Capping, splicing, and 3' processing are independently stimulated by RNA polymerase II: different functions for different segments of the CTD.

Capping, splicing, and cleavage/polyadenylation of pre-mRNAs are interdependent events that are all stimulated in vivo by the carboxy-terminal domain (CTD) of RNA Pol II. We show that the CTD independently enhances splicing and 3' processing and that stimulation of splicing by enhancers is facilitated by the CTD. We provide evidence that stimulation of 3' processing by the CTD requires contact with the 50-kD subunit of the cleavage stimulation factor, CstF. Overexpression of the CTD-binding domain of CstF p50 had a dominant-negative effect on 3' processing without disrupting the CstF complex. The CTD comprises 52 heptad repeats. The CTD carboxyl terminus including heptads 27-52 supported capping, splicing, and 3' processing but the amino terminus supported only capping. We conclude that the CTD independently stimulates all three major pre-mRNA processing steps and that different regions of the CTD can serve distinct functions in pre-mRNA processing.

Transcription elongation: the 'Foggy' is liftingellipsis.

Transcription can be controlled by regulating either the initiation or elongation of RNA chains. Recent studies highlight the importance of an elongation regulator, Spt5, in controlling gene expression in yeast, fruit fly and zebrafish; Spt5 may provide a link between transcriptional elongation and cell fate.

Assessment of white matter injury following prolonged focal cerebral ischaemia in the rat.

The ability of putative neuroprotective compounds to protect against white matter injury remains poorly investigated due to the lack of suitable methods for assessing white matter injury. This study was therefore designed to investigate the utility of Tau 1 (oligodendrocytes/axons), myelin basic protein (MBP; myelin) and amyloid precursor protein (APP; axons) immunohistochemistry in assessing white matter injury at various times following middle cerebral artery occlusion (MCAO) in the rat. Focal cerebral, ischaemia was induced in halothane-anaesthetised rats using an intraluminal thread model. At 24 h, 1 and 2 weeks following MCAO, white matter injury was assessed using Tau 1, APP, MBP and Luxol-fast blue staining and neuronal injury with cresyl fast violet (CFV). In histologically normal tissue MBP immunoreactivity was detected in myelinated fibre tracts, while Tau 1 and APP were axonally located. At 24 h following permanent MCAO, MBP, and Tau 1 staining remained relatively unchanged within the myelin and axonal compartments of the ischaemic region. In contrast, increased Tau 1 staining was apparent in oligodendrocytes within ischaemic tissue, while APP accumulated in axons surrounding the lesion. At 1 and 2 weeks following transient MCAO, Tau 1 and APP staining was markedly decreased within ischaemic tissue. Marked reduction in MBP levels within ischaemic tissue were not detected until 2 weeks following MCAO. The area of axonal injury as assessed by reduced Tau 1 or APP staining correlated with the area of neuronal damage as assessed by CFV staining. This study shows that MBP, Tau 1 and APP immunohistochemistry can be utilised to assess myelin and axonal integrity following sustained ischaemia using standard image analysis techniques.

MCM proteins are associated with RNA polymerase II holoenzyme.

MCMs are a family of proteins related to ATP-dependent helicases that bind to origin recognition complexes and are required for initiation of DNA replication. We report that antibodies against MCM2(BM28) specifically inhibited transcription by RNA polymerase II (Pol II) in microinjected Xenopus oocytes. Consistent with this observation, MCM2 and other MCMs copurified with Pol II and general transcription factors (GTFs) in high-molecular-weight holoenzyme complexes isolated from Xenopus oocytes and HeLa cells. Pol II and GTFs also copurified with MCMs isolated by anti-MCM3 immunoaffinity chromatography. MCMs were specifically displaced from the holoenzyme complex by antibody against the C-terminal domain (CTD) of Pol II. In addition, MCMs bound to a CTD affinity column, suggesting that their association with holoenzyme depends in part on this domain of Pol II. These results suggest a new function for MCM proteins as components of the Pol II transcriptional apparatus.

Optimization of capillary chromatography ion trap-mass spectrometry for identification of gel-separated proteins.

The current paradigm for protein identification using mass spectrometric derived peptide-mass and fragment-ion data employs computer algorithms which match uninterpreted or partially interpreted fragment-ion data to sequence databases, both protein and translated nucleotide sequence databases. Nucleotide sequence databases continue to grow at a rapid rate for some species, providing an unsurpassed resource for protein identification in those species. Ion-trap mass spectrometers with their ability to rapidly generate fragment-ion spectra in a data-dependent manner with high sensitivity and accuracy has led to their increased use for protein identification. We have investigated various parameters on a commercial ion trap-mass spectrometer to enhance our ability to identify peptides separated by capillary reversed phase-high performance liquid chromatography (RP-HPLC) coupled on-line to the mass spectrometer. By systematically evaluating the standard parameters (ion injection time and number of microscans) together with selection of multiple ions from the full mass range, improved tandem mass spectrometry (MS/MS) spectra were generated, facilitating identification of proteins at a low pmol level. Application of this technology to the identification of a standard protein and an unknown from an affinity-enriched mixture are shown.

Regulation of CDK7 substrate specificity by MAT1 and TFIIH.

The cyclin-dependent kinase (CDK)-activating kinase CAK has been proposed to function in the control of cell cycle progression, DNA repair and RNA polymerase II (pol II) transcription. Most CAK exists as complexes of three subunits: CDK7, cyclin H (CycH) and MAT1. This tripartite CAK occurs in a free form and in association with 'core' TFIIH, which functions in both pol II transcription and DNA repair. We investigated the substrate specificities of different forms of CAK. Addition of the MAT1 subunit to recombinant bipartite CDK7-CycH switched its substrate preference to favour the pol II large subunit C-terminal domain (CTD) over CDK2. We suggest that the MAT1 protein, previously shown to function as an assembly factor for CDK7-CycH, also acts to modulate CAK substrate specificity. The substrate specificities of natural TFIIH and free CAK were also compared. TFIIH had a strong preference for the CTD over CDK2 relative to free CAK. TFIIH, but not free CAK, could efficiently hyperphosphorylate the CTD. In the context of TFIIH, the kinase also acquired specificity for the general transcription factors TFIIE and TFIIF which were not recognized by free CAK. We conclude that the substrate preference of the CDK7-CycH kinase is governed by association with both MAT1 and 'core' TFIIH.

Protective monoclonal antibody defines a circumsporozoite-like glycoprotein exoantigen of Cryptosporidium parvum sporozoites and merozoites.

The apicomplexan protozoan parasite Cryptosporidium parvum causes a diarrheal disease in humans and other mammals for which specific therapy and immunoprophylaxis are unavailable. Passive immunization with Abs against whole C. parvum organisms has variable efficacy in immunocompromised or neonatal hosts. Because apical and surface-exposed zoite Ags of the Apicomplexa are critical to infectivity and targets of protective immunity, we examined the ability of mAbs generated against such Ags in C. parvum sporozoites to passively protect against infection and identify biologically relevant parasite molecules. A panel of mAbs was produced against affinity-purified native Ags using sporozoite apical- and surface-reactive mAb C4A1 as binding ligand. One resulting mAb, designated 3E2, elicited prominent morphologic changes in sporozoites and merozoites characterized by rapid and progressive formation, posterior movement, and release of membranous Ag-mAb precipitates. These changes had a striking resemblance to the malarial circumsporozoite precipitate (CSP) reaction. Sporozoite infectivity was completely neutralized after in vitro exposure to 3E2 and the CSP-like reaction. Furthermore, orally administered 3E2 completely prevented or markedly reduced infection in neonatal BALB/c mice. 3E2 bound to apical complex and surface molecules of zoites and was demonstrated in membranous precipitates by immunoelectron microscopy. In Western blots, 3E2 recognized multiple 46 to approximately 770 kDa sporozoite Ags and an approximately 1300-kDa Ag designated CSL, also expressed by merozoites. CSL was characterized as a soluble glycoprotein exoantigen released by infectious sporozoites. Further, CSL was determined to be the molecular species mechanistically involved in the CSP-like reaction by its identification in SDS-PAGE gels and Western blots of purified membranous precipitates. These findings indicate that CSL has a functional role in sporozoite infectivity and is a candidate molecular target for passive or active immunization against cryptosporidiosis.

The C-terminal domain of RNA polymerase II couples mRNA processing to transcription.

Messenger RNA is produced by RNA polymerase II (pol II) transcription, followed by processing of the primary transcript. Transcription, splicing and cleavage-polyadenylation can occur independently in vitro, but we demonstrate here that these processes are intimately linked in vivo. We show that the carboxy-terminal domain (CTD) of the pol II large subunit is required for efficient RNA processing. Splicing, processing of the 3' end and termination of transcription downstream of the poly(A) site, are all inhibited by truncation of the CTD. We found that the cleavage-polyadenylation factors CPSF and CstF specifically bound to CTD affinity columns and copurified with pol II in a high-molecular-mass complex. Our demonstration of an association between the CTD and 3'-processing factors, considered together with reports of a similar interaction with splicing factors, suggests that an mRNA 'factory' exists which carries out coupled transcription, splicing and cleavage-polyadenylation of mRNA precursors.

5'-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II.

We have investigated the role of the RNA Polymerase II (Pol II) carboxy-terminal domain (CTD) in mRNA 5' capping. Transcripts made in vivo by Pol II with a truncated CTD had a lower proportion of capped 5' ends than those made by Pol II with a full-length CTD. In addition, the enzymes responsible for cap synthesis, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase bound directly to the phosphorylated, but not to the nonphosphorylated, form of the CTD in vitro. These results suggest that: (1) Pol II-specific capping of nascent transcripts in vivo is enhanced by recruitment of the capping enzymes to the CTD and (2) capping is co-ordinated with CTD phosphorylation.

Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors.

We developed two solid-phase reagent systems for microscopic visualization and characterization of melanocyte-stimulating hormone (MSH) receptors of melanoma cells. Multiple copies of [Nle-4,D-Phe-7]-alpha-MSH, a potent analog of alpha-MSH, were conjugated to microspheres (latex beads) or macrospheres (polyamide beads) through a thioether or disulfide bond. Binding between the beads and mouse and human melanoma cells was examined by scanning electron microscopy and by light microscopy. Each mouse and human melanoma cell (of all cell lines) evinced binding to the beads. Binding of the melanotropin conjugates was not restricted to any one phase of the cell cycle. Specificity of binding was demonstrated by several studies. Negative controls included cell types of nonmelanocyte origin (e.g., mammary cancer cells) and beads that lacked the melanotropic ligand or had other attached ligands. Beads with a disulfide-linked melanotropin analog served as a direct control. Treatment of these beads with DTT during or before incubation of the beads with melanoma cells (resulting in release of the MSH analog from the beads) eliminated binding of the beads to melanoma cells. Binding interactions between melanoma cells and melanotropin-bound beads also could be abolished by prior incubation with unconjugated MSH analog. During these experiments, certain membrane receptor-hormone associated phenomena, such as capping (aggregation) of the receptor-ligand complex, also were observed. These results provide visual evidence that MSH receptors are a property common to melanoma cells. Normal human epidermal melanocytes and keratinocytes were also shown to express melanotropin receptors by the same criteria established for melanoma cells.

Melanocortin receptors: identification and characterization by melanotropic peptide agonists and antagonists.

Hormones are chemical messengers released from cells to act on and control the activity of other cells. Hormonal ligands initiate their actions by interacting with receptive substances (Langley, 1906) of the target cells. These receptors are proteins that are either integral components of the cell membrane or are localized cytoplasmically within cells. Ligand-receptor interaction results in either the stimulation or inhibition of cellular activity. Since most hormones bind rather specifically to receptors possessed by their target cells, labeling of hormonal ligands can be utilized to identify and localize cells within an animal. In this report we discuss what is presently known about melanocortin receptors (MCRs) as studied by the use of labeled melanotropic peptide ligands.

Human epidermal melanocyte and keratinocyte melanocortin receptors: visualization by melanotropic peptide conjugated microspheres (latex beads).

The objectives of this research were to determine whether melanocortin receptors are characteristic (constant) membrane markers of human epidermal melanocytes. Methodologies were developed to visualize melanotropin receptors by scanning electron microscopy (SEM). Multiple copies (up to a hundred) of [Nle4,D-Phe7]alpha-MSH, a superpotent analog of alpha-melanocyte stimulating hormone (alpha-MSH), were conjugated to a macromolecular carrier (latex beads: microspheres). Incubation in the presence of the melanotropin-conjugated microspheres resulted in binding of human normal epidermal melanocytes to the beads. Almost every (possibly all) melanocyte possesses melanocortin receptors as visualized by SEM. Specificity of binding of the macromolecular conjugate was demonstrated by several studies: 1) Binding of melanocytes to the microspheres was specific since it could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; 2) microspheres lacking bound ligand did not bind to the melanocytes; 3) microspheres that were first treated with reducing agents (e.g., dithiothreitol) did not subsequently bind to melanocytes; 4) another peptide hormone ligand (e.g., a substance-P analog) attached to the latex beads failed to bind to the cells; 5) B16/F10 mouse melanoma cells known to express melanocortin receptors bound to the microspheres; and 6) cells of nonmelanocyte origin (e.g., mammary cancer cells, small-cell lung cancer cells, fibroblasts) did not bind to the macromolecular conjugate. One exception was that human epidermal keratinocytes also expressed melanocortin receptors as determined by all the criteria established above for epidermal melanocytes. Thus, cell specific melanocortin receptors appear to be characteristic cell surface markers of epidermal melanocytes and keratinocytes.

Distinct activated and non-activated RNA polymerase II complexes in yeast.

We used a transcriptional run-on assay in permeabilized yeast cells to study the distribution of RNA polymerase II (pol II) complexes before and after activation by Gal4. Polymerases were found engaged on the gene at the 5' end before activation, but only appeared at the 3' end after activation. Mutations of the pol II C-terminal domain (CTD), the CTD kinase Kin28 and the holoenzyme subunit Srb2 all inhibited the formation of 3' polymerases in response to activator. However, these mutations did not inhibit the establishment of polymerases at the 5' end. The differences between 3' and 5' ternary complexes suggest that they represent qualitatively distinct 'activated' and 'non-activated' forms of polymerase. The results implicate CTD phosphorylation in a switch from 'non-activated' transcription, which is confined to the 5' end, to an 'activated' mode that traverses the length of the gene.

TFIIH functions in regulating transcriptional elongation by RNA polymerase II in Xenopus oocytes.

We investigated the role of TFIIH in transcription by RNA polymerase II (pol II) in vivo by microinjection of antibodies against this factor into Xenopus oocytes. Five different antibodies directed against four subunits of TFIIH were tested for effects on transcription of coinjected human immunodeficiency virus type 2 and c-myc templates. Each of these antibodies severely reduced the efficiency of elongation through human immunodeficiency virus type 2 and c-myc terminator elements. In contrast, an anti-TFIIB antibody did not inhibit elongation. Anti-TFIIH antibodies also had a much smaller inhibitory effect on total transcription than did anti-TFIIB or anti-pol II large subunit. Three inhibitors of TFIIH kinase activity, H-7, H-8, and dichlororibofuranosylbenzimidazole (DRB), inhibited elongation similarly to anti-TFIIH antibodies. These results strongly suggest a role for TFIIH in the stimulation of transcriptional elongation in vivo.

The transcriptional elongation inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits transcription factor IIH-associated protein kinase.

Regulation of chain elongation by RNA polymerase II can have an important effect on gene expression (Bentley, D. (1995) Curr. Opin. Genet. Dev. 5, 210-216; Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759); however the mechanisms that control this step in transcription are not well understood. The adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) has long been used as an inhibitor of RNA polymerase II elongation, but its target is not known. We show that DRB is a potent inhibitor of Cdk-activating kinase, associated with the general transcription factor TFIIH. Two other inhibitors of this kinase, H-7 and H-8, also inhibited transcriptional elongation. Furthermore, TFIIH kinase bound specifically to the herpes simplex virus VP16 activation domain which stimulates polymerase II elongation in addition to initiation (Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759). Our results suggest that DRB affects transcription by inhibiting the TFIIH-associated kinase and that this kinase functions in the control of elongation by RNA polymerase II.

Nitric oxide loading of the salivary nitric-oxide-carrying hemoproteins (nitrophorins) in the blood-sucking bug Rhodnius prolixus.

The salivary glands of the blood-sucking bug Rhodnius prolixus are formed by a single layer of binucleated epithelial cells surrounded by a double layer of transversely oriented smooth muscle cells. The epithelial cells are rich in rough endoplasmic reticulum and mitochondria and have abundant microvillar projections towards the gland lumen. This cell layer surrounds a relatively large cavity where abundant secretory material is stored. Epithelial cells produce an intense and generalized NADPH diaphorase reaction, in contrast to other tissues such as brain, Malpighian tubules and skeletal muscle. Ultrastructural analysis of the osmiophilic reaction product indicates that it is localized within cytoplasmic vacuoles, a similar location to that of NADPH diaphorase (NO synthetase) activity in neuronal cells of vertebrates. Measurements of the time course of protein accumulation, NADPH diaphorase activity and the degree of nitrosylation of hemoproteins (nitrophorins) in the salivary glands of Rhodnius prolixus nymphs after a blood meal indicate that the nitrophorins are synthesized and accumulate when NO production is low (with a 25% loading of the nitrophorins during the fourth- to fifth-instar molt). NO loading of the nitrophorins increases to 90% after the molt, concomitant with a large increase in the salivary NADPH diaphorase activity. It is concluded that synthesis of NO occurs within the epithelial cells while the nitrophorins are stored extracellularly. It is hypothesized that the luminally oriented microvilli may serve as a diffusion bridge to direct intracellularly produced NO into the luminal cavity, where the nitrophorins are stored.

Regulation of transcriptional elongation by RNA polymerase II.

The process of transcription is a cycle of initiation, elongation and termination steps. Initiation and elongation can be rate-limiting for RNA synthesis, and recent evidence shows that transactivators can stimulate both these steps. A stable modification of the RNA polymerase II complex is probably required to convert it from a non-processive to a processive from.

Transcriptional elongation by RNA polymerase II is stimulated by transactivators.

We report that a variety of transactivators stimulate elongation by RNA polymerase II. Activated transcription complexes have high processivity and are able to read through pausing and termination sites efficiently. In contrast, nonactivated and "squelched" transcription mostly arrests prematurely. Activators differ in the extent to which they stimulate processivity; for example, GAL4-VP16 and GAL4-E1a are more effective than GAL4-AH. The stimulation of elongation can be as important as the stimulation of initiation in activating expression of a reporter gene. We suggest that setting the competence of polymerase II to elongate is an integral part of the initiation step that is controlled by activators cooperating with the general transcription factors.

A protein-binding site in the c-myc promoter functions as a terminator of RNA polymerase II transcription.

Termination of transcription not only allows polymerases that have completed RNA synthesis to recycle, but it also has important functions in transcriptional regulation and in preventing promoter interference. The molecular basis for termination by RNA polymerase II (pol II) is unclear, however. We have identified a termination site in the promoter region of the c-myc gene, whose function correlates with DNA binding by a nuclear factor. When the c-myc gene was transcribed in injected Xenopus oocytes or a HeLa nuclear extract, a fraction of RNA initiated at the first promoter, P1, terminated at two positions, T1A and T1B, which flank the TATA box of the second promoter, P2. T1B is a T-rich sequence that resembles previously identified attenuation sites, but T1A appears to represent a different class of termination site. T1A is situated approximately 10 bases upstream of an element that overlaps the P2 TATA box. Mutagenesis of this element affected both the efficiency and the position at which termination occurred. A 28-base sequence including this element caused a low level of termination when inserted into the alpha-globin gene in either orientation. This sequence bound a factor called TBF I (terminator-binding factor), whose binding specificity correlated with T1A terminator function. We suggest that TBF I may function as a pol II termination factor.

Distinct modes of transcription read through or terminate at the c-myc attenuator.

Premature termination of transcription by RNA polymerase II (pol II) occurs in the 5' region of many viral and cellular genes. Modulation of this process, or attenuation, is an important means of transcriptional control, but its mechanism is unknown. Using injected Xenopus oocytes, the efficiency of the mouse c-myc attenuator was tested when it was placed at various distances from the transcription initiation site. The attenuator functioned with each of six different pol II promoters tested; however, termination efficiency declined markedly when it was placed more than approximately 400 bases from the start site. This decline in attenuator function with distance from the start site coincided with increased sensitivity to the pol II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB). Thus transcription complexes situated further from the promoter appear to have a lower ability to recognize the attenuator and a greater sensitivity to DRB. Furthermore, polymerases which have read through one attenuation site have a reduced ability to terminate at a second site. The results imply that a discrete subset of elongation complexes is capable of premature termination, and that this subset exists only within the first few hundred bases of the transcription unit. Regulation of termination efficiency may be effected by changing the balance between the two modes of transcription committed either to read through or to terminate prematurely.