Mechanism Of Splicing Regulation Research Articles

From computational models of the splicing code to regulatory mechanisms and therapeutic implications.

Since the discovery of RNA splicing and its role in gene expression, researchers have sought a set of rules, an algorithm or a computational model that could predict the splice isoforms, and their frequencies, produced from any transcribed gene in a specific cellular context. Over the past 30 years, these models have evolved from simple position weight matrices to deep-learning models capable of integrating sequence data across vast genomic distances. Most recently, new model architectures are moving the field closer to context-specific alternative splicing predictions, and advances in sequencing technologies are expanding the type of data that can be used to inform and interpret such models. Together, these developments are driving improved understanding of splicing regulatory mechanisms and emerging applications of the splicing code to the rational design of RNA- and splicing-based therapeutics.

Immune response stability to the SARS-CoV-2 mRNA vaccine booster is influenced by differential splicing of HLA genes

Many molecular mechanisms that lead to the host antibody response to COVID-19 vaccines remain largely unknown. In this study, we used serum antibody detection combined with whole blood RNA-based transcriptome analysis to investigate variability in vaccine response in healthy recipients of a booster (third) dose schedule of the mRNA BNT162b2 vaccine against COVID-19. The cohort was divided into two groups: (1) low-stable individuals, with antibody concentration anti-SARS-CoV IgG S1 below 0.4 percentile at 180 days after boosting vaccination; and (2) high-stable individuals, with antibody values greater than 0.6 percentile of the range in the same period (median 9525 [185–80,000] AU/mL). Differential gene expression, expressed single nucleotide variants and insertions/deletions, differential splicing events, and allelic imbalance were explored to broaden our understanding of the immune response sustenance. Our analysis revealed a differential expression of genes with immunological functions in individuals with low antibody titers, compared to those with higher antibody titers, underscoring the fundamental importance of the innate immune response for boosting immunity. Our findings also provide new insights into the determinants of the immune response variability to the SARS-CoV-2 mRNA vaccine booster, highlighting the significance of differential splicing regulatory mechanisms, mainly concerning HLA alleles, in delineating vaccine immunogenicity.

The role of alternative splicing in lung cancer.

Aberrant alternative splicing (AS) events are frequently observed in lung cancer, which can be attributed to aberrant gene AS, alterations in splicing regulatory factors, or changes in splicing regulatory mechanisms. Consequently, the dysregulation of alternative RNA splicing is the fundamental cause of lung cancer. In this review, we have summarized the pivotal role of AS in the development, progression, invasion, metastasis, angiogenesis, and drug resistance of lung cancer. Ultimately, this review emphasizes the potential of AS as biomarkers in lung cancer prognosis and diagnosis, and introduces some applications of AS isoform in the treatment of lung cancer. The comprehension of the AS may provide a glimmer of hope for the eradication of lung cancer.

Position-dependent effects of hnRNP A1/A2 in SMN1/2 exon7 splicing

Heterogeneous nuclear ribonucleoprotein A1 and A2 (hnRNP A1/2) is a ubiquitously expressed RNA binding protein known to bind intronic or exonic splicing silencer. Binding of hnRNP A1/2 to survival of motor neuron gene (SMN1/2) exon 7 and flanking sequences strongly inhibits the inclusion of exon 7, which causes spinal muscular atrophy, a common genetic disorder. However, the role of hnRNP A1/2 on the side away from exon 7 is unclear. Here using antisense oligonucleotides, we fished an intronic splicing enhancer (ISE) near the 3′-splice site (SS) of intron 7 of SMN1/2. Mutagenesis identified the efficient motif of the ISE as “UAGUAGG”, coupled with RNA pull down and protein overexpression, we proved that hnRNP A1/2 binding to the ISE promotes the inclusion of SMN1/2 exon 7. Using MS2-tethering array and “UAGGGU” motif walking, we further uncovered that effects of hnRNP A1/2 on SMN1/2 exon 7 splicing are position-dependent: exon 7 inclusion is inhibited when hnRNP A1/2 binds proximal to the 5′SS of intron 7, promoted when its binds proximal to the 3′SS. These data provide new insights into the splicing regulatory mechanism of SMN1/2.

Non-canonical splice junction processing increases the diversity of RBFOX2 splicing isoforms.

The underlying mechanisms of splicing regulation through non-canonical splice junction processing remain largely unknown. Here, we identified two RBFOX2 splicing isoforms by alternative 3' splice site selection of exon 9; the non-canonical splice junction processed RBFOX2 transcript (RBFOX2-N.C.) was expressed by the selection of the 3' splice GG acceptor sequence. The cytoplasmic localization of RBFOX2-C., a canonical splice junction-processed RBFOX2 transcript, was different from that of RBFOX2-N.C., which showed nuclear localization. In addition, we confirmed that RBFOX2-C. showed a significantly stronger localization into stress granules than RBFOX2-N.C. upon sodium arsenite treatment. Next, we investigated the importance of non-canonical 3' splice GG sequence selection of specific cis-regulatory elements using minigene constructs of the RBFOX2 gene. We found that the non-canonical 3' splice GG sequence and suboptimal branch point site adjacent region were critical for RBFOX2-N.C. expression through a non-canonical 3' splice selection. Our results suggest a regulatory mechanism for the non-canonical 3' splice selection in the RBFOX2 gene, providing a basis for studies related to the regulation of alternative pre-mRNA splicing through non-canonical splice junction processing.

Transcriptome-wide changes in gene expression, splicing, and lncRNAs in response to a live attenuated dengue virus vaccine.

SUMMARYThe tetravalent dengue vaccine candidate, TAK-003, induces a functional antibody response, but the titers of antibodies against the four serotypes of the dengue virus (DENV) can vary. Here, through a transcriptomic analysis on whole blood collected from recipients of a two-dose schedule of TAK-003, we examine gene expression, splicing, and transcript isoform-level changes for both protein-coding and noncoding genes to broaden our understanding of the immune response. Our analysis reveals a dynamic pattern of vaccine-associated regulation of long noncoding RNAs (lncRNAs), differential splicing of interferon-stimulated gene exons, and gene expression changes related to multiple signaling pathways that detect viral infection. Co-expression networks isolate immune cell-type-related and interferon-response modules that represent specific biological processes that correlate with more robust antibody responses. These data provide insights into the early determinants of the variable immune response to the vaccine, highlighting the significance of splicing and isoform-level gene regulatory mechanisms in defining vaccine immunogenicity.

Spliceostatin A interaction with SF3B limits U1 snRNP availability and causes premature cleavage and polyadenylation

RNA splicing, a highly conserved process in eukaryotic gene expression, is seen as a promising target for anticancer agents. Splicing is associated with other RNA processing steps, such as transcription and nuclear export; however, our understanding of the interaction between splicing and other RNA regulatory mechanisms remains incomplete. Moreover, the impact of chemical splicing inhibition on long non-coding RNAs (lncRNAs) has been poorly understood. Here, we demonstrate that spliceostatin A (SSA), a chemical splicing modulator that binds to the SF3B subcomplex of the U2 small nuclear ribonucleoprotein particle (snRNP), limits U1 snRNP availability in splicing, resulting in premature cleavage and polyadenylation of MALAT1, a nuclear lncRNA, as well as protein-coding mRNAs. Therefore, truncated transcripts are exported into the cytoplasm and translated, resulting in aberrant protein products. Our work demonstrates that active recycling of the splicing machinery maintains homeostasis of RNA processing beyond intron excision.

JMJD6 Is a Druggable Oxygenase That Regulates AR-V7 Expression in Prostate Cancer.

Endocrine resistance (EnR) in advanced prostate cancer is fatal. EnR can be mediated by androgen receptor (AR) splice variants, with AR splice variant 7 (AR-V7) arguably the most clinically important variant. In this study, we determined proteins key to generating AR-V7, validated our findings using clinical samples, and studied splicing regulatory mechanisms in prostate cancer models. Triangulation studies identified JMJD6 as a key regulator of AR-V7, as evidenced by its upregulation with in vitro EnR, its downregulation alongside AR-V7 by bromodomain inhibition, and its identification as a top hit of a targeted siRNA screen of spliceosome-related genes. JMJD6 protein levels increased (P < 0.001) with castration resistance and were associated with higher AR-V7 levels and shorter survival (P = 0.048). JMJD6 knockdown reduced prostate cancer cell growth, AR-V7 levels, and recruitment of U2AF65 to AR pre-mRNA. Mutagenesis studies suggested that JMJD6 activity is key to the generation of AR-V7, with the catalytic machinery residing within a druggable pocket. Taken together, these data highlight the relationship between JMJD6 and AR-V7 in advanced prostate cancer and support further evaluation of JMJD6 as a therapeutic target in this disease. SIGNIFICANCE: This study identifies JMJD6 as being critical for the generation of AR-V7 in prostate cancer, where it may serve as a tractable target for therapeutic intervention.

Allele-specific expression of Parkinson\u2019s disease susceptibility genes in human brain

Genome-wide association studies have identified genetic variation in genomic loci associated with susceptibility to Parkinson’s disease (PD), the most common neurodegenerative movement disorder worldwide. We used allelic expression profiling of genes located within PD-associated loci to identify cis-regulatory variation affecting gene expression. DNA and RNA were extracted from post-mortem superior frontal gyrus tissue and whole blood samples from PD patients and controls. The relative allelic expression of transcribed SNPs in 12 GWAS risk genes was analysed by real-time qPCR. Allele-specific expression was identified for 9 out of 12 genes tested (GBA, TMEM175, RAB7L1, NUCKS1, MCCC1, BCKDK, ZNF646, LZTS3, and WDHD1) in brain tissue samples. Three genes (GPNMB, STK39 and SIPA1L2) did not show significant allele-specific effects. Allele-specific effects were confirmed in whole blood for three genes (BCKDK, LZTS3 and MCCC1), whereas two genes (RAB7L1 and NUCKS1) showed brain-specific allelic expression. Our study supports the hypothesis that changes to the cis-regulation of gene expression is a major mechanism behind a large proportion of genetic associations in PD. Interestingly, allele-specific expression was also observed for coding variants believed to be causal variants (GBA and TMEM175), indicating that splicing and other regulatory mechanisms may be involved in disease development.

CPEB3 regulates neuron-specific alternative splicing and involves neurogenesis gene expression

In the mammalian brain, alternative pre-mRNA splicing is a fundamental mechanism that modifies neuronal function dynamically where secretion of different splice variants regulates neurogenesis, development, pathfinding, maintenance, migration, and synaptogenesis. Sequence-specific RNA-Binding Protein CPEB3 has distinctive isoform-distinct biochemical interactions and neuronal development assembly roles. Nonetheless, the mechanisms moderating splice isoform options remain unclear. To establish the modulatory trend of CPEB3, we cloned and excessively expressed CPEB3 in HT22 cells. We used RNA-seq to analyze CPEB3-regulated alternative splicing on control and CPEB3-overexpressing cells. Consequently, we used iRIP-seq to identify CPEB-binding targets. We additionally validated CPEB3-modulated genes using RT-qPCR. CPEB3 overexpression had insignificant effects on gene expression in HT22 cells. Notably, CPEB3 partially modulated differential gene splicing enhanced in the modulation of neural development, neuron cycle, neurotrophin, synapse, and specific development pathway, implying an alternative splicing regulatory mechanism associated with neurogenesis. Moreover, qRT-PCR verified the CPEB3-modulated transcription of neurogenesis genes LCN2 and NAV2, synaptogenesis gene CYLD, as well as neural development gene JADE1. Herein, we established that CPEB3 is a critical modulator of alternative splicing in neurogenesis, which remarkably enhances the current understanding of the CPEB3 mediated alternative pre-mRNA splicing.

Defective minor spliceosomes induce SMA-associated phenotypes through sensitive intron-containing neural genes in Drosophila

The minor spliceosome is evolutionarily conserved in higher eukaryotes, but its biological significance remains poorly understood. Here, by precise CRISPR/Cas9-mediated disruption of the U12 and U6atac snRNAs, we report that a defective minor spliceosome is responsible for spinal muscular atrophy (SMA) associated phenotypes in Drosophila. Using a newly developed bioinformatic approach, we identified a large set of minor spliceosome-sensitive splicing events and demonstrate that three sensitive intron-containing neural genes, Pcyt2, Zmynd10, and Fas3, directly contribute to disease development as evidenced by the ability of their cDNAs to rescue the SMA-associated phenotypes in muscle development, neuromuscular junctions, and locomotion. Interestingly, many splice sites in sensitive introns are recognizable by both minor and major spliceosomes, suggesting a new mechanism of splicing regulation through competition between minor and major spliceosomes. These findings reveal a vital contribution of the minor spliceosome to SMA and to regulated splicing in animals.

Phytophthora Effectors Modulate Genome-wide Alternative Splicing of Host mRNAs to Reprogram Plant Immunity

Alternative splicing (AS) of pre-mRNAs increases transcriptome and proteome diversity, regulates gene expression through multiple mechanisms, and plays important roles in plant development and stress responses. However, the prevalence of genome-wide plant AS changes during infection and the mechanisms by which pathogens modulate AS remain poorly understood. Here, we examined the global AS changes in tomato leaves infected with Phytophthora infestans, the infamous Irish famine pathogen. We show that more than 2000 genes exhibiting significant changes in AS are not differentially expressed, indicating that AS is a distinct layer of transcriptome reprogramming during plant–pathogen interactions. Furthermore, our results show that P. infestans subverts host immunity by repressing the AS of positive regulators of plant immunity and promoting the AS of susceptibility factors. To study the underlying mechanism, we established a luminescence-based AS reporter system in Nicotiana benthamiana to screen pathogen effectors modulating plant AS. We identified nine splicing regulatory effectors (SREs) from 87 P. infestans effectors. Further studies revealed that SRE3 physically binds U1-70K to manipulate the plant AS machinery and subsequently modulates AS-mediated plant immunity. Our study not only unveils genome-wide plant AS reprogramming during infection but also establishes a novel AS screening tool to identify SREs from a wide range of plant pathogens, providing opportunities to understand the splicing regulatory mechanisms through which pathogens subvert plant immunity.

Prognostic alternative splicing regulatory network of splicing events in acute myeloid leukemia patients based on SpliceSeq data from 136 cases.

This study aimed to create prognostic signatures to predict AML patients' survival using alternative splicing (AS) events. The AS data, RNA sequencing data, and the survival statistics of 136 AML patients were obtained from The Cancer Genome Atlas (TCGA) and TCGA SpliceSeq databases. Total 34,984 AS events generated from 8,656 genes, 2,583 of which were survival-associated AS events, were identified using univariate Cox regression. The prognostic models constructed using independent survival-associated AS events revealed that low-risk splicing better predicted patients' survival. ROC analysis indicated that the predictive efficacy of the alternate terminator model was best in the area under the curve at 0.781. Enrichment analysis revealed several important genes (TP53, BCL2, AURKB, PPP2R1B, FOS, and BIRC5) and pathways, such as the protein processing pathway in the endoplasmic reticulum, RNA transport pathway, and HTLV-I infection pathway. The splicing network of splicing events and factors revealed interesting interactions, such as the positive correlation between HNRNPH3 and CALHM2-13010-AT, which may indicate the potential splicing regulatory mechanism. Taken together, survival-associated splicing events and the prognostic signatures for predicting survival can help provide an overview of splicing in AML patients and facilitate clinical practice. The splicing regulatory network may improve the understanding of spliceosomes in AML.

Genome-wide profiling of prognosis-related alternative splicing signatures in sarcoma.

Sarcomas (SARCs) are rare malignant tumors with poor prognosis. Increasing evidence has suggested that aberrant alternative splicing (AS) is strongly associated with tumor initiation and progression. We considered whether survival-related AS events might serve as prognosis predictors and underlying targeted molecules in SARC treatment. RNA-Seq data of the SARC cohort were downloaded from The Cancer Genome Atlas (TCGA) database. Survival-related AS events were selected by univariate and multivariate Cox regression analyses. Metascape was used for constructing a gene interaction network and performing functional enrichment analysis. Then, prognosis predictors were established based on statistically significant survival-related AS events and evaluated by receiver operator characteristic (ROC) curve analysis. Finally, the potential regulatory network was analyzed via Pearson's correlation between survival-related AS events and splicing factors (SFs). A total of 3,610 AS events and 2,291 genes were found to be prognosis-related in 261 SARC samples. The focal adhesion pathway was identified as the most critical molecular mechanism corresponding to poor prognosis. Notably, several prognosis predictors based on survival-related AS events showed excellent performance in prognosis prediction. The area under the curve of the ROC of the risk score was 0.85 in the integrated predictor. The splicing network proved complicated regulation between prognosis-related SFs and AS events. Also, driver gene mutations were significantly associated with AS in SARC patients. Survival-related AS events may become ideal indictors for the prognosis prediction of SARCs. Corresponding splicing regulatory mechanisms are worth further exploration.

Differential isoform expression and alternative splicing in sex determination in mice

BackgroundAlternative splicing (AS) may play an important role in gonadal sex determination (GSD) in mammals. The present study was designed to identify differentially expressed isoforms and AS modifications accompanying GSD in mice.ResultsUsing deep RNA-sequencing, we performed a transcriptional analysis of XX and XY gonads during sex determination on embryonic days 11 (E11) and 12 (E12). Analysis of differentially expressed genes (DEG) identified hundreds of genes related to GSD and early sex differentiation that may represent good candidates for sex reversal. Expression at time point E11 in males was significantly enriched in RNA splicing and mRNA processing Gene Ontology terms. Differentially expressed isoform analysis identified hundreds of specific isoforms related to GSD, many of which showed no differences in the DEG analysis. Hundreds of AS events were identified as modified at E11 and E12. Female E11 gonads featured sex-biased upregulation of intron retention (in genes related to regulation of transcription, protein phosphorylation, protein transport and mRNA splicing) and exon skipping (in genes related to chromatin repression) suggesting AS as a post-transcription mechanism that controls sex determination of the bipotential fetal gonad.ConclusionOur data suggests an important role of splicing regulatory mechanisms for sex determination in mice.

The Role of Transposable Elements in Emergence of Metazoa.

Systems initially emerged for protecting genomes against insertions of transposable elements and represented by mechanisms of splicing regulation, RNA-interference, and epigenetic factors have played a key role in the evolution of animals. Many studies have shown inherited transpositions of mobile elements in embryogenesis and preservation of their activities in certain tissues of adult organisms. It was supposed that on the emergence of Metazoa the self-regulation mechanisms of transposons related with the gene networks controlling their activity could be involved in intercellular cell coordination in the cascade of successive divisions with differentiated gene expression for generation of tissues and organs. It was supposed that during evolution species-specific features of transposons in the genomes of eukaryotes could form the basis for creation of dynamically related complexes of systems for epigenetic regulation of gene expression. These complexes could be produced due to the influence of noncoding transposon-derived RNAs on DNA methylation, histone modifications, and processing of alternative splicing variants, whereas the mobile elements themselves could be directly involved in the regulation of gene expression in cis and in trans. Transposons are widely distributed in the genomes of eukaryotes; therefore, their activation can change the expression of specific genes. In turn, this can play an important role in cell differentiation during ontogenesis. It is supposed that transposons can form a species-specific pattern for control of gene expression, and that some variants of this pattern can be favorable for adaptation. The presented data indicate the possible influence of transposons in karyotype formation. It is supposed that transposon localization relative to one another and to protein-coding genes can influence the species-specific epigenetic regulation of ontogenesis.

Development of an in vitro pre-mRNA splicing assay using plant nuclear extract

BackgroundPre-mRNA splicing is an essential post-transcriptional process in all eukaryotes. In vitro splicing systems using nuclear or cytoplasmic extracts from mammalian cells, yeast, and Drosophila have provided a wealth of mechanistic insights into assembly and composition of the spliceosome, splicing regulatory proteins and mechanisms of pre-mRNA splicing in non-plant systems. The lack of an in vitro splicing system prepared from plant cells has been a major limitation in splicing research in plants.ResultsHere we report an in vitro splicing assay system using plant nuclear extract. Several lines of evidence indicate that nuclear extract derived from Arabidopsis seedlings can convert pre-mRNA substrate (LHCB3) into a spliced product. These include: (1) generation of an RNA product that corresponds to the size of expected mRNA, (2) a junction-mapping assay using S1 nuclease revealed that the two exons are spliced together, (3) the reaction conditions are similar to those found with non-plant extracts and (4) finally mutations in conserved donor and acceptor sites abolished the production of the spliced product.ConclusionsThis first report on the plant in vitro splicing assay opens new avenues to investigate plant spliceosome assembly and composition, and splicing regulatory mechanisms specific to plants.

The prevalent deep intronic c. 639+919 G>A GLA mutation causes pseudoexon activation and Fabry disease by abolishing the binding of hnRNPA1 and hnRNP A2/B1 to a splicing silencer.

Fabry disease is an X-linked recessive inborn disorder of the glycosphingolipid metabolism, caused by total or partial deficiency of the lysosomal α-galactosidase A enzyme due to mutations in the GLA gene. The prevalent c.639+919 G>A mutation in GLA leads to pathogenic insertion of a 57bp pseudoexon sequence from intron 4, which is responsible for the cardiac variant phenotype. In this study we investigate the splicing regulatory mechanism leading to GLA pseudoexon activation. Splicing analysis of GLA minigenes revealed that pseudoexon activation is influenced by cell-type. We demonstrate that the wild-type sequence harbors an hnRNP A1 and hnRNP A2/B1-binding exonic splicing silencer (ESS) overlapping the 5'splice site (5'ss) that prevents pseudoexon inclusion. The c.639+919 G>A mutation disrupts this ESS allowing U1 snRNP recognition of the 5'ss. We show that the wild-type GLA 5'ss motif with the ESS is also able to inhibit inclusion of an unrelated pseudoexon in the FGB gene, and that also in the FGB context inactivation of the ESS by the c.639+919 G>A mutation causes pseudoexon activation, underscoring the universal nature of the ESS. Finally, we demonstrate that splice switching oligonucleotide (SSO) mediated blocking of the pseudoexon 3'ss and 5'ss effectively restores normal GLA splicing. This indicates that SSO based splicing correction may be a therapeutic alternative in the treatment of Fabry disease.

The Erythroid Intron Retention Program Encompasses Developmentally Stable and Dynamic Networks and Regulates Diverse Gene Classes

Computational analysis of RNA-seq data from highly purified human erythroblasts has been instrumental in revealing changes in pre-mRNA splicing during terminal erythropoiesis. Here we report updated studies of intron retention (IR), a type of alternative splicing in which specific introns are retained in otherwise efficiently-processed transcripts, allowing post-transcriptional modulation of cellular mRNA levels. Differences in differentiation stage-specificity, degree of retention, nuclear/cytoplasmic localization, and sensitivity to nonsense-mediated decay (NMD) suggest the existence of multiple classes of erythroblast IR subject to distinct regulatory controls. Two clusters comprising ~470 "developmentally dynamic" introns in 354 genes exhibit more efficient splicing in proerythroblasts, but elevated intron retention in orthochromatic erythroblasts prior to enucleation. Dynamic regulation of late erythroblast IR parallels previously described splicing switches involving alternative exons. Gene ontology analysis revealed that the dynamic intron group is highly enriched in genes with RNA processing functions. Among these are several spliceosomal factors including SF3B1, a commonly mutated gene in myelodysplasia patients. We also identified several clusters of "developmentally stable" introns whose IR levels are not substantially modulated during erythropoiesis. Among this latter type are two clusters containing 294 introns that are enriched in functions related to metal ion binding. Key genes include mitoferrin-1 (SC25A37; IR~50%) and mitoferrin-2 (SLC25A28; IR~20-30%), mitochondrial iron importers essential for heme biosynthesis. We observed a correlation between splice site strength and percent IR among developmentally stable but not dynamic intron clusters, indicating that splicing regulatory mechanism(s) for the latter must require additional sequence features. A search for such features revealed that IR was significantly higher adjacent to alternative 'PTC' exons containing premature termination codons than it was adjacent to other exons; moreover, by direct RT-PCR analysis we discovered novel (unannotated) PTC exons in additional retained introns. The proposed role of PTC exons in IR is being studied experimentally using an array of minigene splicing reporter constructs. Finally, we noted that while specific IR events are erythroid specific, e.g., in the alpha spectrin gene SPTA1, computational analysis of public RNA-seq data demonstrated that most erythroblast IR events were also observed in granulocytes and in 16 other tissues surveyed by the human BodyMap project. Intron retention is likely to play critical roles in gene regulation in both hematological and non-hematological tissues. DisclosuresNo relevant conflicts of interest to declare.

Genetic regulation of flowering time in annual and perennial plants

Flowering time plays a significant role in the reproductive success of plants. So far, five major pathways to flowering have been characterized in Arabidopsis, including environmental induction through photoperiod, vernalization, and gibberellins and autonomous floral iation, and aging by sequentially operating miRNAs (typically miR156 and miR172) responding to endogenous cues. The balance of signals from these pathways is integrated by a common set of genes (FLOWERING LOCUS C, FLOWERING LOCUS T, LEAFY, and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1) that determine the flowering time. Recent studies have indicated that epigenetic modification, alternative splicing, antisense RNA and chromatin silencing regulatory mechanisms play an important role in this process by regulating related flowering gene expression. In this review, we discuss the current understanding in genetic regulation of the phase transition from vegetative to reproductive growth by using Arabidopsis as a model. We also describe how this knowledge has been successfully applied for identifying homologous genes from perennial crops. Furthermore, detailed analysis of the similarities and differences between annual and perennial plants flowering will help elucidate the mechanisms of perennial plant maturation and regulation of floral initiation.