Transcriptional Polarity Research Articles

The perinucleolar compartment

The perinucleolar compartment (PNC) is a distinct nuclear body that localizes to the nucleolar periphery. The PNC is predominantly found in cancer cells, and recent evidence suggests that PNC prevalence can be a pan-cancer marker for tumors of solid tissue origin. The PNC is a heritable structure enriched with newly transcribed pol III RNAs and RNA-binding proteins, which exchange rapidly with the surrounding nucleoplasm. The structural integrity of the PNC is dependent upon the continuous transcription of pol III RNA and an intact DNA structure. Although the complete structure and function of the PNC remains to be resolved, much progress has been made in the characterization of the PNC in recent years. Here we summarize our current understanding of the dynamics, structure and function of the PNC.

In Vivo Dynamics of Intracistronic Transcriptional Polarity

Transcriptional polarity occurs in Escherichia coli when cryptic Rho-dependent transcription terminators become activated as a consequence of reduced translation. Increased spacing between RNA polymerase and the leading ribosome allows the transcription termination factor Rho to bind to mRNA, migrate to the RNA polymerase, and induce termination. Transcriptional polarity results in decreased synthesis of inefficiently translated mRNAs and, therefore, in decreased expression not only of downstream genes in the same operon (intercistronic polarity) but also of the cistron in which termination occurs (intracistronic polarity). To quantitatively measure the effect of different levels of translation on intracistronic transcription termination, the polarity-prone lacZ reporter gene was fused to a range of mutated ribosome binding sites, repressed to different degrees by local RNA structure. The results show that polarity gradually increases with decreasing frequency of translational initiation, as expected. Closer analysis, with the help of a newly developed kinetic model, reveals that efficient intracistronic termination requires very low translational initiation frequencies. This finding is unexpected because Rho is a relatively small protein that binds rapidly to its RNA target, but it appears to be true also for other examples of transcriptional polarity reported in the literature. The conclusion must be that polarity is more complex than just an increased exposure of the Rho binding site as the spacing between the polymerase and the leading ribosome becomes larger. Biological consequences and possible mechanisms are discussed.

T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus

Rift Valley fever virus (RVFV) is responsible for large and recurrent outbreaks of acute febrile illness among humans and domesticated animals in Africa. It belongs to the family Bunyaviridae, genus Phlebovirus, and its negative-stranded RNA genome consists of three segments. Here, we report the establishment and characterization of two different systems to rescue the RVFV wild-type strain ZH548. The first system is based on the BHK-21 cell clone BSR-T7/5, which stably expresses T7 RNA polymerase (T7 pol). Rescue of wild-type RVFV was achieved with three T7 pol-driven cDNA plasmids representing the viral RNA segments in the antigenomic sense. The second system involves 293T cells transfected with three RNA pol I-driven plasmids for the viral segments and two RNA pol II-driven support plasmids to express the viral polymerase components L and N. It is known that the 5' triphosphate group of T7 pol transcripts strongly activates the antiviral interferon system via the intracellular RNA receptor RIG-I. Nonetheless, both the T7 pol and the pol I/II system were of similar efficiency. This was even true for the rescue of a RVFV mutant lacking the interferon antagonist nonstructural proteins. Further experiments demonstrated that the unresponsiveness of BHK-21 and BSR-T7/5 cells to T7 pol transcripts is most probably due to a deficiency in the RIG-I pathway. Our reverse genetics systems now enable us to manipulate the genome of RVFV and study its virulence mechanisms. Moreover, the finding that BHK-derived cell lines have a compromised RIG-I pathway may explain their suitability for propagating and rescuing a wide variety of viruses.

The complete mitochondrial genome of Symphylella sp. (Myriapoda: Symphyla): Extensive gene order rearrangement and evidence in favor of Progoneata

We determined the complete 14,667 bp mitochondrial DNA sequence of Symphylella sp., the first representative of the Scolopendrellidae (Arthropoda: Myriapoda: Symphyla). With respect to the ancestral arthropod mitochondrial gene order, two protein-coding genes, the rRNAs and 10 of the tRNAs appear to be rearranged. This rearrangement is novel in the arthropods and genes with identical transcriptional polarity are clustered except for trnE, trnN and putative control region (CR), resembling two previously reported diplopod genomes. A duplication/loss (random and non-random)-recombination model was proposed to account for the generation of the gene order in Symphylella sp. All phylogenetic analysis yielded strong support for a clade of Symphyla plus Diplopoda, i.e., Progoneata. However, the phylogenetic position of Myriapoda within Arthropoda remains unclear. The amino acid dataset gives strong support for an affinity to Pancrustacea, while the nucleotide dataset weakly supports Myriapoda grouped with Chelicerata.

Intracistronic transcriptional polarity enhances translational repression: a new role for Rho

Transcriptional polarity in Escherichia coli occurs when cryptic Rho-dependent transcription terminators become activated as a consequence of reduced translation. Whether this is due to an increased spacing between the RNA polymerase and the leading ribosome or to prior functional inactivation of a subpopulation of the mRNAs has been a matter of discussion. Transcriptional polarity results in decreased synthesis of inefficiently translated mRNAs and therefore in decreased expression of downstream genes in the same operon (intercistronic polarity). By analogy, expression of the gene in which the conditional termination occurs is also expected to decrease, but this has so far not been demonstrated experimentally. To study the relevance of this intracistronic polarity for expression regulation in vivo, the polarity-prone IacZ reporter gene was fused to a range of mutated ribosome binding sites, repressed to different degrees by local RNA structure. Quantitative analysis of protein and mRNA synthesis shows that polarity occurs on functionally active mRNA molecules and that it indeed affects expression of the cistron carrying the terminator, thus enhancing the effect of translational repression. These findings point to a novel regulatory function of transcriptional polarity, reminiscent of transcriptional attenuation but opposite in effect.

Inhibition of translation by a short element in the 5' leader of the herpes simplex virus 1 DNA polymerase transcript.

Many viruses regulate gene expression, both globally and specifically, to achieve maximal rates of replication. During herpes simplex virus 1 infection, translation of the DNA polymerase (Pol) catalytic subunit is inefficient relative to other proteins of the same temporal class (D. R. Yager, A. I. Marcy, and D. M. Coen., J. Virol. 64:2217-2225, 1990). To investigate the mechanisms involved in the inefficient translation of Pol and to determine whether this inefficient translation could affect viral replication, we performed a mutagenic analysis of the 5' end of the pol transcript. We found that a short sequence ( approximately 55 bases) in the 5' leader of the transcript is both necessary and sufficient to inhibit translation in rabbit reticulocyte lysates and sufficient to inhibit reporter gene translation in transfected cells. RNase structure mapping experiments indicated that the inhibitory element adopts a structure that contains regions of a double-stranded nature, which may interfere with ribosomal loading and/or scanning. Pol accumulated to approximately 2- to 3-fold-higher levels per mRNA in cells infected with a mutant virus containing a deletion of the approximately 55-base inhibitory element than in cells infected with a control virus containing this element. Additionally, the mutant virus replicated less efficiently than the control virus. These results suggest that the inhibitory element regulates Pol translation during infection and that its inhibition of Pol translation is beneficial for viral replication.

Lrp6 is required for convergent extension duringXenopusgastrulation

Wnt signaling regulates beta-catenin-mediated gene transcription and planar cell polarity (PCP). The Wnt co-receptor, Lrp6, is required for signaling along the beta-catenin arm. We show that Lrp6 downregulation (by morpholino injection) or overexpression in Xenopus embryos disrupts convergent extension, a hallmark feature of Wnt/PCP components. In embryos with decreased Lrp6 levels, cells of the dorsal marginal zone (DMZ), which undergoes extensive cellular rearrangements during gastrulation, exhibit decreased length:width ratios, decreased migration, and increased numbers of transient cytoplasmic protrusions. We show that Lrp6 opposes Wnt11 activity and localizes to the posterior edge of migrating DMZ cells and that Lrp6 downregulation enhances cortical and nuclear localization of Dsh and phospho-JNK, respectively. Taken together, these data suggest that Lrp6 inhibits Wnt/PCP signaling. Finally, we identify the region of the Lrp6 protein with Wnt/PCP activity to a stretch of 36 amino acids, distinct from regions required for Wnt/beta-catenin signaling. We propose a model in which Lrp6 plays a critical role in the switch from Wnt/PCP to Wnt/beta-catenin signaling.

Polarity of Response to Transforming Growth Factor-β1 in Proximal Tubular Epithelial Cells Is Regulated by β-Catenin

Transforming growth factor-beta1 (TGF-beta1)-mediated loss of proximal tubular epithelial cell-cell interaction is regulated in a polarized fashion. The aim of this study was to further explore the polarity of the TGF-beta1 response and to determine the significance of R-Smad-beta-catenin association previously demonstrated to accompany adherens junction disassembly. Smad3 signaling response to TGF-beta1 was assessed by activity of the Smad3-responsive reporter gene construct (SBE)(4)-Lux and by immunoblotting for phospho-Smad proteins. Similar results were obtained with both methods. Apical application of TGF-beta1 led to increased Smad3 signaling compared with basolateral stimulation. Association of Smad proteins with beta-catenin was greater following basolateral TGFbeta-1 stimulation, as was the expression of cytoplasmic Triton-soluble beta-catenin. Inhibition of beta-catenin expression by small interfering RNA augmented Smad3 signaling. Lithium chloride, a GSK-3 inhibitor, increased expression of beta-catenin and attenuated TGF-beta1-dependent Smad3 signaling. Lithium chloride did not influence degradation of Smad3 but resulted in decreased nuclear translocation. Smad2 activation as assessed by Western blot analysis and activity of the Smad2-responsive reporter constructs ARE/MF1 was also greater following apical as compared with basolateral TGFbeta-1 stimulation, suggesting that this is a generally applicable mechanism for the regulation of TGF-beta1-dependent R-Smads. Caco-2 cells are a colonic carcinoma cell line, with known resistance to the anti-proliferative effects of TGF-beta1 and increased expression of beta-catenin. We used this cell line to address the general applicability of our observations. Inhibition of beta-catenin in this cell line by small interfering RNA resulted in increased TGF-beta1-dependent Smad3 phosphorylation and restoration of TGF-beta1 anti-proliferative effects.

Electron Cryomicroscopy Comparison of the Architectures of the Enveloped Bacteriophages ϕ6 and ϕ8

The enveloped dsRNA bacteriophages phi6 and phi8 are the two most distantly related members of the Cystoviridae family. Their structure and function are similar to that of the Reoviridae but their assembly can be conveniently studied in vitro. Electron cryomicroscopy and three-dimensional icosahedral reconstruction were used to determine the structures of the phi6 virion (14 A resolution), phi8 virion (18 A resolution), and phi8 core (8.5 A resolution). Spikes protrude 2 nm from the membrane bilayer in phi6 and 7 nm in phi8. In the phi6 nucleocapsid, 600 copies of P8 and 72 copies of P4 interact with the membrane, whereas in phi8 it is only P4 and 60 copies of a minor protein. The major polymerase complex protein P1 forms a dodecahedral shell from 60 asymmetric dimers in both viruses, but the alpha-helical fold has apparently diverged. These structural differences reflect the different host ranges and entry and assembly mechanisms of the two viruses.

Analysis of RNase P Protein ( rnpA ) Expression in Bacillus subtilis Utilizing Strains with Suppressible rnpA Expression

Bacterial RNase P is composed of an RNA subunit and a single protein subunit (encoded by the rnpB and rnpA genes, respectively). We constructed Bacillus subtilis mutant strains that conditionally express the RNase P protein under control of the xylose promoter (P(xyl)). In one strain (d7), rnpA expression was efficiently repressed in the absence of the inducer xylose, leading to cell growth arrest. Growth could be restored by a second functional rnpA allele. This is the first RNase P protein knockdown strain, providing the first direct proof that the rnpA gene is essential in B. subtilis and, by inference, in other bacteria. We further show (i) that, in the wild-type context, rnpA expression is attenuated by transcriptional polarity and (ii) that translation of rnpA mRNA in B. subtilis can be initiated at two alternative start codons. His-tagged RNase P protein variants are functional in vivo and permit purification of in vivo-assembled holoenzymes by affinity chromatography. Simultaneous expression of plasmid-encoded RNase P RNA and His-tagged protein increased RNase P holoenzyme yields. Massive overproduction of RNase P protein in strain d7 is compatible with cell viability.

Schnurri transcription factors fromDrosophilaand vertebrates can mediate Bmp signaling through a phylogenetically conserved mechanism

Bone Morphogenetic Proteins (Bmps) are secreted growth factors that play crucial roles in animal development across the phylogenetic spectrum. Bmp signaling results in the phosphorylation and nuclear translocation of Smads, downstream signal transducers that bind DNA. In Drosophila, the zinc finger protein Schnurri (Shn) plays a key role in signaling by the Bmp2/Bmp4 homolog Decapentaplegic (Dpp), by forming a Shn/Smad repression complex on defined promoter elements in the brinker (brk) gene. Brk is a transcriptional repressor that downregulates Dpp target genes. Thus, brk inhibition by Shn results in the upregulation of Dpp-responsive genes. We present evidence that vertebrate Shn homologs can also mediate Bmp responsiveness through a mechanism similar to Drosophila Shn. We find that a Bmp response element (BRE) from the Xenopus Vent2 promoter drives Dpp-dependent expression in Drosophila. However, in sharp contrast to its activating role in vertebrates, the frog BRE mediates repression in Drosophila. Remarkably, despite these opposite transcriptional polarities, sequence changes that abolish cis-element activity in Drosophila also affect BRE function in Xenopus. These similar cis requirements reflect conservation of trans-acting factors, as human Shn1 (hShn1; HIVEP1) can interact with Smad1/Smad4 and assemble an hShn1/Smad complex on the BRE. Furthermore, both Shn and hShn1 activate the BRE in Xenopus embryos, and both repress brk and rescue embryonic patterning defects in shn mutants. Our results suggest that vertebrate Shn proteins function in Bmp signal transduction, and that Shn proteins recruit coactivators and co-repressors in a context-dependent manner, rather than acting as dedicated activators or repressors.

Mitochondrial genome of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa): A linear DNA molecule encoding a putative DNA-dependent DNA polymerase

The 16,937-nuceotide sequence of the linear mitochondrial DNA (mt-DNA) molecule of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa) – the first mtDNA sequence from the class Scypozoa and the first sequence of a linear mtDNA from Metazoa – has been determined. This sequence contains genes for 13 energy pathway proteins, small and large subunit rRNAs, and methionine and tryptophan tRNAs. In addition, two open reading frames of 324 and 969 base pairs in length have been found. The deduced amino-acid sequence of one of them, ORF969, displays extensive sequence similarity with the polymerase [but not the exonuclease] domain of family B DNA polymerases, and this ORF has been tentatively identified as dnab. This is the first report of dnab in animal mtDNA. The genes in A. aurita mtDNA are arranged in two clusters with opposite transcriptional polarities; transcription proceeding toward the ends of the molecule. The determined sequences at the ends of the molecule are nearly identical but inverted and lack any obvious potential secondary structures or telomere-like repeat elements. The acquisition of mitochondrial genomic data for the second class of Cnidaria allows us to reconstruct characteristic features of mitochondrial evolution in this animal phylum.

Transcriptional Control of Activation-Induced Cytidine Deaminase and Error-Prone DNA Polymerases Is Functionally Mature in the B Cells of Infants at Birth

Somatic hypermutation (SHM) of immunoglobulin genes requires activation-induced cytidine deaminase (AID). The error-prone DNA polymerases, such as Pol η, Pol ζ, and Pol ι, also have been implicated in the process. Human adult antibodies directed to microbial pathogens are increased in affinity and function compared with those of infants. Adult antibodies achieve this increased affinity through somatic mutations, which are lacking in the B cells of infants. It is unknown if infant B cells are capable of upregulating the cell machinery needed to introduce mutations after stimulation through the antigen receptor. We show here that infant B cells exhibit similar kinetics and magnitude of transcription of AID and pol η genes and only marginally lower levels of pol ι and pol ζ genes after stimulation through the B cell receptor. These data suggest that the ability to upregulate gene transcription of enzymes mediating SHM is not a limiting determinant of the functional quality of infant antibody responses.

Translation initiation with GUC codon in the archaeon Halobacterium salinarum: implications for translation of leaderless mRNA and strict correlation between translation initiation and presence of mRNA

We have investigated whether anticodon sequence mutant of an archaeal initiator tRNA can initiate protein synthesis using reporter genes carrying mutations in the initiation codon. Halobacterium salinarum was used as the model organism and the bacterio-opsin gene (bop), which encodes the precursor of the protein component of the purple membrane protein bacterio-opsin (Bop), was chosen as the reporter. We demonstrate that a CAU to GAC anticodon sequence mutant of Haloferax volcanii initiator tRNA can initiate Bop protein synthesis using GUC as the initiation codon in H. salinarum. We generated four mutant bop genes, each carrying the AUG to GUC initiation codon mutation, with or without a compensatory mutation to maintain a predicted stem-loop structure at the 5'-end of the bop mRNA, and with or without mutations to test translation initiation at a site corresponding to the amino terminus of mature bacterio-opsin. H. salinarum chromosomal recombinants containing these mutant genes were phenotypically Pum- (purple membrane negative). Upon transformation with a plasmid carrying the mutant initiator tRNA gene, only strains designed to maintain the bop mRNA stem-loop structure produced Bop and were phenotypically Pum+ as indicated by purple colony colour, and immunoblotting and spectral analysis of cell extracts. Thus GUC can serve as an initiation codon in archaea and the stem-loop structure in the bop mRNA is important for translation. Interestingly, for the same mutant mRNA, only transformants that produce Bop protein contain bop mRNA. These results suggest either a strong coupling between translation and mRNA stability or strong transcriptional polarity in H. salinarum.

Cloning and functional expression of glucose dehydrogenase complex of Burkholderia cepacia in Escherichia coli

The thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia sp. SM4 is composed of a catalytic subunit (alpha), an electron transfer subunit (beta), and a small gamma subunit of unknown function. We cloned a 1428-nucleotide gene encoding the beta subunit located immediately downstream of the alpha subunit. This completes the isolation of the genes encoding the three components of the GDH complex, which are clustered very close together with the same transcription polarity in the order gammaalphabeta. The deduced beta subunit amino acid sequence contains three typical heme-binding motifs and was 44-49% identical to the cytochrome c subunits of other FAD-dependent dehydrogenase complexes. The GDHgammaalphabeta complex of B. cepacia was successfully expressed in a fully active form in Escherichia coli by co-expression with cytochrome c maturation genes. Recombinant expression of the GDH complex was also found to restore glucose-dependent respiration in a GDH mutant of E. coli.

An Ising model of transcription polarity in bacterial chromosomes

Bacterial genes form clusters of the same transcription polarity and typically exhibit a preference to be coded on the leading strand of replication. An Ising model is proposed to quantify these two phenomena by analogy to the behavior of magnetic dipoles (spins) in a one-dimensional lattice. Corresponding to magnetic forces that co-orient adjacent spins and align them with an externally applied field, we imagine pseudo-forces that influence transcription polarity. Bonds of uniform strength 1 2 J between adjacent sites will model the adhesive (or repulsive) interactions while a polarity entraining force of strength H has the direction of replication. Ten bacterial chromosomes are reduced to spin configurations from which the model parameters are estimated by the method of maximum likelihood under the assumption of thermal equilibrium, following the application of established methods to locate replication origins and termini. χ 2-tests show that the model fits the data well in about half the cases but cluster size exhibits excess variance in general. These findings lead to a speculative interpretation of the pseudo-forces as the net effects of numerous insertions and deletions that succeed or fail according to their impact on the motions of enzymatic complexes involved in replication and transcription.

Kinetics and Thermodynamics Make Different Contributions to RNA Folding In Vitro and in Yeast

RNAs somehow adopt specific functional structures despite the capacity to form alternative nonfunctional structures with similar stabilities. We analyzed RNA assembly during transcription in vitro and in yeast using hairpin ribozyme self-cleavage to assess partitioning between functional ribozyme structures and nonfunctional stem loops. Complementary insertions located upstream of the ribozyme inhibited ribozyme assembly more than downstream insertions during transcription in vitro, consistent with a sequential folding model in which the outcome is determined by the structure that forms first. In contrast, both upstream and downstream insertions strongly inhibited assembly of the same ribozyme variants when expressed as chimeric mRNAs in yeast, indicating that inhibitory stem loops can form even after the entire ribozyme sequence has been transcribed. Evidently, some feature unique to the intracellular environment modulates the influence of transcription polarity and enhances the contribution of thermodynamic stability to RNA folding in vivo.

Bacteriophage Mu Targets the Trinucleotide Sequence CGG

Target specificity for bacteriophage Mu was studied using a new phage derivative that enabled cloning of Mu-host junctions from phage DNA. Insertions distributed throughout the chromosome showed no orientation bias with respect to transcription or replication polarity. Genes with a high frequency of the triplet CGG were preferred targets.

The ubiquitin ligase SCFGrr1 is necessary for pheromone sensitivity in Saccharomyces cerevisiae

The presence of the appropriate pheromone induces alpha and a cells of the yeast Saccharomyces cerevisiae to activate both changes in transcriptional expression and cell polarity that eventually lead to the mating of alpha and a cells to form a/alpha diploid cells. A third response after exposure to mating pheromone is a transient cell cycle arrest, allowing synchronization of the two cell types in G1 prior to cell fusion. At least in part, this cell cycle arrest requires the inactivation of Cln-kinase activity through transcriptional inactivation of the CLN1 and CLN2 genes, degradation of the Cln proteins and direct inhibition of Cln-kinase complexes. Here we report that GRR1, which encodes a substrate recognition subunit of SCF complexes, is critical for pheromone sensitivity and likely for this arrest. Loss of SCF(Grr1) function by deletion of the GRR1 gene causes pheromone resistance. However, deletion of CLN1 and CLN2 restores pheromone sensitivity to grr1Delta cells. Thus, rapid loss of Cln-kinase activity during mating may require coordinated inactivation of the Cln-kinase complexes, inactivation of CLN transcription and SCF(Grr1)-dependent Cln degradation.

Structural and functional analysis of a 0.5-Mb chicken region orthologous to the imprinted mammalianAscl2/Mash2–Igf2–H19region

Previous studies revealed that Igf2 and Mpr/Igf2r are imprinted in eutherian mammals and marsupials but not in monotremes or birds. Igf2 lies in a large imprinted cluster in eutherians, and its imprinting is regulated by long-range mechanisms. As a step to understand how the imprinted cluster evolved, we have determined a 490-kb chicken sequence containing the orthologs of mammalian Ascl2/Mash2, Ins2 and Igf2. We found that most of the genes in this region are conserved between chickens and mammals, maintaining the same transcriptional polarities and exon-intron structures. However, H19, an imprinted noncoding transcript, was absent from the chicken sequence. Chicken ASCL2/CASH4 and INS, the orthologs of the imprinted mammalian genes, showed biallelic expression, further supporting the notion that imprinting evolved after the divergence of mammals and birds. The H19 imprinting center and many of the local regulatory elements identified in mammals were not found in chickens. Also, a large segment of tandem repeats and retroelements identified between the two imprinted subdomains in mice was not found in chickens. Our findings show that the imprinted genes were clustered before the emergence of imprinting and that the elements associated with imprinting probably evolved after the divergence of mammals and birds.