BackgroundLight is one of the most important factors regulating plant growth and development. Light-sensing photoreceptors tightly regulate gene expression to control photomorphogenic responses. Although many levels of gene expression are modulated by photoreceptors, regulation at the mRNA splicing step remains unclear.ResultsWe performed high-throughput mRNA sequencing to analyze light-responsive changes in alternative splicing in the moss Physcomitrella patens, and found that a large number of alternative splicing events were induced by light in the moss protonema. Light-responsive intron retention preferentially occurred in transcripts involved in photosynthesis and translation. Many of the alternatively spliced transcripts were expressed from genes with a function relating to splicing or light signaling, suggesting a potential impact on pre-mRNA splicing and photomorphogenic gene regulation in response to light. Moreover, most light-regulated intron retention was induced immediately upon light exposure, while motif analysis identified a repetitive GAA motif that may function as an exonic regulatory cis element in light-mediated alternative splicing. Further analysis in gene-disrupted mutants was consistent with a function for multiple red-light photoreceptors in the upstream regulation of light-responsive alternative splicing.ConclusionsOur results indicate that intensive alternative splicing occurs in non-vascular plants and that, during photomorphogenesis, light regulates alternative splicing with transcript selectivity. We further suggest that alternative splicing is rapidly fine-tuned by light to modulate gene expression and reorganize metabolic processes, and that pre-mRNA cis elements are involved in photoreceptor-mediated splicing regulation.

1 ,3 2 -diene system to produce an ethylidene group for assembly with apophytochromes. In this study, we sought to determine the catalytic mechanism of HY2. Data from UV-visible and EPR spectroscopy showed that the HY2-catalyzed BV reaction proceeds via a transient radical intermediate. Site-directed mutagenesis showed several ionizable residues that are involved in the catalytic steps. Detailed analysis of these sitedirected mutants highlighted a pair of aspartate residues central to proton donation and substrate positioning. A mechanistic prediction for the HY2 reaction is proposed. These results support the hypothesis that ferredoxin-dependent bilin reductases reduce BV through a radical mechanism, but their double bond specificity is decided by strategic placement of different protondonating residues surrounding the bilin substrate in the active sites. Phytochromobilin (P⌽B)2 is an open chain tetrapyrrole chromophore critical for light-sensing phytochromes to regulate growth and development of plants. The phytochromebound P⌽B absorbs light energy and proceeds with reversible structural rearrangement to alter the biochemical activities of phytochrome. P⌽B is covalently linked to apophytochrome through a thioether bond between a conserved cysteine residue on the apoprotein and the ethylidene group on the A-ring of P⌽B (1). The biosynthesis of P⌽B has been shown to reside in the plastids, where heme is first linearized by a heme oxygenase into the reaction intermediate biliverdin IX␣ (BV) and then subsequently reduced by a P⌽B synthase (2-5).In Arabidopsis, the HY2 (LONG HYPOCOTYL 2) gene encodes the P⌽B synthase (EC 1.3.7.4), which catalyzes the ferredoxin-dependent reaction of double bond reduction at the A-ring 2,3,3 1 ,3 2 -diene system of BV to yield 3Z/3E-P⌽B (Fig. 1A) (2). Mutations in HY2 have been shown to severely affect photomorphogenetic processes due to the loss of all functional phytochromes (6, 7). Many HY2-related proteins have been identified from various oxygenic photosynthetic organisms and collectively named ferredoxin-dependent bilin reductases (FDBRs) (8). FDBRs utilize reduced ferredoxin as the electron donor to reduce BV into different biliprotein chromophores with different double bond specificities. Phycocyanobilin:ferredoxin oxidoreductase (PcyA; EC 1.3.7.5) catalyzes two double bond reductions of BV on A-and D-rings to yield phycocyanobilin. 15,16-Dihydrobiliverdin:ferredoxin oxidoreductase (PebA; EC 1.3.7.2) and phycoerythrobilin (PEB):ferredoxin oxidoreductase (PebB; EC 1.3.7.3) work together to reduce the C-15-C-16 and A-ring double bond to produce PEB (8). A recently identified PEB synthase (PebS) can itself catalyze the two double bond reductions to yield PEB (9).PcyA is the most extensively studied FDBR enzyme. Previous biochemical analysis has identified a two-electron reduced intermediate, 181 ,18 2 -dihydrobiliverdin IX␣, present in the PcyA reaction, indicating that D-ring reduction precedes A-ring reduction (Fig. 1B) (10). Two organic radical intermediates have also been ...

Plants perceive environmental light conditions and optimize their growth and development accordingly by regulating gene activity at multiple levels. Photoreceptors are important for light sensing and downstream gene regulation. Phytochromes, red/far-red light receptors, are believed to regulate light-responsive alternative splicing, but little is known about the underlying mechanism. Alternative splicing is primarily regulated by transacting factors, such as splicing regulators, and by cis-acting elements in precursor mRNA. In the moss Physcomitrella patens, we show that phytochrome 4 (PpPHY4) directly interacts with a splicing regulator, heterogeneous nuclear ribonucleoprotein F1 (PphnRNP-F1), in the nucleus to regulate light-responsive alternative splicing. RNA sequencing analysis revealed that PpPHY4 and PphnRNP-F1 coregulate 70% of intron retention (IR) events in response to red light. A repetitive GAA motif was identified to be an exonic splicing silencer that controls red light-responsive IR. Biochemical studies indicated that PphnRNP-F1 is recruited by the GAA motif to form RNA-protein complexes. Finally, red light elevates PphnRNP-F1 protein levels via PpPHY4, increasing levels of IR. We propose that PpPHY4 and PphnRNP-F1 regulate alternative splicing through an exonic splicing silencer to control splicing machinery activity in response to light.

Colorectal cancer is one of the most prevalent and lethal malignancies, affecting approximately 900,000 individuals each year worldwide. Patients with colorectal cancer are found with elevated serum interleukin-6 (IL-6), which is associated with advanced tumor grades and is related to their poor survival outcomes. Although IL-6 is recognized as a potent inducer of colorectal cancer progression, the detail mechanisms underlying IL-6-induced colorectal cancer epithelial–mesenchymal transition (EMT), one of the major process of tumor metastasis, remain unclear. In the present study, we investigated the regulatory role of IL-6 signaling in colorectal cancer EMT using HCT116 human colorectal cancer cells. We noted that the expression of epithelial marker E-cadherin was reduced in HCT116 cells exposed to IL-6, along with the increase in a set of mesenchymal cell markers including vimentin and α-smooth muscle actin (α-SMA), as well as EMT transcription regulators—twist, snail and slug. The changes of EMT phenotype were related to the activation of Src, FAK, ERK1/2, p38 mitogen-activated protein kinase (p38MAPK), as well as transcription factors STAT3, κB and C/EBPβ. IL-6 treatment has promoted the recruitment of STAT3, κB and C/EBPβ toward the Twist promoter region. Furthermore, the Src-FAK signaling blockade resulted in the decline of IL-6 induced activation of ERK1/2, p38MAPK, κB, C/EBPβ and STAT3, as well as the decreasing mesenchymal state of HCT116 cells. These results suggested that IL-6 activates the Src-FAK-ERK/p38MAPK signaling cascade to cause the EMT of colorectal cancer cells. Pharmacological approaches targeting Src-FAK signaling may provide potential therapeutic strategies for rescuing colorectal cancer progression.