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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 regulates flowering. (A) Gene structure of HLP1. CDS regions are boxed in black, and the 5′ and 3′ untranslated regions are in green and grey, respectively. Introns are indicated as lines. The T-DNA insertion is indicated with a triangle. Primer pairs (CX578/CX579) for amplifying the full-length CDS of HLP1 are indicated by arrows. Bar = 500 bp. (B) RT-PCR using CX578 and CX579 primer pairs (top panel) and western blot using anti-HLP1 antibody (the third panel) show complete absence of HLP1 full-length transcript and protein in the hlp1-1 mutant. Actin and RLSU (RuBisCo Large SubUnit) were used as loading controls. (C) The hlp1-1 mutant shows late-flowering phenotype. (D) Flowering time of Col and hlp1-1 mutant under different conditions or treatments. Flowering time was assessed by the total leaf number of the plants at bolting under long-day photoperiods (LD), vernalization (LD+Ver), short-day photoperiods (SD) and gibberellin (GA) treatment (SD+GA). SD+EtOH treatment was used as a control for GA treatment. 'X' indicates that counting was terminated after the plants had produced over 100 rosette leaves before bolting. The error bars indicate standard deviation. (E) HLP1 regulates flowering in an FLC-dependent manner. Total leaf number at flowering is shown for each indicated strain. * means statistical significance by t-test. Error bars indicate SD. Expression levels of FLC sense transcripts were detected by northern blot. HLP1 protein levels were assessed by immunoblotting using anti-HLP1 antibody. Actin and HSC70 served as loading controls for the northern blot and immunoblot, respectively.
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fig1: HLP1 regulates flowering. (A) Gene structure of HLP1. CDS regions are boxed in black, and the 5′ and 3′ untranslated regions are in green and grey, respectively. Introns are indicated as lines. The T-DNA insertion is indicated with a triangle. Primer pairs (CX578/CX579) for amplifying the full-length CDS of HLP1 are indicated by arrows. Bar = 500 bp. (B) RT-PCR using CX578 and CX579 primer pairs (top panel) and western blot using anti-HLP1 antibody (the third panel) show complete absence of HLP1 full-length transcript and protein in the hlp1-1 mutant. Actin and RLSU (RuBisCo Large SubUnit) were used as loading controls. (C) The hlp1-1 mutant shows late-flowering phenotype. (D) Flowering time of Col and hlp1-1 mutant under different conditions or treatments. Flowering time was assessed by the total leaf number of the plants at bolting under long-day photoperiods (LD), vernalization (LD+Ver), short-day photoperiods (SD) and gibberellin (GA) treatment (SD+GA). SD+EtOH treatment was used as a control for GA treatment. 'X' indicates that counting was terminated after the plants had produced over 100 rosette leaves before bolting. The error bars indicate standard deviation. (E) HLP1 regulates flowering in an FLC-dependent manner. Total leaf number at flowering is shown for each indicated strain. * means statistical significance by t-test. Error bars indicate SD. Expression levels of FLC sense transcripts were detected by northern blot. HLP1 protein levels were assessed by immunoblotting using anti-HLP1 antibody. Actin and HSC70 served as loading controls for the northern blot and immunoblot, respectively.

Mentions: To investigate the biological roles of HLP1, we isolated a T-DNA insertion line of HLP1, hlp1-1 (Figure 1A). RT-PCR and immunoblot results demonstrated that there was no expression of HLP1 in the hlp1-1 mutant (Figure 1B). Lesions in HLP1 result in late-flowering phenotype with increased FLC transcripts (Figure 1C and 1E). Under both long- and short-day photoperiods, the hlp1-1 mutant flowered late and showed normal responses to vernalization and GA treatments (Figure 1D). The late-flowering phenotype was suppressed when crossed to flc-3, a allele of FLC21, and was reversed by introducing GFP-fused HLP1 full length CDS into hlp1-1 (Figure 1E). Collectively, our data suggest that HLP1 promotes floral transition in an FLC-dependent manner. As HLP1 contains two RRMs, we also investigated the impact of the RRMs on flowering time by overexpressing GFP-HLP1ΔRRM (HLP1 lacking the two RRMs) and GFP-RRM in hlp1-1 mutants, respectively. None of these transgenic plants repressed FLC transcription and rescued the delayed-flowering phenotype of hlp1-1, indicating that the RRMs are required but insufficient for flowering (Supplementary information, Figure S2).


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 regulates flowering. (A) Gene structure of HLP1. CDS regions are boxed in black, and the 5′ and 3′ untranslated regions are in green and grey, respectively. Introns are indicated as lines. The T-DNA insertion is indicated with a triangle. Primer pairs (CX578/CX579) for amplifying the full-length CDS of HLP1 are indicated by arrows. Bar = 500 bp. (B) RT-PCR using CX578 and CX579 primer pairs (top panel) and western blot using anti-HLP1 antibody (the third panel) show complete absence of HLP1 full-length transcript and protein in the hlp1-1 mutant. Actin and RLSU (RuBisCo Large SubUnit) were used as loading controls. (C) The hlp1-1 mutant shows late-flowering phenotype. (D) Flowering time of Col and hlp1-1 mutant under different conditions or treatments. Flowering time was assessed by the total leaf number of the plants at bolting under long-day photoperiods (LD), vernalization (LD+Ver), short-day photoperiods (SD) and gibberellin (GA) treatment (SD+GA). SD+EtOH treatment was used as a control for GA treatment. 'X' indicates that counting was terminated after the plants had produced over 100 rosette leaves before bolting. The error bars indicate standard deviation. (E) HLP1 regulates flowering in an FLC-dependent manner. Total leaf number at flowering is shown for each indicated strain. * means statistical significance by t-test. Error bars indicate SD. Expression levels of FLC sense transcripts were detected by northern blot. HLP1 protein levels were assessed by immunoblotting using anti-HLP1 antibody. Actin and HSC70 served as loading controls for the northern blot and immunoblot, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: HLP1 regulates flowering. (A) Gene structure of HLP1. CDS regions are boxed in black, and the 5′ and 3′ untranslated regions are in green and grey, respectively. Introns are indicated as lines. The T-DNA insertion is indicated with a triangle. Primer pairs (CX578/CX579) for amplifying the full-length CDS of HLP1 are indicated by arrows. Bar = 500 bp. (B) RT-PCR using CX578 and CX579 primer pairs (top panel) and western blot using anti-HLP1 antibody (the third panel) show complete absence of HLP1 full-length transcript and protein in the hlp1-1 mutant. Actin and RLSU (RuBisCo Large SubUnit) were used as loading controls. (C) The hlp1-1 mutant shows late-flowering phenotype. (D) Flowering time of Col and hlp1-1 mutant under different conditions or treatments. Flowering time was assessed by the total leaf number of the plants at bolting under long-day photoperiods (LD), vernalization (LD+Ver), short-day photoperiods (SD) and gibberellin (GA) treatment (SD+GA). SD+EtOH treatment was used as a control for GA treatment. 'X' indicates that counting was terminated after the plants had produced over 100 rosette leaves before bolting. The error bars indicate standard deviation. (E) HLP1 regulates flowering in an FLC-dependent manner. Total leaf number at flowering is shown for each indicated strain. * means statistical significance by t-test. Error bars indicate SD. Expression levels of FLC sense transcripts were detected by northern blot. HLP1 protein levels were assessed by immunoblotting using anti-HLP1 antibody. Actin and HSC70 served as loading controls for the northern blot and immunoblot, respectively.
Mentions: To investigate the biological roles of HLP1, we isolated a T-DNA insertion line of HLP1, hlp1-1 (Figure 1A). RT-PCR and immunoblot results demonstrated that there was no expression of HLP1 in the hlp1-1 mutant (Figure 1B). Lesions in HLP1 result in late-flowering phenotype with increased FLC transcripts (Figure 1C and 1E). Under both long- and short-day photoperiods, the hlp1-1 mutant flowered late and showed normal responses to vernalization and GA treatments (Figure 1D). The late-flowering phenotype was suppressed when crossed to flc-3, a allele of FLC21, and was reversed by introducing GFP-fused HLP1 full length CDS into hlp1-1 (Figure 1E). Collectively, our data suggest that HLP1 promotes floral transition in an FLC-dependent manner. As HLP1 contains two RRMs, we also investigated the impact of the RRMs on flowering time by overexpressing GFP-HLP1ΔRRM (HLP1 lacking the two RRMs) and GFP-RRM in hlp1-1 mutants, respectively. None of these transgenic plants repressed FLC transcription and rescued the delayed-flowering phenotype of hlp1-1, indicating that the RRMs are required but insufficient for flowering (Supplementary information, Figure S2).

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