C-terminal Repeat Domain Research Articles

Cap Completion and C-Terminal Repeat Domain Kinase Recruitment Underlie the Initiation-Elongation Transition of RNA Polymerase II

After transcription initiation, RNA polymerase (Pol) II escapes from the promoter and recruits elongation factors. The molecular basis for the initiation-elongation factor exchange during this transition remains poorly understood. Here, we used chromatin immunoprecipitation (ChIP) to elucidate the initiation-elongation transition of Pol II in the budding yeast Saccharomyces cerevisiae. We show that the early Pol II elongation factor Spt5 contributes to stable recruitment of the mRNA capping enzymes Cet1, Ceg1, and Abd1. Genome-wide occupancy for Cet1 and Ceg1 is restricted to the transcription start site (TSS), whereas occupancy for Abd1 peaks at ~110 nucleotides downstream, and occupancy for the cap-binding complex (CBC) rises subsequently. Abd1 and CBC are important for recruitment of the kinases Ctk1 and Bur1, which promote elongation and capping enzyme release. These results suggest that cap completion stimulates productive Pol II elongation.

The Recruitment of the Saccharomyces cerevisiae Paf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

Transcription elongation factors associate with RNA polymerase II and aid its translocation through chromatin. One such factor is the conserved Paf1 complex (Paf1C), which regulates gene expression through several mechanisms, including the stimulation of cotranscriptional histone modifications. Previous studies revealed a prominent role for the Rtf1 subunit in tethering Paf1C to the RNA polymerase II elongation machinery. Here, we investigated the mechanism by which Rtf1 couples Paf1C to active chromatin. We show that a highly conserved domain of Rtf1 is necessary and sufficient for mediating a physical interaction between Rtf1 and the essential transcription elongation factor Spt5. Mutations that alter this Rtf1 domain or delete the Spt5 C-terminal repeat domain (CTR) disrupt the interaction between Rtf1 and Spt5 and release Paf1C from chromatin. When expressed in cells as the only source of Rtf1, the Spt5-interacting domain of Rtf1 can associate independently with active genes in a pattern similar to that of full-length Rtf1 and in a manner dependent on the Spt5 CTR. In vitro experiments indicate that the interaction between the Rtf1 Spt5-interacting domain and the Spt5 CTR is direct. Collectively, our results provide molecular insight into a key attachment point between Paf1C and the RNA polymerase II elongation machinery.

Rickettsia Sca2 has evolved formin-like activity through a different molecular mechanism

Sca2 (surface cell antigen 2) is the only bacterial protein known to promote both actin filament nucleation and profilin-dependent elongation, mimicking eukaryotic formins to assemble actin comet tails for Rickettsia motility. We show that Sca2's functional mimicry of formins is achieved through a unique mechanism. Unlike formins, Sca2 is monomeric, but has N- and C-terminal repeat domains (NRD and CRD) that interact with each other for processive barbed-end elongation. The crystal structure of NRD reveals a previously undescribed fold, consisting of helix-loop-helix repeats arranged into an overall crescent shape. CRD is predicted to share this fold and might form together with NRD, a doughnut-shaped formin-like structure. In between NRD and CRD, proline-rich sequences mediate the incorporation of profilin-actin for elongation, and WASP-homology 2 (WH2) domains recruit actin monomers for nucleation. Sca2's α-helical fold is unusual among Gram-negative autotransporters, which overwhelmingly fold as β-solenoids. Rickettsia has therefore "rediscovered" formin-like actin nucleation and elongation.

Annexin A1 is regulated by domains cross-talk through post-translational phosphorylation and SUMOYlation

Mouse prostate membrane-associated proteins of the annexin family showed changes in SUMOylation during androgen treatment. Among these the calcium-binding annexin A1 protein (ANXA1) was chosen for further characterization given its role in protein secretion and cancer. SUMOylation of ANXA1 was confirmed by overexpressing SUMO-1 in LNCaP cells. Site-directed mutagenesis indicated that K257 located in a SUMOylation consensus motif in the C-terminal calcium-binding DA3 repeat domain is SUMOylated. Mutation of the N-terminal Y21 decreased markedly the SUMOylation signal while EGF stimulation increased ANXA1 SUMOylation. A structural analysis of ANXA1 revealed that K257 is located in a hot spot where Ca2+ and SUMO-1 bind and where a nuclear export signal and a polyubiquitination site are also present. Also, Y21 is buried inside an α-helix structure in the Ca2+-free conformation implying that Ca2+ binding, and the subsequent expelling of the N-terminal α-helix in a disordered conformation, is permissive for its phosphorylation. These results show for the first time that SUMOylation can be regulated by an external signal (EGF) and indicate the presence of a cross-talk between the N-terminal and C-terminal domains of ANXA1 through post-translational modifications.

Structural and Functional Analysis of the N-terminal Domain of the Streptococcus gordonii Adhesin Sgo0707

The commensal Streptococcus gordonii expresses numerous surface adhesins with which it interacts with other microorganisms, host cells and salivary proteins to initiate dental plaque formation. However, this Gram-positive bacterium can also spread to non-oral sites such as the heart valves and cause infective endocarditis. One of its surface adhesins, Sgo0707, is a large protein composed of a non-repetitive N-terminal region followed by several C-terminal repeat domains and a cell wall sorting motif. Here we present the crystal structure of the Sgo0707 N-terminal domains, refined to 2.1 Å resolution. The model consists of two domains, N1 and N2. The largest domain, N1, comprises a putative binding cleft with a single cysteine located in its centre and exhibits an unexpected structural similarity to the variable domains of the streptococcal Antigen I/II adhesins. The N2-domain has an IgG-like fold commonly found among Gram-positive surface adhesins. Binding studies performed on S. gordonii wild-type and a Sgo0707 deficient mutant show that the Sgo0707 adhesin is involved in binding to type-1 collagen and to oral keratinocytes.

Abstract 1926: Nod-like receptors family member- NLRC5 as a useful prognostic marker in renal cell carcinomas.

Abstract Based on our previous methylated-CpG island recovery assay (MIRA) combined with DNA microarray technique studies, several hypermethylated genes have been proven in renal cell carcinoma (RCC), in which NLRC5 was one of the interested genes. To our knowledge, this is the first study of NLRC5 in RCCs. NLRC5 was a member of the Nod-like receptors family proteins that contains C-terminal leucine-rich repeat (LRR) domain, nucleotide-binding NACHT domain and the CARD domain. It is commonly highly expressed in many immune related tissues, such as bone marrow, lymph node, spleen, thymus; besides, it also presented in normal tissues, such as kidney, liver and prostate. The function of NLRC5 has been widely proven in immunoactivities, it was known may negative regulated NF-κB and type I interferon signaling pathways, however, seldom to be studied in tumorigenesis neoplastic progression. Therefore, it led our attention to the expression of NLRC5 might regulate NF-κB activity, then further promoting the pathogenesis and metastatic behavior of RCC. Herein, we hypothesized that via demethylated NLRC5 might indirect inactivation of NF-κB then result in reducing the cancer cell proliferation. In this study we aimed to investigate the status of methylation and mRNA expression of NLRC5 in RCC. We performed reverse transcription PCR on six RCC cell lines, which included chromophobe type RCC98, sacomatoid type RCC52, clear cell type RCC100, and papillary type HH050 RCC cell lines were derived from Chang Gung Memorial Hospital, Taiwan; 786-O and HEK-293 were commercial available, in which 5-aza-2’deoxycytidine was treated on randomly selected cell lines RCC98, 786-O and HH050 to demethylate the DNA. Real-time PCR were than performed on 75 pairs of clinical RCCs samples (tumor tissue v.s normal tissue), the results were divided into two groups, high expression (T/N≥ 1.5) and low expression (T/N <1.5), the Kaplan-Meier procedure then calculated the survival probability estimate for each of the time periods. The results showed that variant gene expression of the NLRC5 could be observed from all RCC cell lines. Generally, the tumors were considered as poor prognostic types (e.g. RCC100 and RCC52) presented lower level of NLRC5 expression, except 786-O cell line. Survival analysis of the NLRC5 expression of 75 pairs RCC patients, high expression of NLRC5 associated with better disease-free survival and overall survival, p = 0.015 and 0.04, respectively; however, it was not correlated to pT stage and grade. Interestingly, after treated with the demethylated drug on tested cell lines, the expressed level of NLRC5 was restored in all tested cells. We concluded that high expression of NLRC5 was correlated with better patient survival, and it might be used as a prognostic marker to predict patient outcomes in RCC. Besides, the epigenetic demethylation of NLRC5 was suggested it could be a potential way for treatment with RCC. Citation Format: Ying-Tzu Chen, Wen-Hui Weng, See-Tong Pang. Nod-like receptors family member- NLRC5 as a useful prognostic marker in renal cell carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1926. doi:10.1158/1538-7445.AM2013-1926

Regulating CTD‐Interactions by Ess1‐Dependent Prolyl Isomerization

RNA polymerase II directs the synthesis of mRNAs as well as snoRNAs and some snRNAs. The largest subunit, Rpb1, contains an unstructured C‐terminal domain (CTD) composed of multiple repeats of the heptapeptide, Y‐S‐P‐T‐S‐P‐S. These repeats are located near the RNA exit channel and are bound by a variety of protein complexes that function during different stages of transcription and co‐transcriptional RNA processing. Despite the fact that the CTD is relatively long (26 repeats in yeast, 52 in human), different co‐factor complexes are differentially recruited to the CTD during the transcription cycle. Recruitment is regulated by covalent and non‐covalent modification of residues within the CTD repeat. This regulation is referred to as the “CTD code.” Examples of important covalent modifications are phosphorylation of serines at positions 2, 5, and 7.We are studying a non‐covalent modification, the cis/trans isomerization of the peptide bond at phosphorylated Ser5‐Pro6 sites. In yeast, this is carried out by a highly‐conserved enzyme that we discovered called Ess1 (Pin1 in humans). Ess1 binds and isomerizes pSer5‐Pro6, and this has profound consequences for the proper recruitment of proteins (e.g. Nrd1, Pcf11) to the RNAP II complex. Cells that lack Ess1 activity fail to suppress cryptic transcription, and cannot efficiently terminate transcription of small ncRNAs and mRNAs, leading to cell death. Ess1‐dependent isomerization of the CTD also seems to be important for histone modification, as ess1 mutants show reduced levels of H3K4‐ trimethylation. We will discuss potential mechanisms for how Ess1 regulates RNAP II transcription and co‐transcriptional processing.

Conformational Change-Induced Repeat Domain Expansion Regulates Rap Phosphatase Quorum-Sensing Signal Receptors

Author SummaryThe bacterial cell–cell communication process known as quorum sensing regulates important social behaviors including antibiotic production, motility, virulence, biofilm formation, sporulation, bioluminescence, and genetic competence. Gram-positive bacteria secrete oligopeptide quorum-sensing signals that bind to membrane-bound and cytosolic receptors. How oligopeptide quorum-sensing signals regulate the activity of their target receptors was previously largely unknown. Here we show that proteins belonging to the family of bacterial quorum-sensing receptors known as the Rap phosphatases undergo a remarkable regulatory conformational change upon binding oligopeptide signals. More specifically, in the absence of the oligopeptide signal, Rap proteins consist of two distinct domains: an N-terminal domain consisting of a three-helix bundle, and a superhelical C-terminal domain comprising an array of seven similar helix-turn-helix repeats. A flexible helix-containing linker region connects these domains. In complex with the regulatory oligopeptide, however, the Rap protein domains and linker region rearrange, merging to form a single continuous superhelical structure consisting of nine helix-turn-helix repeats. To our knowledge, this represents the first example of conformational change-induced repeat domain expansion. The structure-function studies presented here set the stage for the rational development of antimicrobial peptides and peptide-mimetics capable of targeting cell–cell signaling mediated by Rap proteins and similar bacterial receptors.

Structural Basis of Rap Phosphatase Inhibition by Phr Peptides

Author SummaryIn microorganisms, two component signaling systems are widely used to sense and respond to environmental changes, including quorum-sensing of Phr oligopeptides. Although the minimal machinery required for these systems comprises a sensor histidine kinase and an effector response regulator (RR), ancillary proteins, termed “connectors,” capable of modulating the activity of this machinery, are emerging as additional players in this complex signaling process. Rap proteins are archetypal connectors, able to modulate the activity of RRs either by dephosphorylating them or by physically blocking them. Rap proteins are themselves in turn inhibited by specific Phr peptides, adding an extra level of complexity, but how a Rap protein is regulated by its cognate Phr peptide remains unknown. To answer this question, we solved the structure of RapF, a Rap family member that blocks RR ComA, alone and in the complex with its inhibitory peptide PhrF. Our structural and functional results reveal that PhrF blocks the RapF-ComA interaction by an allosteric mechanism since the PhrF-RapF interaction induces a conformational change that is propagated to the the ComA binding site, disrupting it and triggering the dissociation of ComA from RapF. Using sequence analysis guided by our structure, we pinpointed sets of residues responsible for peptide anchor and specificity, respectively, and were able to relax RapF-Phr specificity simply by changing a single residue. Knowledge of these key residues and the Rap inhibition mechanism opens up the possibility of re-engineering Rap proteins, and paves the way to reprogramming signaling pathways for biological and biotechnological applications.

The FKBP-Type Domain of the Human Aryl Hydrocarbon Receptor-Interacting Protein Reveals an Unusual Hsp90 Interaction

The aryl hydrocarbon receptor-interacting protein (AIP) has been predicted to consist of an N-terminal FKBP-type peptidyl-prolyl cis/trans isomerase (PPIase) domain and a C-terminal tetratricopeptide repeat (TPR) domain, as typically found in FK506-binding immunophilins. AIP, however, exhibited no inherent FK506 binding or PPIase activity. Alignment with the prototypic FKBP12 showed a high sequence homology but indicated inconsistencies with regard to the secondary structure prediction derived from chemical shift analysis of AIP(2-166). NMR-based structure determination of AIP(2-166) now revealed a typical FKBP fold with five antiparallel β-strands forming a half β-barrel wrapped around a central α-helix, thus permitting AIP to be also named FKBP37.7 according to FKBP nomenclature. This PPIase domain, however, features two structure elements that are unusual for FKBPs: (i) an N-terminal α-helix, which additionally stabilizes the domain, and (ii) a rather long insert, which connects the last two β-strands and covers the putative active site. Diminution of the latter insert did not generate PPIase activity or FK506 binding capability, indicating that the lack of catalytic activity in AIP is the result of structural differences within the PPIase domain. Compared to active FKBPs, a diverging conformation of the loop connecting β-strand C' and the central α-helix apparently is responsible for this inherent lack of catalytic activity in AIP. Moreover, Hsp90 was identified as potential physiological interaction partner of AIP, which revealed binding contacts not only at the TPR domain but uncommonly also at the PPIase domain.

Selective constraint and the evolution of the RNA Polymerase II C-Terminal Domain

The C-Terminal Domain (CTD) of the large subunit (Rpb1) of RNA Polymerase II has a Tyrosine-Serine-Proline-Threonine-Serine-Proline-Serine repeat structure in many eukaryotes. Chemical modifications of these residues play a central role in the regulation and coordination of the events of transcription. However, substantial variability in the presence and regularity of repeat arrays exists between eukaryote taxa. Following a survey of CTD structure from diverse eukaryote species, two hypotheses were tested relating to repeat structure and the action of selection on the CTD. First, it was found that degenerated repeat structure is associated with lower serine and proline frequencies in some eukaryote taxa but not in others. Second, maximum likelihood models of the evolution of Rpb1 in a number of species groups found that purifying selection on the non-repetitive CTD of several Leishmania species was substantially lower than for the rest of Rpb1, whereas purifying selection in a number of species groups containing repeat arrays was usually as high or nearly as high as for the rest of Rpb1. Characterization of CTD structure for a larger number of species than has been completed previously also revealed a greater diversity of CTD structures in eukaryotes than previously known, along with loss of repeat structure in the animals and fungi, two taxa where it has not previously been known. These results suggest that loss of CTD repeat structure has been an important aspect of RNA Polymerase II evolution in diverse eukaryotes.

Crystal structure of Cex1p reveals the mechanism of tRNA trafficking between nucleus and cytoplasm

In all eukaryotes, transcribed precursor tRNAs are maturated by processing and modification processes in nucleus and are transported to the cytoplasm. The cytoplasmic export protein (Cex1p) captures mature tRNAs from the nuclear export receptor (Los1p) on the cytoplasmic side of the nuclear pore complex, and it delivers them to eukaryotic elongation factor 1α. This conserved Cex1p function is essential for the quality control of mature tRNAs to ensure accurate translation. However, the structural basis of how Cex1p recognizes tRNAs and shuttles them to the translational apparatus remains unclear. Here, we solved the 2.2 Å resolution crystal structure of Saccharomyces cerevisiae Cex1p with C-terminal 197 disordered residues truncated. Cex1p adopts an elongated architecture, consisting of N-terminal kinase-like and a C-terminal α-helical HEAT repeat domains. Structure-based biochemical analyses suggested that Cex1p binds tRNAs on its inner side, using the positively charged HEAT repeat surface and the C-terminal disordered region. The N-terminal kinase-like domain acts as a scaffold to interact with the Ran-exportin (Los1p·Gsp1p) machinery. These results provide the structural basis of Los1p·Gsp1p·Cex1p·tRNA complex formation, thus clarifying the dynamic mechanism of tRNA shuttling from exportin to the translational apparatus.

Identification of RNA targets for the nuclear multidomain cyclophilin atCyp59 and their effect on PPIase activity

AtCyp59 is a multidomain cyclophilin containing a peptidyl-prolyl cis/trans isomerase (PPIase) domain and an evolutionarily highly conserved RRM domain. Deregulation of this class of cyclophilins has been shown to affect transcription and to influence phosphorylation of the C-terminal repeat domain of the largest subunit of the RNA polymerase II. We used a genomic SELEX method for identifying RNA targets of AtCyp59. Analysis of the selected RNAs revealed an RNA-binding motif (G[U/C]N[G/A]CC[A/G]) and we show that it is evolutionarily conserved. Binding to this motif was verified by gel shift assays in vitro and by RNA immunopreciptation assays of AtCyp59 in vivo. Most importantly, we show that binding also occurs on unprocessed transcripts in vivo and that binding of specific RNAs inhibits the PPIase activity of AtCyp59 in vitro. Surprisingly, genome-wide analysis showed that the RNA motif is present in about 70% of the annotated transcripts preferentially in exons. Taken together, the available data suggest that these cyclophilins might have an important function in transcription regulation.

Characterization of three Nod-like receptors and their role in antimicrobial responses of goldfish (Carassius auratus L.) macrophages to Aeromonas salmonicida and Mycobacterium marinum

The nucleotide-binding oligomerization domain proteins Nod1, Nod2 and Nlrx1 are cytoplasmic pathogen recognition receptors (PRRs) of the Nod-like receptor (NLR) family. In this report, goldfish Nod1 (gfNod1), Nod2 (gfNod2) and Nlrx1 (gfNlrx1) genes were cloned and characterized. The full length of gfNod1, gfNod2 and gfNlrx1 were 3234bp, 3129bp and 4900bp, encoding 937, 982 and 1008 amino acids, respectively. The three Nod-like receptors have a NACHT domain and C-terminal leucine rich repeat (LRR) domains. In addition to these, gfNod1 and gfNod2 also had an N-terminal CARD domain (two in gfNod2). Phylogenetic analysis showed that the three NLRs are highly conserved. Quantitative gene expression analysis of the three receptors revealed the greatest mRNA levels in the spleen, and in isolated neutrophils and splenocytes. Furthermore, treatment of goldfish macrophages with LPS, Poly I:C, MDP, PGN, heat-killed Aeromonas salmonicida or Mycobacterium marinum differentially altered the expression of the Nod-like receptors. Our results indicate that Nod-like receptors are functionally highly conserved and that they play a pivotal role in recognition of fish pathogens such as A. salmonicida and M. marinum.

Fast-Forward Genetics Identifies Plant CPL Phosphatases as Regulators of miRNA Processing Factor HYL1

MicroRNAs (miRNAs) are processed from primary transcripts that contain partially self-complementary foldbacks. As in animals, the core microprocessor in plants is a Dicer protein, DICER-LIKE1 (DCL1). Processing accuracy and strand selection is greatly enhanced through the RNA binding protein HYPONASTIC LEAVES 1 (HYL1) and the zinc finger protein SERRATE (SE). We have combined a luciferase-based genetic screen with whole-genome sequencing for rapid identification of new regulators of miRNA biogenesis and action. Among the first six mutants analyzed were three alleles of C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1)/FIERY2 (FRY2). In the miRNA processing complex, SE functions as a scaffold to mediate CPL1 interaction with HYL1, which needs to be dephosphorylated for optimal activity. In the absence of CPL1, HYL1 dephosphorylation and hence accurate processing and strand selection from miRNA duplexes are compromised. Our findings thus define a new regulatory step in plant miRNA biogenesis.

DSIF Restricts NF-κB Signaling by Coordinating Elongation with mRNA Processing of Negative Feedback Genes

NF-κB is central for immune response and cell survival, and its deregulation is linked to chronic inflammation and cancer through poorly defined mechanisms. IκBα and A20 are NF-κB target genes and negative feedback regulators. Upon their activation by NF-κB, DSIF is recruited, P-TEFb is released, and their elongating polymerase II (Pol II) C-terminal domain (CTD) remains hypophosphorylated. We show that upon DSIF knockdown, mRNA levels of a subset of NF-κB targets are not diminished; yet much less IκBα and A20 protein are synthesized, and NF-κB activation is abnormally prolonged. Further analysis of IκBα and A20 mRNA revealed that a significant portion is uncapped, unspliced, and retained in the nucleus. Interestingly, the Spt5 C-terminal repeat (CTR) domain involved in elongation stimulation through P-TEFb is dispensable for IκBα and A20 regulation. These findings assign a function for DSIF in cotranscriptional mRNA processing when elongating Pol II is hypophosphorylated and define DSIF as part of the negative feedback regulation of NF-κB.

The Complex NOD-Like Receptor Repertoire of the Coral Acropora digitifera Includes Novel Domain Combinations

Innate immunity in corals is of special interest not only in the context of self-defense but also in relation to the establishment and collapse of their obligate symbiosis with dinoflagellates of the genus Symbiodinium. In innate immunity system of vertebrates, approximately 20 tripartite nucleotide oligomerization domain (NOD)-like receptor proteins that are defined by the presence of a NAIP, CIIA, HET-E and TP1 (NACHT) domain, a C-terminal leucine-rich repeat (LRR) domain, and one of three types of N-terminal effector domain, are known to function as the primary intracellular pattern recognition molecules. Surveying the coral genome revealed not only a larger number of NACHT- and related domain nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)-encoding loci (~500) than in other metazoans but also surprising diversity of domain combinations among the coral NACHT/NB-ARC-containing proteins; N-terminal effector domains included the apoptosis-related domains caspase recruitment domain (CARD), death effector domain (DED), and Death, and C-terminal repeat domains included LRRs, tetratricopeptide repeats, ankyrin repeats, and WD40 repeats. Many of the predicted coral proteins that contain a NACHT/NB-ARC domain also contain a glycosyl transferase group 1 domain, a novel domain combination first found in metazoans. Phylogenetic analyses suggest that the NACHT/NB-ARC domain inventories of various metazoan lineages, including corals, are largely products of lineage-specific expansions. Many of the NACHT/NB-ARC loci are organized in pairs or triplets in the Acropora genome, suggesting that the large coral NACHT/NB-ARC repertoire has been generated at least in part by tandem duplication. In addition, shuffling of N-terminal effector domains may have occurred after expansions of specific NACHT/NB-ARC-repeat domain types. These results illustrate the extraordinary complexity of the innate immune repertoire of corals, which may in part reflect adaptive evolution to a symbiotic lifestyle in a uniquely complex and challenging environment.

Biochemical, Conformational, and Immunogenic Analysis of Soluble Trimeric Forms of Henipavirus Fusion Glycoproteins

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are paramyxoviruses discovered in the mid- to late 1990s that possess a broad host tropism and are known to cause severe and often fatal disease in both humans and animals. HeV and NiV infect cells by a pH-independent membrane fusion mechanism facilitated by their attachment (G) and fusion (F) glycoproteins. Here, several soluble forms of henipavirus F (sF) were engineered and characterized. Recombinant sF was produced by deleting the transmembrane (TM) and cytoplasmic tail (CT) domains and appending a glycosylphosphatidylinositol (GPI) anchor signal sequence followed by GPI-phospholipase D digestion, appending a trimeric coiled-coil (GCNt) domain (sF(GCNt)), or deleting the TM, CT, and fusion peptide domain. These sF glycoproteins were produced as F(0) precursors, and all were apparent stable trimers recognized by NiV-specific antisera. Surprisingly, however, only the GCNt-appended constructs (sF(GCNt)) could elicit cross-reactive henipavirus-neutralizing antibody in mice. In addition, sF(GCNt) constructs could be triggered in vitro by protease cleavage and heat to transition from an apparent prefusion to postfusion conformation, transitioning through an intermediate that could be captured by a peptide corresponding to the C-terminal heptad repeat domain of F. The pre- and postfusion structures of sF(GCNt) and non-GCNt-appended sF could be revealed by electron microscopy and were distinguishable by F-specific monoclonal antibodies. These data suggest that only certain sF constructs could serve as potential subunit vaccine immunogens against henipaviruses and also establish important tools for further structural, functional, and diagnostic studies on these important emerging viruses.

Arabidopsis Fused kinase and the Kinesin‐12 subfamily constitute a signalling module required for phragmoplast expansion

The conserved Fused kinase plays vital but divergent roles in many organisms from Hedgehog signalling in Drosophila to polarization and chemotaxis in Dictyostelium. Previously we have shown that Arabidopsis Fused kinase termed TWO-IN-ONE (TIO) is essential for cytokinesis in both sporophytic and gametophytic cell types. Here using in vivo imaging of GFP-tagged microtubules in dividing microspores we show that TIO is required for expansion of the phragmoplast. We identify the phragmoplast-associated kinesins, PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B, as TIO-interacting proteins and determine TIO-Kinesin-12 interaction domains and their requirement in male gametophytic cytokinesis. Our results support the role of TIO as a functional protein kinase that interacts with Kinesin-12 subfamily members mainly through the C-terminal ARM repeat domain, but with a contribution from the N-terminal kinase domain. The interaction of TIO with Kinesin proteins and the functional requirement of their interaction domains support the operation of a Fused kinase signalling module in phragmoplast expansion that depends upon conserved structural features in diverse Fused kinases.

Pol II CTD kinases Bur1 and Kin28 promote Spt5 CTR-independent recruitment of Paf1 complex

Paf1 complex (Paf1C) is a transcription elongation factor whose recruitment is stimulated by Spt5 and the CDKs Kin28 and Bur1, which phosphorylate the Pol II C-terminal domain (CTD) on Serines 2, 5, and 7. Bur1 promotes Paf1C recruitment by phosphorylating C-terminal repeats (CTRs) in Spt5, and we show that Kin28 enhances Spt5 phosphorylation by promoting Bur1 recruitment. It was unclear, however, whether CTD phosphorylation by Kin28 or Bur1 also stimulates Paf1C recruitment. We find that Paf1C and its Cdc73 subunit bind diphosphorylated CTD repeats (pCTD) and phosphorylated Spt5 CTRs (pCTRs) in vitro, and that cdc73 mutations eliminating both activities reduce Paf1C recruitment in vivo. Phosphomimetic (acidic) substitutions in the Spt5 CTR sustain high-level Paf1C recruitment in otherwise wild-type cells, but not following inactivation of Bur1 or Kin28. Furthermore, inactivating the pCTD/pCTR-interaction domain (PCID) in Cdc73 decreases Paf1C-dependent histone methylation in cells containing non-phosphorylatable Spt5 CTRs. These results identify an Spt5 pCTR-independent pathway of Paf1C recruitment requiring Kin28, Bur1, and the Cdc73 PCID. We propose that pCTD repeats and Spt5 pCTRs provide separate interaction surfaces that cooperate to ensure high-level Paf1C recruitment.