Limits...
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 preferentially binds to the A- and U-rich elements in the 3′-UTR and introns of targeted transcripts. (A) Immunoprecipitation of the HLP1-RNA complex. Positions of the 32P-labeled HLP1ΔRRM-RNA complex (left panel) and the HLP1-RNA complex (right panel) after MNase over-digestion (*) are indicated. The protein-RNA complex in dashed box was purified and used to prepare the library for high-throughput sequencing. (B) Percentages of 5′-UTR, 3′-UTR, intron, CDS, pseudogene, ncexon and intergenic region in Arabidopsis genome (dark grey) and HLP1 CLIP-seq tags (light grey). (C) HLP1 CLIP tags are significantly enriched in 3′-UTR and intron. (D) Distribution and percentage of HLP1 binding sites in genes. Binding sites are shown as wiggle plots on the left (blue for the HLP1 library and dark grey for the ΔRRM library). 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-RT-PCR amplified region. The x axis indicates genome location 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. Right panels show validation of binding by RIP-RT-PCR. (E) HLP1 binds to transcripts of small RNA genes. Binding sites are shown as wiggle plots on the left (blue for the HLP1 library and grey for the ΔRRM library). CDS regions are boxed in black. 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. Right panels show validation of binding by RIP-RT-PCR. (F) Distribution of the two overrepresented binding motifs (insert) relative to the poly(A) site (PAS, indicated as 0) are indicated by blue and red curves, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493284&req=5

fig2: HLP1 preferentially binds to the A- and U-rich elements in the 3′-UTR and introns of targeted transcripts. (A) Immunoprecipitation of the HLP1-RNA complex. Positions of the 32P-labeled HLP1ΔRRM-RNA complex (left panel) and the HLP1-RNA complex (right panel) after MNase over-digestion (*) are indicated. The protein-RNA complex in dashed box was purified and used to prepare the library for high-throughput sequencing. (B) Percentages of 5′-UTR, 3′-UTR, intron, CDS, pseudogene, ncexon and intergenic region in Arabidopsis genome (dark grey) and HLP1 CLIP-seq tags (light grey). (C) HLP1 CLIP tags are significantly enriched in 3′-UTR and intron. (D) Distribution and percentage of HLP1 binding sites in genes. Binding sites are shown as wiggle plots on the left (blue for the HLP1 library and dark grey for the ΔRRM library). 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-RT-PCR amplified region. The x axis indicates genome location 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. Right panels show validation of binding by RIP-RT-PCR. (E) HLP1 binds to transcripts of small RNA genes. Binding sites are shown as wiggle plots on the left (blue for the HLP1 library and grey for the ΔRRM library). CDS regions are boxed in black. 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. Right panels show validation of binding by RIP-RT-PCR. (F) Distribution of the two overrepresented binding motifs (insert) relative to the poly(A) site (PAS, indicated as 0) are indicated by blue and red curves, respectively.

Mentions: To further delineate the underlying mechanism of HLP1 in flowering regulation, we identified HLP1 in vivo targets, using a modified unbiased high-throughput sequencing of RNAs isolated by the cross-linking immunoprecipitation (HITS-CLIP)/CLIP-Seq method31,32, which was first established in animal system to capture and identify RNA target sites bound by a specific RBP. To this end, Arabidopsis seedlings were UV cross-linked to fix the protein-nucleic acid interaction. HLP1-RNA complex was then immunoprecipitated from the GFP-HLP1 transgenic plants using the GFP antibody followed by MNase digestion. A narrow smear above the HLP1 over-digestion control was separated in NuPAGE gel and was used to generate the HITS-CLIP/CLIP-Seq library for high-throughput sequencing. HITS-CLIP/CLIP-Seq reads obtained from both HLP1 and HLP1ΔRRM libraries were analyzed in parallel (Figure 2A). Overall, ∼29 million and ∼24 million raw reads were collected from the HLP1- and ΔRRM-CLIP library, respectively. After subtraction of adaptors, barcodes, multiple mapped reads and PCR duplicates, a total of 2 324 041 final tags from HLP1-CLIP were uniquely mapped to the Arabidopsis genome (TAIR10), whereas only 61 156 final tags from ΔRRM-CLIP were unique (Supplementary information, Table S1). The drastic reduction of final tags in the ΔRRM-CLIP library indicated that the vast majority of RNA binding capacity of HLP1 is contributed by the two RRMs.


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 preferentially binds to the A- and U-rich elements in the 3′-UTR and introns of targeted transcripts. (A) Immunoprecipitation of the HLP1-RNA complex. Positions of the 32P-labeled HLP1ΔRRM-RNA complex (left panel) and the HLP1-RNA complex (right panel) after MNase over-digestion (*) are indicated. The protein-RNA complex in dashed box was purified and used to prepare the library for high-throughput sequencing. (B) Percentages of 5′-UTR, 3′-UTR, intron, CDS, pseudogene, ncexon and intergenic region in Arabidopsis genome (dark grey) and HLP1 CLIP-seq tags (light grey). (C) HLP1 CLIP tags are significantly enriched in 3′-UTR and intron. (D) Distribution and percentage of HLP1 binding sites in genes. Binding sites are shown as wiggle plots on the left (blue for the HLP1 library and dark grey for the ΔRRM library). 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-RT-PCR amplified region. The x axis indicates genome location 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. Right panels show validation of binding by RIP-RT-PCR. (E) HLP1 binds to transcripts of small RNA genes. Binding sites are shown as wiggle plots on the left (blue for the HLP1 library and grey for the ΔRRM library). CDS regions are boxed in black. 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. Right panels show validation of binding by RIP-RT-PCR. (F) Distribution of the two overrepresented binding motifs (insert) relative to the poly(A) site (PAS, indicated as 0) are indicated by blue and red curves, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: HLP1 preferentially binds to the A- and U-rich elements in the 3′-UTR and introns of targeted transcripts. (A) Immunoprecipitation of the HLP1-RNA complex. Positions of the 32P-labeled HLP1ΔRRM-RNA complex (left panel) and the HLP1-RNA complex (right panel) after MNase over-digestion (*) are indicated. The protein-RNA complex in dashed box was purified and used to prepare the library for high-throughput sequencing. (B) Percentages of 5′-UTR, 3′-UTR, intron, CDS, pseudogene, ncexon and intergenic region in Arabidopsis genome (dark grey) and HLP1 CLIP-seq tags (light grey). (C) HLP1 CLIP tags are significantly enriched in 3′-UTR and intron. (D) Distribution and percentage of HLP1 binding sites in genes. Binding sites are shown as wiggle plots on the left (blue for the HLP1 library and dark grey for the ΔRRM library). 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-RT-PCR amplified region. The x axis indicates genome location 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. Right panels show validation of binding by RIP-RT-PCR. (E) HLP1 binds to transcripts of small RNA genes. Binding sites are shown as wiggle plots on the left (blue for the HLP1 library and grey for the ΔRRM library). CDS regions are boxed in black. 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. Right panels show validation of binding by RIP-RT-PCR. (F) Distribution of the two overrepresented binding motifs (insert) relative to the poly(A) site (PAS, indicated as 0) are indicated by blue and red curves, respectively.
Mentions: To further delineate the underlying mechanism of HLP1 in flowering regulation, we identified HLP1 in vivo targets, using a modified unbiased high-throughput sequencing of RNAs isolated by the cross-linking immunoprecipitation (HITS-CLIP)/CLIP-Seq method31,32, which was first established in animal system to capture and identify RNA target sites bound by a specific RBP. To this end, Arabidopsis seedlings were UV cross-linked to fix the protein-nucleic acid interaction. HLP1-RNA complex was then immunoprecipitated from the GFP-HLP1 transgenic plants using the GFP antibody followed by MNase digestion. A narrow smear above the HLP1 over-digestion control was separated in NuPAGE gel and was used to generate the HITS-CLIP/CLIP-Seq library for high-throughput sequencing. HITS-CLIP/CLIP-Seq reads obtained from both HLP1 and HLP1ΔRRM libraries were analyzed in parallel (Figure 2A). Overall, ∼29 million and ∼24 million raw reads were collected from the HLP1- and ΔRRM-CLIP library, respectively. After subtraction of adaptors, barcodes, multiple mapped reads and PCR duplicates, a total of 2 324 041 final tags from HLP1-CLIP were uniquely mapped to the Arabidopsis genome (TAIR10), whereas only 61 156 final tags from ΔRRM-CLIP were unique (Supplementary information, Table S1). The drastic reduction of final tags in the ΔRRM-CLIP library indicated that the vast majority of RNA binding capacity of HLP1 is contributed by the two RRMs.

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