Molecular Dissection Of Arabidopsis RNA Binding Proteins HLP1 And HLP2 In Regulation Of Flowering Time

Flowering is a critical developmental process that sets the transition from vegetative to reproductive development in plants.The time of flowering not only guarantees seed production for propagation of population but also affects crops yield and quality.Regulation of flowering time in plants is complicated and elaborative,it refers to integrating environmental signals such as temperature and photoperiod with endogenous stimuli of plant development.The expression of a series of genes contributes to regulating the timing of flowering,ensuring that the plants accomplish the floral transition at favourable conditions.According to recent literatures,in addition to four classical flowering pathways,a large variety of RNA binding proteins or RNA processing factors that are involved in RNA post-transcriptional processing also play a role in flowering time control.RNA binding proteins(RBPs)are abundant in eukaryotes and have crucial roles in almost all aspects of RNA metabolisms,including 5?-capping,alternative splicing,RNA editing,3?-end polyadenylation,the shuttle of nucleus/cytoplasm and stability.Due to their roles in controlling all aspects of RNA fate,RBPs are of great importance in gene expression regulation and organism development.The Arabidopsis(Arabidopsis thaliana)genome encodes about 200 RBPs and more and more researches focusing on plant RBPs cleraly showed that,RBPs have an important role in flowering time transition,stress response and chromatin modification.Pre-mRNA 3?-end polyadenylation is a critical step in eukaryotic genes expression and regulation.The event of 3?-end polyadenylation is composed of two close steps: an endonucleolytic cleavege at pre-mRNA ploy(A)sites followed by the addition of a poly(A)tail.The poly(A)sites are versatile and this phenomenon is called alternative polyadenylation(APA).Pre-m RNA can generate transcript isoforms with different coding sequences or alternative 3?-UTR via APA,to enlarge the diversity and complexity of transcriptome.A number of recent discoveries using transcriptome-wide sequencing have revealed that APA is widely existing in multiple organisms and most genes contain more than one poly(A)sites,and more than 70% Arabidopsis genes have been reported to display APA.Increasing evidence support the notion that APA-directed gene expression participates in flowering time control and environmental response.The molecular mechanisms of APA regulation is intricated,and refers to numerous and dynamic cross-talks between 3?-processing factors and cis-elements,but remains to be elucidated in plants.To understand the molecular basis of plant flowering,a T-DNA knockout mutant with At5G40490 mutation was screened,and this gene encodes a RNA binding protein.Phylogenetic analysis revealed that it shares similarity with human hnRNP A1 and yeast Hrp1.This protein was designated as HLP1.To understand the underlying mechanism of HLP1 function,CLIP-seq was carried out to identify HLP1 in vivo targets at a transcriptome-wide level.Bioinformatics analysis indicated that HLP1-tags showed a broad range of binding sites to transcripts,and the authenticity of HLP1-RNA binding was confirmed by RIP-qRT-PCR.Markedly,HLP1-tags were significantly enriched in 3?-UTR and introns.Unbiased search for motifs enriched in HLP1 binding sites revealed over-represented A-rich(5?-AGAAAA-3?)and U-rich(5?-UUUUCU-3?)sequences predominantly near the poly(A)sites.Gene Ontology analysis of the HLP1 binding targets revealed significantly enriched terms in flower development.HLP1 binding motifs feature is extremely similar to poly(A)signals,indicating that HLP1 may participate in APA regulation.PAS-seq revealed that the defects of HLP1 mutation caused plentiful APA shifts.Bioinformatics analysis combined with CLIP-seq showed that a portion of transcripts with APA shift were HLP1 binding targets,implying that HLP1 directly regulates APA.Among the HLP1 targets with APA shift,HLP1 bound directly to the 3?-UTR and the third intron of FCA transcripts,and that promotes the distal poly(A)sites usage to generate functional transcript,and eventually promotes plant flowering by repressing FLC expression.Sequence alignment revealed that there was a paralog of HLP1 in Arabidopsis.Both of them contained two RRM domains in N terminal,so we named the paralog as HLP2.Sub-cellular location assay indicated that both HLP1 and HLP2 located in nucleus,and GUS activity analysis found that HLP1 and HLP2 had the same expression pattern,predicting there is a functional correlation between HLP1 and HLP2.Phenotype analysis showed FLC expression level was up-regulated in hlp2-1 mutant,indicating that HLP2 regulates flowering time in a FLC-dependent way.In addition,we failed to obtain hlp1hlp2 double mutant beacause the embryo was lethal in hlp1hlp2 double mutant,implying the significance of HLP1 and HLP2 in plant development.Taking the role of HLP1 in APA regulation into consideration,and taking At3G15450 and At3G23030 as APA shift cases,we found that the mutation of HLP2 led to similar APA shift with that of hlp1-1 mutant,suggesting that both HLP1 and HLP2 are invovled in APA regulation in a synergetic manner.Further analysis demonstrated that both HLP1 and HLP2 bound directly to the 3?-UTR and the third intron of FCA transcripts,cooperatively promoting the distal poly(A)sites usage to generate functional transcripts.Biochemistry assay proved HLP1 and HLP2 could form a heterodimer to perform biological function in vivo.Interestingly,HLP1 negatively modulated HLP2 expression by regulating HLP2 transcript APA.HLP2 pre-mRNAdisplays APA phenomenon.HLP1 bound directly to HLP2 pre-mRNA and supressed selection of distal poly(A)sites,resulting in the reduction of the functional transcript isoform production.In summary,our results demonstrated that two novel RNA binding proteins HLP1 and HLP2 in Arabidopsis bound directly to certain RNA targets to control the selection of poly(A)sites in a synergetic mode to regulate flowering time.The results in this study shed new light on the role of RBPs-directed RNA processing in plant development.