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Integrative genome-wide analysis reveals HLP1, a novel RNA-binding protein, regulates plant flowering by targeting alternative polyadenylation.

Zhang Y, Gu L, Hou Y, Wang L, Deng X, Hang R, Chen D, Zhang X, Zhang Y, Liu C, Cao X - Cell Res. (2015)

Bottom Line: We show HLP1 is significantly enriched at transcripts involved in RNA metabolism and flowering.A distal-to-proximal poly(A) site shift in the flowering regulator FCA, a direct target of HLP1, leads to upregulation of FLC and delayed flowering.Our results elucidate that HLP1 is a novel factor involved in 3'-end processing and controls reproductive timing via targeting APA.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
Alternative polyadenylation (APA) is a widespread mechanism for gene regulation and has been implicated in flowering, but the molecular basis governing the choice of a specific poly(A) site during the vegetative-to-reproductive growth transition remains unclear. Here we characterize HLP1, an hnRNP A/B protein as a novel regulator for pre-mRNA 3'-end processing in Arabidopsis. Genetic analysis reveals that HLP1 suppresses Flowering Locus C (FLC), a key repressor of flowering in Arabidopsis. Genome-wide mapping of HLP1-RNA interactions indicates that HLP1 binds preferentially to A-rich and U-rich elements around cleavage and polyadenylation sites, implicating its role in 3'-end formation. We show HLP1 is significantly enriched at transcripts involved in RNA metabolism and flowering. Comprehensive profiling of the poly(A) site usage reveals that HLP1 mutations cause thousands of poly(A) site shifts. A distal-to-proximal poly(A) site shift in the flowering regulator FCA, a direct target of HLP1, leads to upregulation of FLC and delayed flowering. Our results elucidate that HLP1 is a novel factor involved in 3'-end processing and controls reproductive timing via targeting APA.

No MeSH data available.


Related in: MedlinePlus

Enriched HLP1 binding to floral genes. (A) Gene Ontology (GO) analysis of HLP1 binding targets. Significantly enriched GO terms of genes with HLP1 binding were identified using the BiNGO software (hypergeometric test with Benjamini and Hochberg false discovery rate correction). The x axis indicates the enrichment P-value on a −log10 scale; the y axis indicates number of genes with HLP1 binding on a log2 scale. The size of each point is proportional to the ratio of HLP1-bound genes associated with one GO term to all genes associated with this GO term. (B) Circos diagram shows enriched HLP1 binding sites at flowering-related transcripts: circle 1, Arabidopsis chromosomes (indicated as chr01-05 with different colors); circle 2, heat map displaying all of Arabidopsis genes; circle 3, binding density of HLP1ΔRRM showing very few binding peaks of the truncated HLP1; circle 4, binding density of HLP1 indicating all the HLP1 binding sites across the transcriptome; circle 5, heat map view of genes with HLP1 binding; circle 6, red link lines indicating flowering-related transcripts with HLP1 binding. (C) Examples and validations of flowering associated genes bound by HLP1. Binding sites are shown as wiggle plots on the left. CDS regions are boxed in black and the 5′-UTR and 3′-UTR are boxed in green and grey, respectively. Introns are indicated as lines. Blue line above gene structure indicates RIP-RT-PCR amplified region. The x axis indicates genome site in chromosome. The y axis indicates normalized HITS-CLIP/CLIP-seq abundance. HITS-CLIP/CLIP-seq tag counts were normalized to tag per 10 million (TP10M) to adjust for differences of two HITS-CLIP/CLIP-seq libraries in sequencing depth. Blue for binding peaks at FPA, FLK, GRP7 sense transcripts and orange for peaks at FLC antisense transcripts. Right panels show validation of binding by RIP-RT-PCR.
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fig3: Enriched HLP1 binding to floral genes. (A) Gene Ontology (GO) analysis of HLP1 binding targets. Significantly enriched GO terms of genes with HLP1 binding were identified using the BiNGO software (hypergeometric test with Benjamini and Hochberg false discovery rate correction). The x axis indicates the enrichment P-value on a −log10 scale; the y axis indicates number of genes with HLP1 binding on a log2 scale. The size of each point is proportional to the ratio of HLP1-bound genes associated with one GO term to all genes associated with this GO term. (B) Circos diagram shows enriched HLP1 binding sites at flowering-related transcripts: circle 1, Arabidopsis chromosomes (indicated as chr01-05 with different colors); circle 2, heat map displaying all of Arabidopsis genes; circle 3, binding density of HLP1ΔRRM showing very few binding peaks of the truncated HLP1; circle 4, binding density of HLP1 indicating all the HLP1 binding sites across the transcriptome; circle 5, heat map view of genes with HLP1 binding; circle 6, red link lines indicating flowering-related transcripts with HLP1 binding. (C) Examples and validations of flowering associated genes bound by HLP1. Binding sites are shown as wiggle plots on the left. CDS regions are boxed in black and the 5′-UTR and 3′-UTR are boxed in green and grey, respectively. Introns are indicated as lines. Blue line above gene structure indicates RIP-RT-PCR amplified region. The x axis indicates genome site in chromosome. The y axis indicates normalized HITS-CLIP/CLIP-seq abundance. HITS-CLIP/CLIP-seq tag counts were normalized to tag per 10 million (TP10M) to adjust for differences of two HITS-CLIP/CLIP-seq libraries in sequencing depth. Blue for binding peaks at FPA, FLK, GRP7 sense transcripts and orange for peaks at FLC antisense transcripts. Right panels show validation of binding by RIP-RT-PCR.

Mentions: Gene Ontology analysis of the HLP1 sense binding targets revealed significantly enriched terms in RNA metabolism, flower development, various stimuli and stress responses, suggesting that HLP1 may have profound regulatory roles in these physiological processes (Figure 3A). Consistent with its role in floral transition, HLP1 binds to many transcripts of flowering-related genes, some of which encode RNA-processing factors or RBPs involved in RNA processing (Figure 3B, 3C and Supplementary information, Table S2). We showed that HLP1 binds to FLC antisense transcripts around the proximal polyadenylation site, suggesting a role in FLC antisense processing (Figure 3C, bottom panel).


Integrative genome-wide analysis reveals HLP1, a novel RNA-binding protein, regulates plant flowering by targeting alternative polyadenylation.

Zhang Y, Gu L, Hou Y, Wang L, Deng X, Hang R, Chen D, Zhang X, Zhang Y, Liu C, Cao X - Cell Res. (2015)

Enriched HLP1 binding to floral genes. (A) Gene Ontology (GO) analysis of HLP1 binding targets. Significantly enriched GO terms of genes with HLP1 binding were identified using the BiNGO software (hypergeometric test with Benjamini and Hochberg false discovery rate correction). The x axis indicates the enrichment P-value on a −log10 scale; the y axis indicates number of genes with HLP1 binding on a log2 scale. The size of each point is proportional to the ratio of HLP1-bound genes associated with one GO term to all genes associated with this GO term. (B) Circos diagram shows enriched HLP1 binding sites at flowering-related transcripts: circle 1, Arabidopsis chromosomes (indicated as chr01-05 with different colors); circle 2, heat map displaying all of Arabidopsis genes; circle 3, binding density of HLP1ΔRRM showing very few binding peaks of the truncated HLP1; circle 4, binding density of HLP1 indicating all the HLP1 binding sites across the transcriptome; circle 5, heat map view of genes with HLP1 binding; circle 6, red link lines indicating flowering-related transcripts with HLP1 binding. (C) Examples and validations of flowering associated genes bound by HLP1. Binding sites are shown as wiggle plots on the left. CDS regions are boxed in black and the 5′-UTR and 3′-UTR are boxed in green and grey, respectively. Introns are indicated as lines. Blue line above gene structure indicates RIP-RT-PCR amplified region. The x axis indicates genome site in chromosome. The y axis indicates normalized HITS-CLIP/CLIP-seq abundance. HITS-CLIP/CLIP-seq tag counts were normalized to tag per 10 million (TP10M) to adjust for differences of two HITS-CLIP/CLIP-seq libraries in sequencing depth. Blue for binding peaks at FPA, FLK, GRP7 sense transcripts and orange for peaks at FLC antisense transcripts. Right panels show validation of binding by RIP-RT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493284&req=5

fig3: Enriched HLP1 binding to floral genes. (A) Gene Ontology (GO) analysis of HLP1 binding targets. Significantly enriched GO terms of genes with HLP1 binding were identified using the BiNGO software (hypergeometric test with Benjamini and Hochberg false discovery rate correction). The x axis indicates the enrichment P-value on a −log10 scale; the y axis indicates number of genes with HLP1 binding on a log2 scale. The size of each point is proportional to the ratio of HLP1-bound genes associated with one GO term to all genes associated with this GO term. (B) Circos diagram shows enriched HLP1 binding sites at flowering-related transcripts: circle 1, Arabidopsis chromosomes (indicated as chr01-05 with different colors); circle 2, heat map displaying all of Arabidopsis genes; circle 3, binding density of HLP1ΔRRM showing very few binding peaks of the truncated HLP1; circle 4, binding density of HLP1 indicating all the HLP1 binding sites across the transcriptome; circle 5, heat map view of genes with HLP1 binding; circle 6, red link lines indicating flowering-related transcripts with HLP1 binding. (C) Examples and validations of flowering associated genes bound by HLP1. Binding sites are shown as wiggle plots on the left. CDS regions are boxed in black and the 5′-UTR and 3′-UTR are boxed in green and grey, respectively. Introns are indicated as lines. Blue line above gene structure indicates RIP-RT-PCR amplified region. The x axis indicates genome site in chromosome. The y axis indicates normalized HITS-CLIP/CLIP-seq abundance. HITS-CLIP/CLIP-seq tag counts were normalized to tag per 10 million (TP10M) to adjust for differences of two HITS-CLIP/CLIP-seq libraries in sequencing depth. Blue for binding peaks at FPA, FLK, GRP7 sense transcripts and orange for peaks at FLC antisense transcripts. Right panels show validation of binding by RIP-RT-PCR.
Mentions: Gene Ontology analysis of the HLP1 sense binding targets revealed significantly enriched terms in RNA metabolism, flower development, various stimuli and stress responses, suggesting that HLP1 may have profound regulatory roles in these physiological processes (Figure 3A). Consistent with its role in floral transition, HLP1 binds to many transcripts of flowering-related genes, some of which encode RNA-processing factors or RBPs involved in RNA processing (Figure 3B, 3C and Supplementary information, Table S2). We showed that HLP1 binds to FLC antisense transcripts around the proximal polyadenylation site, suggesting a role in FLC antisense processing (Figure 3C, bottom panel).

Bottom Line: We show HLP1 is significantly enriched at transcripts involved in RNA metabolism and flowering.A distal-to-proximal poly(A) site shift in the flowering regulator FCA, a direct target of HLP1, leads to upregulation of FLC and delayed flowering.Our results elucidate that HLP1 is a novel factor involved in 3'-end processing and controls reproductive timing via targeting APA.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
Alternative polyadenylation (APA) is a widespread mechanism for gene regulation and has been implicated in flowering, but the molecular basis governing the choice of a specific poly(A) site during the vegetative-to-reproductive growth transition remains unclear. Here we characterize HLP1, an hnRNP A/B protein as a novel regulator for pre-mRNA 3'-end processing in Arabidopsis. Genetic analysis reveals that HLP1 suppresses Flowering Locus C (FLC), a key repressor of flowering in Arabidopsis. Genome-wide mapping of HLP1-RNA interactions indicates that HLP1 binds preferentially to A-rich and U-rich elements around cleavage and polyadenylation sites, implicating its role in 3'-end formation. We show HLP1 is significantly enriched at transcripts involved in RNA metabolism and flowering. Comprehensive profiling of the poly(A) site usage reveals that HLP1 mutations cause thousands of poly(A) site shifts. A distal-to-proximal poly(A) site shift in the flowering regulator FCA, a direct target of HLP1, leads to upregulation of FLC and delayed flowering. Our results elucidate that HLP1 is a novel factor involved in 3'-end processing and controls reproductive timing via targeting APA.

No MeSH data available.


Related in: MedlinePlus