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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

HLP1 directs poly(A) site choice at the FCA transcripts. (A) The genomic structures of FCA transcripts are shown in the top panel. CDS regions are boxed in black. 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-qPCR amplified region. Wiggle plots of two HITS-CLIP/CLIP-seq (CLIP-1 and CLIP-2) and two PAS-seq (PAS-1 and PAS-2) replicates are shown below the gene structure. HLP1 binding peaks in the third intron and 3′-UTR of FCA transcripts are indicated by blue and purple wiggle plots. The x axis indicates genome site in chromosome. The y axis indicates normalized HITS-CLIP/CLIP-seq or PAS-seq abundance. HITS-CLIP/CLIP-seq or PAS-seq tag counts were normalized to tag per 10 million (TP10M) to adjust for differences of two libraries (wild-type and mutant) in sequencing depth. PAS-Seq analyses show decreased PAC numbers at the distal 3′-UTR poly(A) site in hlp1-1 mutant (red plots in PAS-1 and brown plots in PAS-2) compared with Col (light blue plots in PAS-1 and green plots in PAS-2). (B) RIP-qPCR validation of HLP1 binding to the third intron and 3′-UTR of FCA transcripts. (C) Quantitative PCR results show decreased APA at the distal poly(A) site of FCA in hlp1-1 mutant. (D) Working model for HLP1-directed APA of FCA in the regulation of flowering.
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fig5: HLP1 directs poly(A) site choice at the FCA transcripts. (A) The genomic structures of FCA transcripts are shown in the top panel. CDS regions are boxed in black. 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-qPCR amplified region. Wiggle plots of two HITS-CLIP/CLIP-seq (CLIP-1 and CLIP-2) and two PAS-seq (PAS-1 and PAS-2) replicates are shown below the gene structure. HLP1 binding peaks in the third intron and 3′-UTR of FCA transcripts are indicated by blue and purple wiggle plots. The x axis indicates genome site in chromosome. The y axis indicates normalized HITS-CLIP/CLIP-seq or PAS-seq abundance. HITS-CLIP/CLIP-seq or PAS-seq tag counts were normalized to tag per 10 million (TP10M) to adjust for differences of two libraries (wild-type and mutant) in sequencing depth. PAS-Seq analyses show decreased PAC numbers at the distal 3′-UTR poly(A) site in hlp1-1 mutant (red plots in PAS-1 and brown plots in PAS-2) compared with Col (light blue plots in PAS-1 and green plots in PAS-2). (B) RIP-qPCR validation of HLP1 binding to the third intron and 3′-UTR of FCA transcripts. (C) Quantitative PCR results show decreased APA at the distal poly(A) site of FCA in hlp1-1 mutant. (D) Working model for HLP1-directed APA of FCA in the regulation of flowering.

Mentions: Among the genes whose APA is regulated by HLP1, we found that HLP1 binds directly to the 3′-UTR and the third intron of FCA transcripts (Figure 5A and 5B). Lesion in HLP1 caused upregulated retention of the third intron, which contains the proximal poly(A) site (Figure 5A). PAS-Seq analysis showed decreased polyadenylation at the distal poly(A) site in FCA transcripts in hlp1-1 mutant, resulting in reduced FCA-γ functional isoform, and this shift was rescued in a GFP-HLP1 complementary line (Figure 5A and 5C). This reduction releases the suppression of the floral repressor FLC in hlp1-1 mutant, which leads to delayed flowering (Figures 1E and 5D).


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)

HLP1 directs poly(A) site choice at the FCA transcripts. (A) The genomic structures of FCA transcripts are shown in the top panel. CDS regions are boxed in black. 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-qPCR amplified region. Wiggle plots of two HITS-CLIP/CLIP-seq (CLIP-1 and CLIP-2) and two PAS-seq (PAS-1 and PAS-2) replicates are shown below the gene structure. HLP1 binding peaks in the third intron and 3′-UTR of FCA transcripts are indicated by blue and purple wiggle plots. The x axis indicates genome site in chromosome. The y axis indicates normalized HITS-CLIP/CLIP-seq or PAS-seq abundance. HITS-CLIP/CLIP-seq or PAS-seq tag counts were normalized to tag per 10 million (TP10M) to adjust for differences of two libraries (wild-type and mutant) in sequencing depth. PAS-Seq analyses show decreased PAC numbers at the distal 3′-UTR poly(A) site in hlp1-1 mutant (red plots in PAS-1 and brown plots in PAS-2) compared with Col (light blue plots in PAS-1 and green plots in PAS-2). (B) RIP-qPCR validation of HLP1 binding to the third intron and 3′-UTR of FCA transcripts. (C) Quantitative PCR results show decreased APA at the distal poly(A) site of FCA in hlp1-1 mutant. (D) Working model for HLP1-directed APA of FCA in the regulation of flowering.
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Related In: Results  -  Collection

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fig5: HLP1 directs poly(A) site choice at the FCA transcripts. (A) The genomic structures of FCA transcripts are shown in the top panel. CDS regions are boxed in black. 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-qPCR amplified region. Wiggle plots of two HITS-CLIP/CLIP-seq (CLIP-1 and CLIP-2) and two PAS-seq (PAS-1 and PAS-2) replicates are shown below the gene structure. HLP1 binding peaks in the third intron and 3′-UTR of FCA transcripts are indicated by blue and purple wiggle plots. The x axis indicates genome site in chromosome. The y axis indicates normalized HITS-CLIP/CLIP-seq or PAS-seq abundance. HITS-CLIP/CLIP-seq or PAS-seq tag counts were normalized to tag per 10 million (TP10M) to adjust for differences of two libraries (wild-type and mutant) in sequencing depth. PAS-Seq analyses show decreased PAC numbers at the distal 3′-UTR poly(A) site in hlp1-1 mutant (red plots in PAS-1 and brown plots in PAS-2) compared with Col (light blue plots in PAS-1 and green plots in PAS-2). (B) RIP-qPCR validation of HLP1 binding to the third intron and 3′-UTR of FCA transcripts. (C) Quantitative PCR results show decreased APA at the distal poly(A) site of FCA in hlp1-1 mutant. (D) Working model for HLP1-directed APA of FCA in the regulation of flowering.
Mentions: Among the genes whose APA is regulated by HLP1, we found that HLP1 binds directly to the 3′-UTR and the third intron of FCA transcripts (Figure 5A and 5B). Lesion in HLP1 caused upregulated retention of the third intron, which contains the proximal poly(A) site (Figure 5A). PAS-Seq analysis showed decreased polyadenylation at the distal poly(A) site in FCA transcripts in hlp1-1 mutant, resulting in reduced FCA-γ functional isoform, and this shift was rescued in a GFP-HLP1 complementary line (Figure 5A and 5C). This reduction releases the suppression of the floral repressor FLC in hlp1-1 mutant, which leads to delayed flowering (Figures 1E and 5D).

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