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PubMed1995

Cell size regulation, a mechanism that controls cellular RNA accumulation: consequences on regulation of the ubiquitous transcription factors Oct1 and NF-Y and the liver-enriched transcription factor DBP.

Cell sizes can differ vastly between cell types in individual metazoan organisms. In rat liver, spleen, and thymus, differences in average cell size roughly reflect differences in RNA:DNA ratios. For example, hepatocytes were found to have a cytoplasmic:nuclear volume ratio and an RNA:DNA ratio which were 34- and 21-fold higher, respectively, than those in thymocytes. RNA synthesis per DNA-equivalent in the hepatocytes was 25-fold greater than that in thymocytes, suggesting that differences in overall transcriptional activity, not differences in overall RNA stability, were primarily responsible for determining cellular RNA:DNA ratios. The mechanisms determining the capacity of large cells to synthesize and accumulate more ubiquitous cytoplasmic macromolecules, such as ribosomes, than smaller cells is entitled "cell size regulation." Cell size regulation may have important consequences on the tissue distribution of transcription factors. Thus, in liver, lung, kidney, spleen, and brain, cellular levels of the mRNA encoding the leucine zipper protein DBP correlate closely to cellular RNA:DNA ratios. Our results suggest that DBP mRNA levels, like rRNA levels, are transcriptionally determined. Thus the dbp gene, like the ribosomal genes, may be subject to cell size regulation. As a consequence, nuclei from liver, a tissue with a very large average cell size, accumulated higher levels of DBP protein than nuclei from small-celled tissues, such as spleen or lung. In contrast to DBP, the ubiquitous transcription factors Oct1 and NF-Y escaped cell size control. Nuclei from most tissues contained similar amounts of these factors irrespective of cell size. Likewise, tissues with large or small average cell sizes contained similar levels of the mRNAs encoding Oct1 or NF-Ya, one of the subunits of the heteromeric CCAAT-binding factor NF-Y, per DNA-equivalent. Interestingly, mRNA encoding NF-Yb, another subunit of NF-Y, was subject to cell size regulation. Our results suggest that NF-Yb protein escapes cell size regulation at a posttranslational level.

Animals↗
PubMed2002

Editing machines: the complexities of trypanosome RNA editing.

The assembly and disassembly of ribonucleoprotein complexes containing substrate precursor mRNAs and guide RNAs is crucial to the initiation and propagation of RNA editing. We discuss here the composition of these complexes and how their assembly may regulate RNA editing.

Animals↗
PubMed1999

Ribonuclease III processing of coaxially stacked RNA helices.

The RNase III family of endoribonucleases participates in maturation and decay of cellular and viral transcripts by processing of double-stranded RNA. RNase III degradation is inherent to most antisense RNA-regulated gene systems in Escherichia coli. In the hok/sok system from plasmid R1, Sok antisense RNA targets the hok mRNA for RNase III-mediated degradation. An intermediate in the pairing reaction between Sok RNA and hok mRNA forms a three-way junction. A complex between a chimeric antisense RNA and hok mRNA that mimics the three-way junction was cleaved by RNase III both in vivo and in vitro. Footprinting using E117A RNase III binding to partially complementary RNAs showed protection of the 13 base pairs of interstrand duplex and of the bottom part of the transcriptional terminator hairpin of the antisense RNA. This suggests that the 13 base pairs of RNA duplex are coaxially stacked on the antisense RNA terminator stem-loop and that each stem forms a monomer half-site, allowing symmetrical binding of the RNase III dimer. This processing scheme shows an unanticipated diversity in RNase III substrates and may have a more general implication for RNA metabolism.

Bacterial Proteins↗
PubMed2003

Iron uptake by Escherichia coli.

Ferric iron is transported into Escherichia coli by a number of chelating compounds. Iron transport through the outer membrane by citrate, ferrichrome, enterobactin, aerobactin, yersiniabactin, and heme is catalyzed by highly specific proteins and across the cytoplasmic membrane by ABC transport systems with lower specificity. Transport across the outer membrane requires energy, which is provided by the proton motive force of the cytoplasmic membrane and transmitted to the outer membrane via the TonB-ExbB-ExbD proteins. Binding of substrates induces large long-range structural changes in the transport proteins, but does not open the channel. It is thought that the channel is opened by energy input from the cytoplasmic membrane. Although a basic understanding of how the transport proteins might function has been obtained from the crystal structures of three outer membrane proteins of E. coli and from many genetic and biochemical experiments, numerous fundamental questions still remain open. Transcription of the transport protein genes is regulated by the Fur protein, which when loaded with ferrous iron functions as a repressor. Fur also positively regulates genes of iron-containing proteins by repressing synthesis of an anti-sense RNA. Regulation of ferric citrate transport genes via a transmembrane device has become the paradigm of the regulation of a variety of systems, including the hypersensitivity response of plants to bacterial infections.

Biological Transport, Active↗
PubMed2026

Distinct STRIPAK subunits drive conserved and subunit-specific signaling programs in Cryptococcus neoformans.

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a conserved protein phosphatase 2A (PP2A)-associated signaling hub that integrates kinase-phosphatase networks, yet its roles in human fungal pathogens remain poorly defined. Here, we dissected STRIPAK functions in the opportunistic pathogen Cryptococcus neoformans by combining genetic, genomic, virulence, and phosphoproteomic analyses across mutants lacking individual STRIPAK subunits. Loss of the core STRIPAK components via PPH22, FAR8, FAR9, or FAR11 mutations caused severe defects in growth, stress adaptation, cell cycle progression, and morphogenesis, accompanied by widespread aneuploidy and genome instability. In murine infection models, far11Δ strains were avirulent, whereas far9Δ mutants caused delayed but ultimately fatal disease and underwent host-associated genome remodeling, with recovered isolates exhibiting chromosome 11 amplification despite no consistent in vitro fitness advantage. In contrast, deletion of MOB3 produced a hypervirulent phenotype. mob3Δ cells exhibited enhanced transmigration across an in vitro blood-brain barrier model, increased survival in macrophages, and generated small-cell morphotypes, features associated with increased dissemination. Phosphoproteomic profiling revealed extensive and overlapping phosphorylation changes among core STRIPAK mutants, affecting pathways involved in signaling, cytoskeletal, cell cycle control, chromatin regulation, RNA metabolism, and stress responses. Conversely, mob3Δ mutants displayed a smaller, largely distinct phosphoproteomic signature. Network and functional enrichment analyses highlighted STRIPAK-dependent regulation of TORC2-associated signaling, MAPK/GTPase signaling, autophagy, nuclear transport, RNA processing, DNA replication, and ribosome biogenesis. Together, these findings establish STRIPAK as a coordinator of genome stability, morphological plasticity, stress adaptation, and virulence in C. neoformans, and demonstrate that individual STRIPAK subunits drive shared yet divergent signaling outputs that shape host-pathogen interactions.IMPORTANCEFungal pathogens must rapidly adapt their growth, morphology, and stress responses to survive within the host, requiring precise coordination of cellular signaling pathways. The conserved striatin-interacting phosphatase and kinase (STRIPAK) complex controls key developmental programs in eukaryotes, but its roles in fungal pathogenesis are not fully defined. We previously showed that STRIPAK is important for genome stability, development, and virulence in the opportunistic human fungal pathogen Cryptococcus neoformans. Here, we define how individual STRIPAK subunits differentially regulate fungal morphogenesis, genome plasticity, host adaptation, and virulence, revealing both shared and subunit-specific functions within this conserved signaling complex. Core STRIPAK mutants exhibit severe growth and stress-response defects and attenuation of virulence, whereas loss of the Mob3 subunit promotes hypervirulence by enhancing dissemination and persistence within the host. Phosphoproteomic profiling reveals that individual STRIPAK components exert shared yet distinct control over phosphorylation networks that shape host-pathogen interactions, establishing STRIPAK as a central signaling hub and a potential target for antifungal intervention.

Cryptococcus neoformans↗
PubMed2005

twin, a CCR4 homolog, regulates cyclin poly(A) tail length to permit Drosophila oogenesis.

Cyclins regulate progression through the cell cycle. Control of cyclin levels is essential in Drosophila oogenesis for the four synchronous divisions that generate the 16 cell germ line cyst and for ensuring that one cell in each cyst, the oocyte, is arrested in meiosis, while the remaining fifteen cells become polyploid nurse cells. Changes in cyclin levels could be achieved by regulating transcription, translation or protein stability. The proteasome limits cyclin protein levels in the Drosophila ovary, but the mechanisms regulating RNA turnover or translation remain largely unclear. Here, we report the identification of twin, a homolog of the yeast CCR4 deadenylase. We show that twin is important for the number and synchrony of cyst divisions and oocyte fate. Consistent with the deadenylase activity of CCR4 in yeast, our data suggest that Twin controls germ line cyst development by regulating poly(A) tail lengths of several targets including Cyclin A (CycA) RNA. twin mutants exhibit very low expression of Bag-of-marbles (Bam), a regulator of cyst division, indicating that Twin/Ccr4 activity is necessary for wild-type Bam expression. Lowering the levels of CycA or increasing the levels of Bam suppresses the defects we observe in twin ovaries, implicating CycA and Bam as downstream effectors of Twin. We propose that Twin/Ccr4 functions during early oogenesis to coordinate cyst division, oocyte fate specification and egg chamber maturation.

Adenosine↗
PubMed2026

Distinct STRIPAK subunits drive conserved and subunit-specific signaling programs in Cryptococcus neoformans.

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a conserved PP2A-associated signaling hub that integrates kinase-phosphatase networks, yet its roles in human fungal pathogens remain poorly defined. Here, we dissected STRIPAK functions in the opportunistic pathogen Cryptococcus neoformans by combining genetic, genomic, virulence, and phosphoproteomic analyses across mutants lacking individual STRIPAK subunits. Loss of the core STRIPAK components via PPH22, FAR8, FAR9, or FAR11 mutations caused severe defects in growth, stress adaptation, cell-cycle progression, and morphogenesis, accompanied by widespread aneuploidy and genome instability. In murine infection models, far11Δ strains were avirulent, whereas far9Δ mutants caused delayed but ultimately fatal disease and underwent host-associated genome remodeling, with recovered isolates exhibiting chromosome 11 amplification despite no consistent in vitro fitness advantage. In contrast, deletion of MOB3 produced a hypervirulent phenotype. mob3Δ cells exhibited enhanced transmigration across an in vitro blood-brain barrier model, increased survival in macrophages, and generated small-cell morphotypes, features associated with increased dissemination. Phosphoproteomic profiling revealed extensive and overlapping phosphorylation changes among core STRIPAK mutants, affecting pathways involved in signaling, cytoskeletal and cell-cycle control, chromatin regulation, RNA metabolism, and stress responses. Conversely, mob3Δ mutants displayed a smaller, largely distinct phosphoproteomic signature. Network and functional enrichment analyses highlighted STRIPAK-dependent regulation of TORC2-associated signaling, MAPK/GTPase signaling, autophagy, nuclear transport, RNA processing, DNA replication, and ribosome biogenesis. Together, these findings establish STRIPAK as a coordinator of genome stability, morphological plasticity, stress adaptation, and virulence in C. neoformans, and demonstrate that individual STRIPAK subunits drive shared yet divergent signaling outputs that shape host-pathogen interactions.

Journal Article↗
PubMed2001

fusilli, an essential gene with a maternal role in Drosophila embryonic dorsal-ventral patterning.

The Drosophila fusilli (fus) gene was identified in a genetic screen for dominant maternal enhancers of an unusual dorsalizing mutation in the cactus gene, cact(E10). While females that are heterozygous for the cact(E10) allele produce embryos with wild-type dorsal-ventral patterning, more than 90% of the embryos produced by females that are heterozygous for both cact(E10) and the fus(1) mutation are weakly dorsalized. Loss of fusilli activity causes lethality during embryogenesis but not dorsal-ventral patterning defects, indicating that fusilli is important in more than one developmental process. The fusilli gene encodes a protein with RNA binding motifs related to those in mammalian hnRNP F and H, which play roles in regulated RNA splicing. The fusilli RNA is not present in the oocyte or early embryo, and germ-line clones of fusilli mutations have no maternal effect on dorsal-ventral patterning, indicating that the fusilli maternal effect does not depend on germ-line expression of the gene. Because the fusilli RNA is present in ovarian follicle cells, we propose that fusilli acts downstream of the Drosophila EGF receptor to control the biogenesis of follicle cell transcripts that control the initial dorsal-ventral asymmetry of the embryo.

Amino Acid Sequence↗
PubMed2002

Temperature sensitivity of RNA editing and intron splicing reactions in the plastid ndhB transcript.

Primary transcripts in higher plant cell organelles undergo a series of essential RNA processing steps, including 5'- and 3'-terminal maturation, intron splicing and RNA editing. Most of the protein factors involved in these RNA processing mechanisms are still unknown. Also, little is known about the environmental and developmental factors regulating RNA processing events in plastids. Here we have tested the influence of the environmental factor temperature on RNA processing in the ndhB mRNA from tobacco plastids. We find that, whereas temperature increase to 37 degrees C fully inhibits RNA editing at only one out of nine sites in the tobacco ndhB transcript, further temperature increase to 42 degrees C selectively blocks editing at two additional sites. As these temperature effects are strictly site-specific, our findings provide evidence for the existence of site-specific editing factors which exhibit differential temperature sensitivity. Furthermore, splicing of the group II intron present in the ndhB pre-mRNA is largely blocked at 42 degrees C, suggesting that chloroplast RNA processing steps are more sensitive to high temperature than chloroplast transcription. Our findings may also suggest that impaired plastid RNA processing contributes to the loss of chloroplast function upon plant growth at high temperature.

Hot Temperature↗
PubMed2005

Nowa1p and Nowa2p: novel putative RNA binding proteins involved in trans-nuclear crosstalk in Paramecium tetraurelia.

BACKGROUND: The germline genome of ciliates is extensively rearranged during development of a new somatic macronucleus from the germline micronucleus, a process that follows sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) and multicopy transposons are eliminated, whereas cellular genes are amplified to approximately 800 n. For a subset of IESs, introduction of the IES sequence into the maternal (prezygotic) macronucleus specifically inhibits excision of the homologous IES in the developing zygotic macronucleus. This and other homology-dependent maternal effects have suggested that rearrangement patterns are epigenetically determined by an RNA-mediated, trans-nuclear comparison, involving the RNA interference pathway, of germline and somatic genomes. RESULTS: We report the identification of novel developmentally regulated RNA binding proteins, Nowa1p and Nowa2p, which are required for the survival of sexual progeny. Green fluorescent protein (GFP) fusions show that Nowa1p accumulates into the maternal macronucleus shortly before meiosis of germline micronuclei and is later transported to developing macronuclei. Nowa1p/2p depletion impairs the elimination of transposons and of those IESs that are controlled by maternal effects, confirming the existence of distinct IES classes. CONCLUSIONS: The results indicate that Nowa proteins are essential components of the trans-nuclear-crosstalk mechanism that is responsible for epigenetic programming of genome rearrangements. We discuss implications for the current models of genome scanning in ciliates, a process related to the formation of heterochromatin by RNA interference in other eukaryotes.

Amino Acid Sequence↗
PubMed2003

Gene regulation at the RNA layer: RNA binding proteins in intercellular signaling networks.

Transcriptional regulators are sometimes believed to be the only targets through which signal transduction pathways regulate gene expression. Although it is certainly true that many well-characterized intercellular signaling pathways operate by modifying the activity of specific transcription factors, an increasing body of evidence indicates that external signals can modulate gene expression by posttranscriptional mechanisms. RNA binding motifs are combined with other conserved domains, such as protein-interaction domains and consensus phosphorylation motifs, to allow gene expression to be regulated at the level of the RNA in response to extracellular signals. In this review, I discuss evidence that reveals how a particular family of RNA binding proteins, called signal transduction and activation of RNA (STAR) proteins, function in signaling and in the development of multicellular organisms. Furthermore, insulin and related growth factors regulate cell growth, at least in part, by moderating the activity of eukaryotic initiation factor 4E (eIF4E)-binding protein (4EBP), a protein that does not bind RNA directly but inhibits the activity of eIF4E, which is an mRNA cap-binding protein. I discuss the evidence linking insulin signaling to 4EBP phosphorylation. Finally, several other genes have been identified from invertebrate model organisms that encode RNA binding proteins and whose mutant phenotypes implicate them in intercellular signaling, but for which the mechanisms of function currently are unclear. The study of these and other similar genes is likely to uncover a diversity of roles for RNA binding proteins in signal transduction.

Amino Acid Motifs↗
PubMed2005

Effect of RyhB small RNA on global iron use in Escherichia coli.

RyhB is a noncoding RNA regulated by the Fur repressor. It has previously been shown to cause the rapid degradation of a number of mRNAs that encode proteins that utilize iron. Here we examine the effect of ectopic RyhB production on global gene expression by microarray analysis. Many of the previously identified targets were found, as well as other mRNAs encoding iron-binding proteins, bringing the total number of regulated operons to at least 18, encoding 56 genes. The two major operons involved in Fe-S cluster assembly showed different behavior; the isc operon appears to be a direct target of RyhB action, while the suf operon does not. This is consistent with previous findings suggesting that the suf genes but not the isc genes are important for Fe-S cluster synthesis under iron-limiting conditions, presumably for essential iron-binding proteins. In addition, we observed repression of Fur-regulated genes upon RyhB expression, interpreted as due to intracellular iron sparing resulting from reduced synthesis of iron-binding proteins. Our results demonstrate the broad effects of a single noncoding RNA on iron homeostasis.

Bacterial Proteins↗
PubMed2003

Gene expression and viral prodution in latently infected, resting CD4+ T cells in viremic versus aviremic HIV-infected individuals.

The presence of HIV-1 in latently infected, resting CD4(+) T cells has been clearly demonstrated in infected individuals; however, the extent of viral expression and the underlying mechanisms of the persistence of HIV-1 in this viral reservoir have not been fully delineated. Here, we show that resting CD4(+) T cells from the majority of viremic patients are capable of producing cell-free HIV-1 spontaneously ex vivo. The levels of HIV-1 released by resting CD4(+) T cells were not significantly reduced in the presence of inhibitors of cellular proliferation and viral replication. However, resting CD4(+) T cells from the majority of aviremic patients failed to produce virions, despite levels of HIV-1 proviral DNA and cell-associated HIV-1 RNA comparable to viremic patients. The DNA microarray analysis demonstrated that a number of genes involving transcription regulation, RNA processing and modification, and protein trafficking and vesicle transport were significantly upregulated in resting CD4(+) T cells of viremic patients compared to those of aviremic patients. These results suggest that active viral replication has a significant impact on the physiologic state of resting CD4(+) T cells in infected viremic patients and, in turn, allows release of HIV-1 without exogenous activation stimuli. In addition, given that no quantifiable virions were produced by the latent viral reservoir in the majority of aviremic patients despite the presence of cell-associated HIV-1 RNA, evidence for transcription of HIV-1 RNA in resting CD4(+) T cells of aviremic patients should not necessarily be taken as direct evidence for ongoing viral replication during effective therapy.

Base Sequence↗
PubMed1998

The helC gene encodes a putative DEAD-box RNA helicase required for development in Dictyostelium discoideum.

DEAD-box RNA helicases, defined by the sequence Asp-Glu-Ala-Asp (DEAD, in single-letter amino-acid code), regulate RNA unwinding and secondary structure in an ATP-dependent manner in vitro [1] and control mRNA stability and protein translation. Both yeast and mammals have large families of DEAD-box proteins, many of unknown function. We have disrupted a Dictyostelium discoideum gene, helC, which encodes helicase C, a member of the DEAD-box family of RNA helicases that shows strong homology to the product of the essential Saccharomyces cerevisiae gene dbp5 [2] and to related helicases in mouse and Schizosaccharomyces pombe. The HelC protein also shows weaker homology to the translation initiation factor elF-4a. Other DEAD-box-containing proteins, which are less closely related to HelC, have been implicated in developmental roles in Drosophila [3] and Xenopus laevis; one example is the Xenopus Vasa-like protein (XVLP) [4-6]. In Drosophila and Xenopus, Vasa and XVLP, respectively, are required for the establishment of tissue polarity during development. In yeast, DEAD-box helicases such as Prp8 [7] are components of the spliceosome and connect pre-mRNA splicing with the cell cycle. Disruption of the helC gene in D. discoideum led to developmental asynchrony, failure to differentiate and aberrant morphogenesis. We postulate that one reason for the existence of large families of homologous DEAD-box proteins in yeast, mammals and Dictyostelium could be that some DEAD-box proteins have developmentally specific roles regulating protein translation or mRNA stability.

Animals↗
PubMed1997

Identification of elongin C sequences required for interaction with the von Hippel-Lindau tumor suppressor protein.

Elongin C is a 112-amino acid protein that is found in mammalian cells as a positive regulatory subunit of heterotrimeric RNA polymerase II elongation factor Elongin (SIII) and as a component of a multiprotein complex containing the von Hippel-Lindau (VHL) tumor suppressor protein. As a subunit of the Elongin complex, Elongin C interacts directly with the transcriptionally active Elongin A subunit and potently induces its elongation activity; in addition, Elongin C interacts with the ubiquitin-like Elongin B subunit, which regulates the interaction of Elongin C with Elongin A. As a component of the VHL complex, Elongin C interacts directly with both Elongin B and the VHL protein. Binding of the VHL protein to Elongin C was found to prevent Elongin C from interacting with and activating Elongin A in vitro, leading to the proposal that one function of the VHL protein may be to regulate RNA polymerase II elongation by negatively regulating the Elongin complex. In this report, we identify Elongin C sequences required for its interaction with the VHL protein. We previously demonstrated that the ability of Elongin C to bind and activate Elongin A is sensitive to mutations in the C-terminal half of Elongin C, as well as to mutations in an N-terminal Elongin C region needed for formation of the Elongin BC complex. Here we show that interaction of Elongin C with the VHL tumor suppressor protein depends strongly on sequences in the C terminus of Elongin C but is independent of the N-terminal Elongin C region required for binding to Elongin B and for binding and activation of Elongin A. Taken together, our results are consistent with the proposal that the VHL protein negatively regulates Elongin C activation of the Elongin complex by sterically blocking the interaction of C-terminal Elongin C sequences with Elongin A. In addition, our finding that only a subset of Elongin C sequences required for its interaction with Elongin A are critical for binding to VHL may offer the opportunity to develop reagents that selectively interfere with Elongin and VHL function.

Amino Acid Sequence↗
PubMed1990

micF RNA binds to the 5' end of ompF mRNA and to a protein from Escherichia coli.

micF RNA regulates the levels of outer membrane protein F (OmpF) in Escherichia coli in response to temperature increase and other stress conditions by decreasing the levels of ompF mRNA (Andersen et al., 1989). A 93-nucleotide micF RNA was synthesized in vitro directly from polymerase chain reaction generated DNA which was designed to contain a functional T7 RNA polymerase promoter upstream of the micF RNA gene and an appropriate restriction site for transcription termination. A transcript (150 nucleotides) containing the ribosomal binding domain of ompF mRNA messenger was synthesized in vitro from the ompF gene cloned into a T7 expression vector. A stable duplex was formed between micF RNA and the 150-nucleotide 5' transcript of ompF mRNA after incubation at 37 degrees C in a physiological buffer. The melting curve of the duplex formed by micF RNA and 150-nucleotide transcript revealed a Tm of 56 degrees C and a delta Tm that spans about 20 degrees C; both are consistent with the proposed structure for the micF/ompF duplex. In addition, as determined by competition studies and UV cross-linking/label-transfer analyses, an E. coli protein was found to bind specifically to micF RNA. The protein also bound weakly to the 150-nucleotide ompF transcript. The data are the first to demonstrate the complex between micF RNA and the 5' end of ompF mRNA and suggest that in vivo a micF ribonucleoprotein (RNP) particle may participate in the destabilization ompF mRNA during thermoregulation of OmpF porin.

Bacterial Outer Membrane Proteins↗
PubMed2006

Characterization of a vaccinia virus mutant with a deletion of the D10R gene encoding a putative negative regulator of gene expression.

The D9 and D10 proteins of vaccinia virus are 25% identical to each other, contain a mutT motif characteristic of nudix hydrolases, and are conserved in all sequenced poxviruses. Previous studies indicated that overexpression of D10 and, to a lesser extent, D9 decreased the levels of capped mRNAs and their translation products. Here, we further characterized the D10 protein and showed that only trace amounts are associated with purified virions and that it is expressed exclusively at late times after vaccinia virus infection. A viable deletion mutant (vdeltaD10) produced smaller plaques and lower virus yields than either wild-type virus or a D9R deletion mutant (vdeltaD9). Purified vdeltaD10 virions appeared normal by microscopic examination and biochemical analysis but produced 6- to 10-fold-fewer plaques at the same concentration as wild-type or vdeltaD9 virions. When 4 PFU per cell of wild-type or vdeltaD9 virions or equal numbers of vdeltaD10 virions were used for inoculation, nearly all cells were infected in each case, but viral early and late transcription was initiated more slowly in vdeltaD10-infected cells than in the others. However, viral early transcripts accumulated to higher levels in vdeltaD10-infected cells than in cells infected with the wild type or vdeltaD9. In addition, viral early and late mRNAs and cellular actin mRNA persisted longer in vdeltaD10-infected cells than in others. Furthermore, analysis of pulse-labeled proteins indicated prolonged synthesis of cellular and viral early proteins. These results are consistent with a role for D10 in regulating RNA levels in poxvirus-infected cells.

Actins↗
PubMed2002

Movement of potato spindle tuber viroid reveals regulatory points of phloem-mediated RNA traffic.

Increasing evidence indicates that the phloem mediates traffic of selective RNAs within a plant. How an RNA enters, moves in, and exits the phloem is poorly understood. Potato spindle tuber viroid (PSTVd) is a pathogenic RNA that does not encode proteins and is not encapsidated, and yet it replicates autonomously and traffics systemically within an infected plant. The viroid RNA genome must interact directly with cellular factors to accomplish these functions and is, therefore, an excellent probe to study mechanisms that regulate RNA traffic. Our analyses of PSTVd traffic in Nicotiana benthamiana yielded evidence that PSTVd movement within sieve tubes does not simply follow mass flow from source to sink organs. Rather, this RNA is transported into selective sink organs. Furthermore, two PSTVd mutants can enter the phloem to spread systemically but cannot exit the phloem in systemic leaves of tobacco (Nicotiana tabacum). A viroid most likely has evolved structural motifs that mimic endogenous plant RNA motifs so that they are recognized by cellular factors for traffic. Thus, analysis of PSTVd traffic functions may provide insights about endogenous mechanisms that control phloem entry, transport, and exit of RNAs.

Biological Transport↗