Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5' ETS molecule using three distinct methods and located the acceptor site between two known 5' ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5' ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5' ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5' ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

The intronic long noncoding RNA ANRASSF1 recruits PRC2 to the RASSF1A promoter, reducing the expression of RASSF1A and increasing cell proliferation

The down-regulation of the tumor-suppressor gene RASSF1A has been shown to increase cell proliferation in several tumors. RASSF1A expression is regulated through epigenetic events involving the polycomb repressive complex 2 (PRC2); however, the molecular mechanisms modulating the recruitment of this epigenetic modifier to the RASSF1 locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand on the RASSF1 gene locus in several cell lines and tissues and binds PRC2. ANRASSF1 is transcribed through RNA polymerase II and is 5'-capped and polyadenylated; it exhibits nuclear localization and has a shorter half-life compared with other lncRNAs that bind PRC2. ANRASSF1 endogenous expression is higher in breast and prostate tumor cell lines compared with non-tumor, and an opposite pattern is observed for RASSF1A. ANRASSF1 ectopic overexpression reduces RASSF1A abundance and increases the proliferation of HeLa cells, whereas ANRASSF1 silencing causes the opposite effects. These changes in ANRASSF1 levels do not affect the RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase in both PRC2 occupancy and histone H3K27me3 repressive marks, specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression was detected on PRC2 occupancy and histone H3K27me3 at the promoter regions of RASSF1C and the four other neighboring genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrated that ANRASSF1 forms an RNA/DNA hybrid and recruits PRC2 to the RASSF1A promoter. Together, these results demonstrate a novel mechanism of epigenetic repression of the RASSF1A tumor suppressor gene involving antisense unspliced lncRNA, in which ANRASSF1 selectively represses the expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome might contribute to a location-specific epigenetic modulation of genes.

A single chain antibody against a viral RNA polymerase (TBSV-BS3-Statice)

Expression of antibodies in plant against essential viral proteins could provide an alternative approach to engineered viral resistance. Engineered single chain Fv antibodies scFV are particularly suitable for expression in plant because of their small size and the lack of assembly requirements. RNA-dependent RNA polymerases (RdRps) function as the catalytic subunit of viral replicases required for the replication of all positive strand RNA viruses. By using Phage technology we selected scFvs from a phage library using purified E.coli expressed TBSV(Tomato bushy stunt virus) replicase as antigen. The scFvs mediated-inhibition of RdRp activity was studied in vitro and in planta. In vitro experiments showed the inhibition of CNV(Cucumber necrosis virus) and TCV(Turnip crinkle virus) RdRp. Transient in planta assays based on agroinfiltration and an infectious clone of TBSV demonstrated the inhibition of the replication of TBSV(Tomato bushy stunt virus). Epitope mapping showed that the selected scFvs target the motif E of RdRp which is involved in template binding.Moreover T1 plants of transgenic lines of N. benthamiana expressing different scFvs either in the cytoplasm or the ER (endoplasmic reticulum) showed a high level of resistance against infection with TBSV and RCNMV(Red clover necrotic mosaic virus) upon inoculation with virus particles. This is the first report that scFvs against a RdRp of a plant viruses can inhibit viral replication in vivo. The resistance is even efficient against viruses belonging to different virus families.

Ribosome Biogenesis and cell cycle regulation: Effect of RNA Polymerase III inhibition

In cycling cells positive stimuli like nutrient, growth factors and mitogens increase ribosome biogenesis rate and protein synthesis to ensure both growth and proliferation. In contrast, under stress situation, proliferating cells negatively modulate ribosome production to reduce protein synthesis and block cell cycle progression. The main strategy used by cycling cell to coordinate cell proliferation and ribosome biogenesis is to share regulatory elements, which participate directly in ribosome production and in cell cycle regulation. In fact, there is evidence that stimulation or inhibition of cell proliferation exerts direct effect on activity of the RNA polymerases controlling the ribosome biogenesis, while several alterations in normal ribosome biogenesis cause changes of the expression and the activity of the tumor suppressor p53, the main effector of cell cycle progression inhibition. The available data on the cross-talk between ribosome biogenesis and cell proliferation have been until now obtained in experimental model in which changes in ribosome biogenesis were obtained either by reducing the activity of the RNA polymerase I or by down-regulating the expression of the ribosomal proteins. The molecular pathways involved in the relationship between the effect of the inhibition of RNA polymerase III (Pol III) activity and cell cycle progression have been not yet investigated. In eukaryotes, RNA Polymerase III is responsible for transcription of factors involved both in ribosome assembly (5S rRNA) and rRNA processing (RNAse P and MRP).Thus, the aim of this study is characterize the effects of the down-regulation of RNA Polymerase III activity, or the specific depletion of 5S rRNA. The results that will be obtained might lead to a deeper understanding of the molecular pathway that controls the coordination between ribosome biogenesis and cell cycle, and might give useful information about the possibility to target RNA Polymerase III for cancer treatment.

Sendai virus RNA polymerase scanning for mRNA start sites at gene junctions

Mini-genomes expressing two reporter genes and a variable gene junction were used to study Sendai virus RNA polymerase (RdRp) scanning for the mRNA start signal of the downstream gene (gs2). We found that RdRp could scan the template efficiently as long as the initiating uridylate of gs2 (3' UCCCnnUUUC) was preceded by the conserved intergenic region (3' GAA) and the last 3 uridylates of the upstream gene end signal (ge1; 3' AUUCUUUUU). The end of the leader sequence (3' CUAAAA, which precedes gs1) could also be used for gene2 expression, but this sequence was considerably less efficient. Increasing the distance between ge1 and gs2 (up to 200 nt) led to the progressive loss of gene2 expression, in which half of gene2 expression was lost for each 70 nucleotides of intervening sequence. Beyond 200 nt, gene2 expression was lost more slowly. Our results suggest that there may be two populations of RdRp that scan at gene junctions, which can be distinguished by the efficiency with which they can scan the genome template for gs.

Trypanosoma brucei RRM1 is a nuclear RNA-binding protein and modulator of chromatin structure

TbRRM1 of Trypanosoma brucei is a nucleoprotein that was previously identified in a search for splicing factors in T. brucei. We show that TbRRM1 associates with mRNAs and with the auxiliary splicing factor polypyrimidine tract-binding protein 2, but not with components of the core spliceosome. TbRRM1 also interacts with several retrotransposon hot spot (RHS) proteins and histones. RNA immunoprecipitation of a tagged form of TbRRM1 from procyclic (insect) form trypanosomes identified ca. 1,500 transcripts that were enriched and 3,000 transcripts that were underrepresented compared to cellular mRNA. Enriched transcripts encoded RNA-binding proteins, including TbRRM1 itself, several RHS transcripts, mRNAs with long coding regions, and a high proportion of stage-regulated mRNAs that are more highly expressed in bloodstream forms. Transcripts encoding ribosomal proteins, other factors involved in translation, and procyclic-specific transcripts were underrepresented. Knockdown of TbRRM1 by RNA interference caused widespread changes in mRNA abundance, but these changes did not correlate with the binding of the protein to transcripts, and most splice sites were unchanged, negating a general role for TbRRM1 in splice site selection. When changes in mRNA abundance were mapped across the genome, regions with many downregulated mRNAs were identified. Two regions were analyzed by chromatin immunoprecipitation, both of which exhibited increases in nucleosome occupancy upon TbRRM1 depletion. In addition, subjecting cells to heat shock resulted in translocation of TbRRM1 to the cytoplasm and compaction of chromatin, consistent with a second role for TbRRM1 in modulating chromatin structure. IMPORTANCE: Trypanosoma brucei, the parasite that causes human sleeping sickness, is transmitted by tsetse flies. The parasite progresses through different life cycle stages in its two hosts, altering its pattern of gene expression in the process. In trypanosomes, protein-coding genes are organized as polycistronic units that are processed into monocistronic mRNAs. Since genes in the same unit can be regulated independently of each other, it is believed that gene regulation is essentially posttranscriptional. In this study, we investigated the role of a nuclear RNA-binding protein, TbRRM1, in the insect stage of the parasite. We found that TbRRM1 binds nuclear mRNAs and also affects chromatin status. Reduction of nuclear TbRRM1 by RNA interference or heat shock resulted in chromatin compaction. We propose that TbRRM1 regulates RNA polymerase II-driven gene expression both cotranscriptionally, by facilitating transcription and efficient splicing, and posttranscriptionally, via its interaction with nuclear mRNAs.

DnaA-stimulated transcriptional activation of oriλ: Escherichia coli RNA polymerase β subunit as a transcriptional activator contact site

We present evidence that Escherichia coli RNA polymerase β subunit may be a transcriptional activator contact site. Stimulation of the activity of the pR promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage λ. We found that DnaA activates the pR promoter in vitro. Particular mutations in the rpoB gene were able to suppress negative effects that certain dnaA mutations had on the replication of λ plasmids; this suppression was allele-specific. When a potential DnaA-binding sequence located several base pairs downstream of the pR promoter was scrambled by in vitro mutagenesis, the pR promoter was no longer activated by DnaA both in vivo and in vitro. Therefore, we conclude that DnaA may contact the β subunit of RNA polymerase during activation of the pR promoter. A new classification of prokaryotic transcriptional activators is proposed.

Mammalian capping enzyme binds RNA and uses protein tyrosine phosphatase mechanism

Mammalian capping enzymes are bifunctional proteins with both RNA 5′-triphosphatase and guanylyltransferase activities. The N-terminal 237-aa triphosphatase domain contains (I/V)HCXXGXXR(S/T)G, a sequence corresponding to the conserved active-site motif in protein tyrosine phosphatases (PTPs). Analysis of point mutants of mouse RNA 5′-triphosphatase identified the motif Cys and Arg residues and an upstream Asp as required for activity. Like PTPs, this enzyme was inhibited by iodoacetate and VO43− and independent of Mg2+, providing additional evidence for phosphate removal from RNA 5′ ends by a PTP-like mechanism. The full-length, 597-aa mouse capping enzyme and the C-terminal guanylyltransferase fragment (residues 211–597), unlike the triphosphatase domain, bound poly (U) and were nuclear in transfected cells. RNA binding was increased by GTP, and a guanylylation-defective, active-site mutant was not affected. Ala substitution at positions required for the formation of the enzyme-GMP capping intermediate (R315, R530, K533, or N537) also eliminated poly (U) binding, while proteins with conservative substitutions at these sites retained binding but not guanylyltransferase activity. These results demonstrate that the guanylyltransferase domain of mammalian capping enzyme specifies nuclear localization and RNA binding. Association of capping enzyme with nascent transcripts may act in synergy with RNA polymerase II binding to ensure 5′ cap formation.

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

RNA secondary structures (hairpins) that form as the nascent RNA emerges from RNA polymerase are important components of many signals that regulate transcription, including some pause sites, all ρ-independent terminators, and some antiterminators. At the his leader pause site, a 5-bp-stem, 8-nt-loop pause RNA hairpin forms 11 nt from the RNA 3′ end and stabilizes a transcription complex conformation slow to react with NTP substrate. This stabilization appears to depend at least in part on an interaction with RNA polymerase. We tested for RNA hairpin interaction with the paused polymerase by crosslinking 5-iodoUMP positioned specifically in the hairpin loop. In the paused conformation, strong and unusual crosslinking of the pause hairpin to β904–950 replaced crosslinking to β′ and to other parts of β that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3′ end in the paused complex and be related to events at ρ-independent terminators.

Multiple pathways to bypass the enhancer requirement of sigma 54 RNA polymerase: Roles for DNA and protein determinants

Sigma 54 is a required factor for bacterial RNA polymerase to respond to enhancers and directs a mechanism that is a hybrid between bacterial and eukaryotic transcription. Three pathways were found that bypass the enhancer requirement in vitro. These rely on either deletion of the sigma 54 N terminus or destruction of the DNA consensus −12 promoter recognition element or altering solution conditions to favor transient DNA melting. Each of these allows unstable heparin-sensitive pre-initiation complexes to form that can be driven to transcribe in the absence of both enhancer protein and ATP β–γ hydrolysis. These disparate pathways are proposed to have a common basis in that multiple N-terminal contacts may mediate the interactions between the polymerase and the DNA region where melting originates. The results raise possibilities for common features of open complex formation by different RNA polymerases.

Sequence-specific recognition of a subgenomic RNA promoter by a viral RNA polymerase

RNA templates of 33 nucleotides containing the brome mosaic virus (BMV) core subgenomic promoter were used to determine the promoter elements recognized by the BMV RNA-dependent RNA polymerase (RdRp) to initiate RNA synthesis. Nucleotides at positions −17, −14, −13, and −11 relative to the subgenomic initiation site must be maintained for interaction with the RdRp. Changes to every other nucleotide at these four positions allow predictions for the base-specific functional groups required for RdRp recognition. RdRp contact of the nucleotide at position −17 was suggested with a template competition assay. Comparison of the BMV subgenomic promoter to those from other plant and animal alphaviruses shows a remarkable degree of conservation of the nucleotides required for BMV subgenomic RNA synthesis. We show that the RdRp of the plant-infecting BMV is capable of accurately, albeit inefficiently, initiating RNA synthesis from the subgenomic promoter of the animal-infecting Semliki Forest virus. The sequence-specific recognition of RNA by the BMV RdRp is analogous to the recognition of DNA promoters by DNA-dependent RNA polymerases.

Positioning of two alpha subunit carboxy-terminal domains of RNA polymerase at promoters by two transcription factors

Interactions between the cAMP receptor protein (CRP) and the carboxy-terminal regulatory domain (CTD) of Escherichia coli RNA polymerase α subunit were analyzed at promoters carrying tandem DNA sites for CRP binding using a chemical nuclease covalently attached to α. Each CRP dimer was found to direct the positioning of one of the two α subunit CTDs. Thus, the function of RNA polymerase may be subject to regulation through protein–protein interactions between the two α subunits and two different species of transcription factors.