Lateral roots are initiated postembryonically in response to environmental cues, enabling plants to explore efficiently their underground environment. However, the mechanisms by which the environment determines the position of lateral root formation are unknown. In this study, we demonstrate that in Arabidopsis thaliana lateral root initiation can be induced mechanically by either gravitropic curvature or by the transient bending of a root by hand. The plant hormone auxin accumulates at the site of lateral root induction before a primordium starts to form. Here we describe a subcellular relocalization of PIN1, an auxin transport protein, in a single protoxylem cell in response to gravitropic curvature. This relocalization precedes auxin-dependent gene transcription at the site of a new primordium. Auxin-dependent nuclear signaling is necessary for lateral root formation; arf7/19 double knock-out mutants normally form no lateral roots but do so upon bending when the root tip is removed. Signaling through arf7/19 can therefore be bypassed by root bending. These data support a model in which a root-tip-derived signal acts on downstream signaling molecules that specify lateral root identity.

In dicotyledonous plants, the apical region of the embryo shifts from radial to bilateral symmetry as the two cotyledon primordia develop on opposite sides of the shoot meristem. To further elucidate the mechanisms regulating this patterning process, we analyzed functions of two Arabidopsis genes, PIN-FORMED1 (PIN1) and MONOPTEROS (MP), encoding a putative auxin efflux carrier and a transcription factor thought to mediate auxin signaling, respectively. The corresponding mutants show similar defects in apical patterning, including cotyledon fusion and dissymmetric organ positioning. Both mutations perturb the spatial expression patterns of CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, which are redundantly required for cotyledon separation and meristem formation. During early embryogenesis, both CUC genes are affected differently: the area of CUC1 expression is expanded while that of CUC2 expression is reduced. In addition, genetic analysis indicates that PIN1 and MP are required for the activity of CUC2 while CUC1 activity is only slightly affected by both mutations. These results suggest a differential regulation of the CUC genes by PIN1 and MP. Furthermore, genetic analysis suggests that SHOOT MERISTEMLESS (STM), another regulator for cotyledon separation and meristem formation, promotes CUC1 activity in parallel with PIN1. Our results suggest a model where PIN1 and MP regulate apical patterning partially through the control of CUC gene expression.

The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here we describe the implementation of 193 nm ultraviolet photodissociation (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimer's disease and in cancer pathogenesis. The 5 ns, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision- and electron-based dissociation methods.

Protein phosphorylation on certain serine or threonine residues preceding proline (Ser/Thr-Pro) is a pivitol signaling mechanism in diverse cellular processes and its deregulation can lead to human disease. However, little is known about how these phosphorylation events actually control cell signaling. Pin1 is a highly conserved enzyme that isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins, thereby inducing conformational changes. Recent results indicate that such conformational changes following phosphorylation are a novel signaling mechanism pivotal in regulating many cellular functions. This mechanism also offers new insights into the pathogenesis and treatment of human disease, most notably cancer and Alzheimer's disease. Thus, Pin1 plays a key role in linking signal transduction to the pathogenesis of cancer and Alzheimer's disease - two major age-related diseases.

Phosphorylation on certain Ser/Thr-Pro motifs is a major oncogenic mechanism. The conformation and function of phosphorylated Ser/Thr-Pro motifs are further regulated by the prolyl isomerase Pin1. Pin1 is prevalently overexpressed in human cancers and implicated in oncogenesis. However, the role of Pin1 in oncogenesis in vivo is not known. We have shown that Pin1 ablation is highly effective in preventing oncogenic Neu or Ras from inducing cyclin D1 and breast cancer in mice, although it neither affects transgene expression nor mammary gland development. Moreover, we have developed an ex vivo assay to uncover that a significant fraction of primary mammary epithelial cells from Neu or Ras mice display various malignant properties long before they develop tumors in vivo. Importantly, these early transformed properties are effectively suppressed by Pin1 deletion, which can be fully rescued by overexpression of cyclin D1. Thus, Pin1 is essential for tumorigenesis and is an attractive anticancer target. Our ex vivo assay can be used to study early events of breast cancer development in genetically predisposed mice.

Myeloid cell leukemia-1 (Mcl-1), a Bcl-2-like antiapoptotic protein, plays a role in cell immortalization and chemoresistance in a number of human malignancies. A peptidyl-prolyl cis/trans isomerase, Pin1 is involved in many cellular events, such as cell cycle progression, cell proliferation, and differentiation through isomerizing prophosphorylated substrates. It has been reported that down-regulation of Pin1 induces apoptosis, and that Erk phosphorylates and up-regulates Mcl-1; however, the underlying mechanisms for the two phenomena are not clear yet. Here, we showed that Pin 1 stabilizes Mcl-1, which is required for Mcl-1 posphorylation by Erk. First, we found expression of Mcl-1 and Pin1 were positively correlated and associated with poor survival in human breast cancer. We then showed that Erk could phosphorylate Mcl-1 at two consensus residues, Thr 92 and 163, which is required for the association of Mcl-1 and Pin1, resulting in stabilization of Mcl-1. Moreover, Pin1 is also required for the up-regulation of Mcl-1 by Erk activation. Based on this newly identified mechanism of Mcl-1 stabilization, two strategies were used to overcome Mcl-1-mediated chemoresistance: inhibiting Erk by Sorafenib, an approved clinical anticancer drug, or knocking down Pin1 by using a SiRNA technique. In conclusion, the current report not only unravels a novel mechanism to link Erk/Pin1 pathway and Mcl-1-mediated chemoresistance but also provides a plausible combination therapy, Taxol (Paclitaxel) plus Sorafenib, which was shown to be effective in killing breast cancer cells.

Nitric oxide (NO) is considered a key regulator of plant developmental processes and defense, although the mechanism and direct targets of NO action remain largely unknown. We used phenotypic, cellular, and genetic analyses in Arabidopsis thaliana to explore the role of NO in regulating primary root growth and auxin transport. Treatment with the NO donors S-nitroso-N-acetylpenicillamine, sodium nitroprusside, and S-nitrosoglutathione reduces cell division, affecting the distribution of mitotic cells and meristem size by reducing cell size and number compared with NO depletion by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Interestingly, genetic backgrounds in which the endogenous NO levels are enhanced [chlorophyll a/b binding protein underexpressed 1/NO overproducer 1 (cue1/nox1) mirror this response, together with an increased cell differentiation phenotype. Because of the importance of auxin distribution in regulating primary root growth, we analyzed auxin-dependent response after altering NO levels. Both elevated NO supply and the NO-overproducing Arabidopsis mutant cue1/nox1 exhibit reduced expression of the auxin reporter markers DR5pro:GUS/GFP. These effects were accompanied by a reduction in auxin transport in primary roots. NO application and the cue1/nox1 mutation caused decreased PIN-FORMED 1 (PIN1)-GFP fluorescence in a proteasome-independent manner. Remarkably, the cue1/nox1-mutant root phenotypes resemble those of pin1 mutants. The use of both chemical treatments and mutants with altered NO levels demonstrates that high levels of NO reduce auxin transport and response by a PIN1-dependent mechanism, and root meristem activity is reduced concomitantly.

Embryonic stem cells (ESCs) can undergo unlimited self-renewal and retain the pluripotency to differentiate into all cell types in the body, thus holding great promise as a renewable source of cells for human therapy. The mechanisms that maintain self-renewal of ESCs remain unclear. Here we show that Nanog, a transcription factor crucial for the self-renewal of ESCs, is phosphorylated at multiple Ser/Thr-Pro motifs. This phosphorylation promotes the interaction between Nanog and the prolyl isomerase Pin1, leading to Nanog stabilization by suppressing its ubiquitination. Inhibition of Pin1 activity or disruption of Pin1-Nanog interaction in ESCs suppresses their capability to self-renew and to form teratomas in immunodeficient mice. Therefore, in addition to the stringent transcriptional regulation of Nanog, the expression level of Nanog is also modulated by posttranslational mechanisms.

Plasma membrane proteins internalized by endocytosis and targeted for degradation are sorted into lumenal vesicles of multivesicular bodies (MVBs) by the endosomal sorting complexes required for transport (ESCRT) machinery. Here, we show that the Arabidopsis thaliana ESCRT-related CHARGED MULTIVESICULAR BODY PROTEIN/CHROMATIN MODIFYING PROTEIN1A (CHMP1A) and CHMP1B proteins are essential for embryo and seedling development. Double homozygous chmp1a chmp1b mutant embryos showed limited polar differentiation and failed to establish bilateral symmetry. Mutant seedlings show disorganized apical meristems and rudimentary true leaves with clustered stomata and abnormal vein patterns. Mutant embryos failed to establish normal auxin gradients. Three proteins involved in auxin transport, PINFORMED1 (PIN1), PIN2, and AUXIN-RESISTANT1 (AUX1) mislocalized to the vacuolar membrane of the mutant. PIN1 was detected in MVB lumenal vesicles of control cells but remained in the limiting membrane of chmp1a chmp1b MVBs. The chmp1a chmp1b mutant forms significantly fewer MVB lumenal vesicles than the wild type. Furthermore, CHMP1A interacts in vitro with the ESCRT-related proteins At SKD1 and At LIP5. Thus, Arabidopsis CHMP1A and B are ESCRT-related proteins with conserved endosomal functions, and the auxin carriers PIN1, PIN2, and AUX1 are ESCRT cargo proteins in the MVB sorting pathway.

The peptidyl prolil cis/trans isomerase Ess1/Pin1 is essential for mitosis progression in yeast cells and is hypothesized to perform the same role in mammalian cells. To investigate the function of Pin1 in mammalian cells, we created mice lacking Pin1. These mice underwent normal development. Although the embryonic Pin1-/- fibroblasts grew normally, they proved significantly deficient in their ability to restart proliferation in response to serum stimulation after G(0) arrest. These results suggest that Pin1 is required for cell cycle progression from G(0) arrest as well as mitosis progression in normal mammalian cells.

The plant hormone auxin plays a crucial role in regulating plant development and plant architecture. The directional auxin distribution within tissues depends on PIN transporters that are polarly localized on the plasma membrane. The PIN polarity and the resulting auxin flow directionality are mediated by the antagonistic actions of PINOID kinase and protein phosphatase 2A. However, the contribution of the PIN phosphorylation to the polar PIN sorting is still unclear. Here, we identified an evolutionarily conserved phosphorylation site within the central hydrophilic loop of PIN proteins that is important for the apical and basal polar PIN localizations. Inactivation of the phosphorylation site in PIN1(Ala) resulted in a predominantly basal targeting and increased the auxin flow to the root tip. In contrast, the outcome of the phosphomimic PIN1(Asp) manipulation was a constitutive, PINOID-independent apical targeting of PIN1 and an increased auxin flow in the opposite direction. Furthermore, the PIN1(Asp) functionally replaced PIN2 in its endogenous expression domain, revealing that the phosphorylation-dependent polarity regulation contributes to functional diversification within the PIN family. Our data suggest that PINOID-independent PIN phosphorylation at one single site is adequate to change the PIN polarity and, consequently, to redirect auxin fluxes between cells and provide the conceptual possibility and means to manipulate auxin-dependent plant development and architecture.

Key steps in a plant's development and adaptation to the environment are the initiation and development of lateral roots (LRs). LR development is regulated by auxin, the major plant hormone promoting LR formation, its counteracting hormones cytokinin, and abscisic acid (ABA). Here, we show that mutating ABSCISIC ACID INSENSITIVE4 (ABI4), which encodes an ABA-regulated AP2 domain transcription factor, results in an increased number of LRs. We show that ABI4 is expressed in roots and that its overexpression impairs LR development. Root expression of ABI4 is enhanced by ABA, and cytokinin and is repressed by auxin. Using hormone response promoters, we show that ABI4 also affects auxin and cytokinin profiles in the root. Furthermore, LR development in abi4 mutants is not altered or inhibited by cytokinin or ABA. Expression of the auxin-efflux carrier protein PIN1 is reduced in ABI4 overexpressors, enhanced in abi4 mutants, and is less sensitive to inhibition by cytokinin and ABA in abi4 mutants than in wild-type plants. Transport levels of exogenously applied auxin were elevated in abi4 mutants and reduced in ABI4 overexpressors. We therefore suggest that ABI4 mediates ABA and cytokinin inhibition of LR formation via reduction of polar auxin transport and that the resulting decrease in root auxin leads to a reduction in LR development.

Activation of p73 upon genotoxic treatment triggers apoptosis of tumor cells lacking functional p53 and involves the activities of c-Abl and p300. Here, we demonstrate that conformational changes of p73 catalyzed by the prolyl isomerase Pin1 are crucial in this pathway. Lack of Pin1 reduces p73 stability, hampering its accumulation upon genotoxic stress. Indeed, we show that upon treatment with chemotherapeutic drugs c-Abl enhances the phosphorylation-dependent interaction between Pin1 and p73, and this in turn promotes p73 acetylation by p300. Consistently, the ability of c-Abl and p300 to increase p73 stability and transcriptional activity requires Pin1. As a consequence, Pin1 appears to be essential for activation of the apoptotic response by endogenous p73.

Maize (Zea mays) plants make different types of vegetative or reproductive branches during development. Branches develop from axillary meristems produced on the flanks of the vegetative or inflorescence shoot apical meristem. Among these branches are the spikelets, short grass-specific structures, produced by determinate axillary spikelet-pair and spikelet meristems. We investigated the mechanism of branching in maize by making transgenic plants expressing a native expressed endogenous auxin efflux transporter (ZmPIN1a) fused to yellow fluorescent protein and a synthetic auxin-responsive promoter (DR5rev) driving red fluorescent protein. By imaging these plants, we found that all maize branching events during vegetative and reproductive development appear to be regulated by the creation of auxin response maxima through the activity of polar auxin transporters. We also found that the auxin transporter ZmPIN1a is functional, as it can rescue the polar auxin transport defects of the Arabidopsis (Arabidopsis thaliana) pin1-3 mutant. Based on this and on the groundbreaking analysis in Arabidopsis and other species, we conclude that branching mechanisms are conserved and can, in addition, explain the formation of axillary meristems (spikelet-pair and spikelet meristems) that are unique to grasses. We also found that BARREN STALK1 is required for the creation of auxin response maxima at the flanks of the inflorescence meristem, suggesting a role in the initiation of polar auxin transport for axillary meristem formation. Based on our results, we propose a general model for branching during maize inflorescence development.

Heterologous expression systems based on tobacco BY-2 cells, Arabidopsis cell cultures, Xenopus oocytes, Saccharomyces cerevisiae, and human HeLa cells have been used to express and characterize PIN, ABCB (PGP), and AUX/LAX auxin transporters from Arabidopsis. However, no single system has been identified that can be used for effective comparative analyses of these proteins. We have developed an accessible Schizosaccharomyces pombe system for comparative studies of plant transport proteins. The system includes knockout mutants in all ABC and putative auxin transport genes and Gateway((R))-compatible expression vectors for functional analysis and subcellular localization of recombinant proteins. We expressed Arabidopsis ABCB1 and ABCB19 in mam1pdr1 host lines under the inducible nmt41 promoter. ABCB19 showed a higher (3)H-IAA export activity than ABCB1. Arabidopsis PIN proteins were expressed in a mutant lacking the auxin effluxer like 1 (AEL1) gene. PIN1 showed higher activity than PIN2 with similar protein expression levels. Expression of AUX1 in a permease-deficient vat3 mutant resulted in increased net auxin uptake activity. Finally, ABCB4 expressed in mam1pdr1 displayed a concentration-dependent reversal of (3)H-IAA transport that is consistent with its observed activity in planta. Structural modelling suggests that ABCB4 has three substrate interaction sites rather than the two found in ABCB19, thus providing a rationale for the observed substrate activation. Taken together, these results suggest that the S. pombe system described here can be employed for comparative analyses and subsequent structural characterizations of plant transport proteins.

Fbw7 is the substrate recognition component of the Skp1-Cullin-F-box (SCF)-type E3 ligase complex and a well-characterized tumor suppressor that targets numerous oncoproteins for destruction. Genomic deletion or mutation of FBW7 has been frequently found in various types of human cancers; however, little is known about the upstream signaling pathway(s) governing Fbw7 stability and cellular functions. Here we report that Fbw7 protein destruction and tumor suppressor function are negatively regulated by the prolyl isomerase Pin1. Pin1 interacts with Fbw7 in a phoshorylation-dependent manner and promotes Fbw7 self-ubiquitination and protein degradation by disrupting Fbw7 dimerization. Consequently, overexpressing Pin1 reduces Fbw7 abundance and suppresses Fbw7's ability to inhibit proliferation and transformation. By contrast, depletion of Pin1 in cancer cells leads to elevated Fbw7 expression, which subsequently reduces Mcl-1 abundance, sensitizing cancer cells to Taxol. Thus, Pin1-mediated inhibition of Fbw7 contributes to oncogenesis, and Pin1 may be a promising drug target for anticancer therapy.

Cell polarization via asymmetrical distribution of structures or molecules is essential for diverse cellular functions and development of organisms, but how polarity is developmentally controlled has been poorly understood. In plants, the asymmetrical distribution of the PIN-FORMED (PIN) proteins involved in the cellular efflux of the quintessential phytohormone auxin plays a central role in developmental patterning, morphogenesis, and differential growth. Recently we showed that auxin promotes cell interdigitation by activating the Rho family ROP GTPases in leaf epidermal pavement cells. Here we found that auxin activation of the ROP2 signaling pathway regulates the asymmetric distribution of PIN1 by inhibiting its endocytosis. ROP2 inhibits PIN1 endocytosis via the accumulation of cortical actin microfilaments induced by the ROP2 effector protein RIC4. Our findings suggest a link between the developmental auxin signal and polar PIN1 distribution via Rho-dependent cytoskeletal reorganization and reveal the conservation of a design principle for cell polarization that is based on Rho GTPase-mediated inhibition of endocytosis.

Signalling through Notch receptors requires ligand-induced cleavage to release the intracellular domain, which acts as a transcriptional activator in the nucleus. Deregulated Notch1 signalling has been implicated in mammary tumorigenesis; however the mechanisms underlying Notch activation in breast cancer remain unclear. Here, we demonstrate that the prolyl-isomerase Pin1 interacts with Notch1 and affects Notch1 activation. Pin1 potentiates Notch1 cleavage by gamma-secretase, leading to an increased release of the active intracellular domain and ultimately enhancing Notch1 transcriptional and tumorigenic activity. We found that Notch1 directly induces transcription of Pin1, thereby generating a positive loop. In human breast cancers, we observed a strong correlation between Pin1 overexpression and high levels of activated Notch1. Thus, the molecular circuitry established by Notch1 and Pin1 may have a key role in cancer.

OBJECTIVE

Colorectal cancer is one of the most common cancers worldwide. We tested the hypothesis that differences in the expression of certain molecular markers of colon cancer may account for different clinical outcomes.

METHODS

Tissue microarray technology was used to assay the expression of 17 biological markers [beta-catenin, CD44v7, c-myc, cyclin D1, estrogen receptor beta, mitogen-activated protein kinase/extracellular signal-regulated kinase, maspin, matrix metalloproteinase-7 (MMP7), p53, Pin1, peroxisome proliferators-activated receptor-gamma, survivin, T cell transcription factor 4 (TCF4), transforming growth factor beta receptor II (TGFbetaR II), TGFbeta, TROP2, and Wnt] by immunohistochemistry in 620 colon cancer patients. The Cox proportional hazards regression model was applied to analyze the lifetime data, including time to death, time to recurrence, and time to liver metastasis.

RESULTS

All the markers were present at significantly higher expression levels in tumor specimens than in normal colonic specimens. Kaplan-Meier analysis showed that high expression of TROP2, MMP7, and survivin were related to decreased survival; TCF4 and TROP2 were related to disease recurrence; and CD44v7, cyclin D1, MMP7, p53, survivin, and TCF4 were related to liver metastasis. However, the results of the multivariate analysis only showed that expression of MMP7, survivin, and TROP2 were significant predictors of lower patient survival, while TROP2 and MMP7 were significantly related to disease recurrence and liver metastasis, respectively.

CONCLUSIONS

We conclude that elevated survivin, MMP7, and TROP2 expression levels are related to decreased survival. In addition, elevated MMP7 and TROP2 expression levels are predictors of disease recurrence and liver metastasis, respectively.

We have established a detailed framework for the process of shoot regeneration from Arabidopsis root and hypocotyl explants grown in vitro. Using transgenic plant lines in which the GUS or GFP genes were fused to promoters of developmental genes (WUS, CLV1, CLV3, STM, CUC1, PLT1, RCH1, QC25), or to promoters of genes encoding indicators of the auxin response (DR5) or transport (PIN1), cytokinin (CK) response (ARR5) or synthesis (IPT5), or mitotic activity (CYCB1), we showed that regenerated shoots originated directly or indirectly from the pericycle cells adjacent to xylem poles. In addition, shoot regeneration appeared to be partly similar to the formation of lateral root meristems (LRMs). During pre-culture on a 2, 4-dichlorophenoxyacetic acid (2, 4-D)-rich callus-inducing medium (CIM), xylem pericycle reactivation established outgrowths that were not true calli but had many characteristics of LRMs. Transfer to a CK-rich shoot-inducing medium (SIM) resulted in early LRM-like primordia changing to shoot meristems. Direct origin of shoots from the xylem pericycle occurred upon direct culture on CK-containing media without prior growth on CIM. Thus, it appeared that the xylem pericycle is more pluripotent than previously thought. This pluripotency was accompanied by the ability of pericycle derivatives to retain diploidy, even after several rounds of cell division. In contrast, the phloem pericycle did not display such developmental plasticity, and responded to CKs with only periclinal divisions. Such observations reinforce the view that the pericycle is an 'extended meristem' that comprises two types of cell populations. They also suggest that the founder cells for LRM initiation are not initially fully specified for this developmental pathway.

Endocytic vesicle trafficking is crucial for regulating activity and localization of plasma membrane components, but the process is still poorly genetically defined in plants. Membrane proteins of the PIN-FORMED (PIN) family exhibit polar localization in plant cells and facilitate cellular efflux of the plant hormone auxin, thereby regulating multiple developmental processes. PIN proteins undergo constitutive endocytosis and GNOM ARF GEF-dependent recycling, and their localization is under extensive regulation by developmental and environmental cues. We designed a fluorescence imaging-based screen to identify Arabidopsis thaliana mutants defective in internalization of proteins including PINs from the plasma membrane. We identified three mutant loci, BFA-visualized endocytic trafficking defective1 (ben1) through ben3 that do not efficiently accumulate PIN1-GFP in intracellular compartments after inhibition of recycling and secretion by fungal toxin brefeldin A (BFA). Fine mapping revealed that BEN1 encodes an ARF GEF vesicle trafficking regulator from the functionally uncharacterized BIG class. ben1 mutant has been previously implicated in pathogen response and shows cell polarity, BFA sensitivity, and growth defects. BEN1 is involved in endocytosis of plasma membrane proteins and localizes to early endocytic compartments distinct from GNOM-positive endosomes. Our results identify BEN1 as the ARF GEF mediating early endosomal traffic.

Strigolactones (SLs) were originally identified through their activities as root exudates in the rhizosphere; however, it is now clear that they have many endogenous signalling roles in plants. In this review we discuss recent progress in understanding SL action in planta, particularly in the context of the regulation of shoot branching, one of the best-characterized endogenous roles for SLs. Rapid progress has been made in understanding SL biosynthesis, but many questions remain unanswered. There are hints of as yet unidentified sources of SL, as well as unknown SL-like molecules with important signalling functions. SL signalling is even more enigmatic. Although a likely receptor has been identified, along with some candidate immediate downstream targets, our understanding of how these targets mediate SL signalling is limited. There is still considerable uncertainty about whether the targets of SL signalling are primarily transcriptional or not. There is at least one non-transcriptional target, because a rapid primary response to SL is the removal of PIN1 auxin exporter proteins from the plasma membrane in vascular-associated cells of the stem. We discuss how the various early events in SL signalling could result in the observed changes in shoot branching.

DORNROSCHEN (DRN) (also known as ENHANCER OF SHOOT REGENERATION1; ESR1) and DRN-LIKE (DRNL; also known as ESR2) are two linked paralogues encoding AP2 domain-containing proteins. drn mutants show embryo cell patterning defects and, similarly to drnl mutants, disrupt cotyledon development at incomplete penetrance. drn drnl double mutants with weak or strong drnl alleles show more highly penetrant and extreme phenotypes, including a pin-like embryo without cotyledons, confirming a high degree of functional redundancy for the two genes in embryo patterning. Altered expression of PIN1::PIN1-GFP and DR5::GFP in drn mutant embryos places DRN upstream of auxin transport and response. A yeast two-hybrid screen with DRN followed by co-immunoprecipitation and bimolecular fluorescence complementation revealed PHAVOLUTA (PHV) to be a protein interaction partner in planta. drn phv double mutants show an increased penetrance of embryo cell division defects. DRNL can also interact with PHV and both DRN and DRNL can heterodimerise with additional members of the class III HD-ZIP family, PHABULOSA, REVOLUTA, CORONA and ATHB8. Interactions involve the PAS-like C-terminal regions of these proteins and the DRN/DRNL AP2 domain.

The C-terminal domain of the RNA polymerase (RNAP) II largest subunit (CTD) plays critical roles both in transcription of mRNA precursors and in the processing reactions needed to form mature mRNAs. The CTD undergoes dynamic changes in phosphorylation during the transcription cycle, and this plays a significant role in coordinating its multiple activities. But how these changes themselves are regulated is not well understood. Here we show that the peptidyl-prolyl isomerase Pin1 influences the phosphorylation status of the CTD in vitro by inhibiting the CTD phosphatase FCP1 and stimulating CTD phosphorylation by cdc2/cyclin B. This is reflected in vivo by accumulation of hypophosphorylated RNAP II in pin1-/- cells, and of a novel hyper-hyperphosphorylated form in cells induced to overexpress Pin1. This hyper-hyperphosphorylated form of RNAP II also accumulates in M-phase cells, in a Pin1-dependent manner, and associates specifically with Pin1. Functionally, we find that Pin1 overexpression specifically inhibits ongoing transcription of mRNA precursors in vivo and both transcription and RNAP II-stimulated pre-mRNA splicing in cell extracts. Pin1 thus plays a significant role in regulating RNAP II CTD structure and function.