription in gene regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Texas Tech University, Lubbock, Texas, USA.

ABSTRACT
Antisense transcription is a pervasive phenomenon, but its source and functional significance is largely unknown. We took an expression-based approach to explore microRNA (miRNA)-related antisense transcription by computational analyses of published whole-genome tiling microarray transcriptome and deep sequencing small RNA (smRNA) data. Statistical support for greater abundance of antisense transcription signatures and smRNAs was observed for miRNA targets than for paralogous genes with no miRNA cleavage site. Antisense smRNAs were also found associated with MIRNA genes. This suggests that miRNA-associated "transitivity" (production of small interfering RNAs through antisense transcription) is more common than previously reported. High-resolution (3 nt) custom tiling microarray transcriptome analysis was performed with probes 400 bp 5' upstream and 3' downstream of the miRNA cleavage sites (direction relative to the mRNA) for 22 select miRNA target genes. We hybridized RNAs labeled from the smRNA pathway mutants, including hen1-1, dcl1-7, hyl1-2, rdr6-15, and sgs3-14. Results showed that antisense transcripts associated with miRNA targets were mainly elevated in hen1-1 and sgs3-14 to a lesser extent, and somewhat reduced in dcl11-7, hyl11-2, or rdr6-15 mutants. This was corroborated by semi-quantitative reverse transcription PCR; however, a direct correlation of antisense transcript abundance in MIR164 gene knockouts was not observed. Our overall analysis reveals a more widespread role for miRNA-associated transitivity with implications for functions of antisense transcription in gene regulation. HEN1 and SGS3 may be links for miRNA target entry into different RNA processing pathways.

Show MeSH