Arabidopsis retains vertebrate-type telomerase accessory proteins via a plant-specific assembly

The recent discovery of the bona-fide telomerase RNA (TR) from plants reveals conserved and unique secondary structure elements and the opportunity for new insight into the telomerase RNP. Here we examine how two highly conserved proteins previously implicated in Arabidopsis telomere maintenance, AtPOT1a and AtNAP57 (dyskerin), engage plant telomerase. We report that AtPOT1a associates with Arabidopsis telomerase via interaction with TERT. While loss of AtPOT1a does not impact AtTR stability, the templating domain is more accessible in pot1a mutants, supporting the conclusion that AtPOT1a stimulates telomerase activity but does not facilitate telomerase RNP assembly. We also show, that despite the absence of a canonical H/ACA binding motif within AtTR, dyskerin binds AtTR with high affinity and specificity in vitro via a plant specific three-way junction (TWJ). A core element of the TWJ is the P1a stem, which unites the 5′ and 3′ ends of AtTR. P1a is required for dyskerin-mediated stimulation of telomerase repeat addition processivity in vitro, and for AtTR accumulation and telomerase activity in vivo. The deployment of vertebrate-like accessory proteins and unique RNA structural elements by Arabidopsis telomerase provides a new platform for exploring telomerase biogenesis and evolution.     

Inhibition of telomerase by 2'-O-(2-methoxyethyl) RNA oligomers: effect of length, phosphorothioate substitution and time inside cells

2′-O-(2-methoxyethyl) (2′-MOE) RNA possesses favorable pharmocokinetic properties that make it a promising option for the design of oligonucleotide drugs. Telomerase is a ribonucleoprotein that is up-regulated in many types of cancer, but its potential as a target for chemotherapy awaits the development of potent and selective inhibitors. Here we report inhibition of human telomerase by 2′-MOE RNA oligomers that are complementary to the RNA template region. Fully complementary oligomers inhibited telomerase in a cell extract with IC₅₀ values of 5–10 nM at 37°C. IC₅₀ values for mismatch-containing oligomers varied with length and phosphorothioate substitution. After introduction into DU 145 prostate cancer cells inhibition of telomerase activity persisted for up to 7 days, equivalent to six population doublings. Inside cells discrimination between complementary and mismatch-containing oligomers increased over time. Our results reveal two oligomers as especially promising candidates for initiation of in vivo preclinical trials and emphasize that conclusions regarding oligonucleotide efficacy and specificity in cell extracts do not necessarily offer accurate predictions of activity inside cells.     

3'-box-dependent processing of human pre-U1 snRNA requires a combination of RNA and protein co-factors

Using an in vitro system we have recently shown that the 3′ ends of human pre-snRNAs synthesized by RNA polymerase II are produced by RNA processing directed by the snRNA gene-specific 3′ box. Towards a complete characterization of this processing reaction we have further investigated the in vitro requirements for proper 3′ end formation of pre-U1 snRNA. Here we show that the 5′ cap plays a stimulatory role and processing requires creatine phosphate. Our results also indicate that the pre-U1 processing activity is heat sensitive and that an RNA component is required. In addition, the exact sequence adjacent to the 3′ box influences the position of the pre-U1 3′ end produced in vitro. Interestingly, the processing extract active for 3′-box-dependent processing also contains an activity that converts the 3′ end of RNA containing the U1 Sm protein binding site and the 3′ terminal stem–loop into the mature form.     

Dynamics of human telomerase RNA structure revealed by antisense oligonucleotide technique

Telomeres are the nucleoprotein complexes that cap the linear chromosome ends. Telomerase is a ribonucleoprotein that maintains telomere length in stem, embryonic and cancer cells. Somatic cells don't contain active telomerase and telomere function as mitotic clock and telomere length determines the number of cell divisions. Telomerase RNA (TER) contains the template for telomere synthesis and serves as a structural scaffold for holoenzyme assembly. We compared different oligonucleotide based methods for telomerase RNA inhibition, such as antisense oligonucleotides, knockdown by transient siRNA transfection and silencing by miRNA derived from short expressed RNA hairpin in HEK293 cells. All of these methods were applied to different TER regions. Our results revealed that CR2/CR3 domain of TER is accessible in vitro and in vivo and could serve as an optimal site for oligonucleotide-based telomerase silencing.     

3'-Azido-2',3'-dideoxynucleoside 5'-triphosphates inhibit telomerase activity in vitro, and the corresponding nucleosides cause telomere shortening in human HL60 cells

Telomerase adds telomeric DNA repeats to the ends of linear chromosomal DNA. 3′-Azido-3′-deoxythymidine 5′-triphosphate (AZTTP) is a known telomerase inhibitor. To obtain more selective and potent inhibitors that can be employed as tools for studying telomerase, we investigated the telomerase-inhibitory effects of purine nucleosides bearing a 3′-down azido group: 3′-azido-2′,3′-dideoxyguanosine (AZddG) 5′-triphosphate (AZddGTP), 3′-azido-2′,3′-dideoxy-6-thioguanosine (AZddSG) 5′-triphosphate (AZddSGTP), 3′-azido-2′,3′-dideoxyadenosine (AZddA) 5′-triphosphate (AZddATP) and 3′-azido-2′,3′-dideoxy-2-aminoadenosine (AZddAA) 5′-triphosphate (AZddAATP). Of these, AZddGTP showed the most potent inhibitory activity against HeLa cell telomerase. AZddGTP was significantly incorporated into the 3′-terminus of DNA by partially purified telomerase. However, AZddGTP did not exhibit significant inhibitory activity against DNA polymerases α and δ, suggesting that AZddGTP is a selective inhibitor of telomerase.

We also investigated whether long-term treatment with these nucleosides could alter telomere length and growth rates of human HL60 cells in culture. Southern hybridization analysis of genomic DNA prepared from cells cultured in the presence of AZddG and AZddAA revealed reproducible telomere shortening.

     

The 5′ Terminal Trailer Region of Vesicular Stomatitis Virus Contains a Position-Dependent cis-Acting Signal for Assembly of RNA into Infectious Particles

The cis-acting genomic RNA requirements for the assembly of vesicular stomatitis virus (VSV) ribonucleocapsids into infectious particles were investigated. Using a biological assay based on particle infectivity, we demonstrated that subgenomic replicons that contained all four possible combinations of the natural genomic termini, the 3′ leader (Le) and 5′ trailer (Tr) regions, were replication competent; however, a 3′ copyback replicon (3′CB), containing the natural 3′ terminus but having the 5′ Tr replaced by a sequence complementary to the 3′ Le for 46 nucleotides, was unable to assemble infectious particles, despite efficient replication. When a copy of Tr was inserted 51 nucleotides from the 5′ end of 3′CB, infectious particles were produced. However, analysis of the replication products of these particles showed that the 51 nucleotides which corresponded to the Le complement sequences at the 5′ terminus were removed during RNA replication, thus restoring the wild-type 5′ Tr to the exact 5′ terminus. These data showed that a cis-acting signal was necessary for assembly of VSV RNAs into infectious particles and that this signal was supplied by Tr when located at the 5′ end. The regions within Tr required for assembly were analyzed by a series of deletions and exchanges for Le complement sequences, which demonstrated that the 5′ terminal 29 nucleotides of Tr allowed assembly of infectious particles but that the 5′ terminal 22 nucleotides functioned poorly. Deletions in Tr also altered the balance between negative- and positive-strand genomic RNA and affected levels of replication. RNAs that retained fewer than 45 but at least 22 nucleotides of the 5′ terminus could replicate but were impaired in RNA replication, and RNAs that retained only 14 nucleotides of the 5′ terminus were severely reduced in ability to replicate. These data define the VSV Tr as a position-dependent, cis-acting element for the assembly of RNAs into infectious particles, and they delineate RNA sequences that are essential for negative-strand RNA synthesis. These observations are consistent with, and offer an explanation for, the absence of 3′ copyback defective interfering particles in nature.     

Arabidopsis thaliana exosome subunit AtRrp4p is a hydrolytic 3′→5′ exonuclease containing S1 and KH RNA-binding domains

The exosome, an evolutionarily conserved complex of multiple 3′→5′ exoribonucleases, is responsible for a variety of RNA processing and degradation events in eukaryotes. In this report Arabidopsis thaliana AtRrp4p is shown to be an active 3′→5′ exonuclease that requires a free 3′-hydroxyl and degrades RNA hydrolytically and distributively, releasing nucleoside 5′-monophosphate products. AtRrp4p behaves as an ~500 kDa species during sedimentation through a 10–30% glycerol gradient, co-migrating with AtRrp41p, another exosome subunit, and it interacts in vitro with AtRrp41p, suggesting that it is also present in the plant cell as a subunit of the exosome. We found that, in addition to a previously reported S1-type RNA-binding domain, members of the Rrp4p family of proteins contain a KH-type RNA-binding domain in the C-terminal half and show that either domain alone can bind RNA. However, only the full-length protein is capable of degrading RNA and interacting with AtRrp41p.     

In vitro selection, characterization, and application of deoxyribozymes that cleave RNA

Over the last decade, many catalytically active DNA molecules (deoxyribozymes; DNA enzymes) have been identified by in vitro selection from random-sequence DNA pools. This article focuses on deoxyribozymes that cleave RNA substrates. The first DNA enzyme was reported in 1994 and cleaves an RNA linkage. Since that time, many other RNA-cleaving deoxyribozymes have been identified. Most but not all of these deoxyribozymes require a divalent metal ion cofactor such as Mg²⁺ to catalyze attack by a specific RNA 2′-hydroxyl group on the adjacent phosphodiester linkage, forming a 2′,3′-cyclic phosphate and a 5′-hydroxyl group. Several deoxyribozymes that cleave RNA have utility for in vitro RNA biochemistry. Some DNA enzymes have been applied in vivo to degrade mRNAs, and others have been engineered into sensors. The practical impact of RNA-cleaving deoxyribozymes should continue to increase as additional applications are developed.     

Identification of DEAD-box RNA Helicase 6 (DDX6) as a Cellular Modulator of Vascular Endothelial Growth Factor Expression under Hypoxia

Vascular endothelial growth factor A (VEGF) is a crucial proangiogenic factor, which regulates blood vessel supply under physiologic and pathologic conditions. The VEGF mRNA 5′-untranslated region (5′-UTR) bears internal ribosome entry sites (IRES), which confer sustained VEGF mRNA translation under hypoxia when 5′-cap-dependent mRNA translation is inhibited. VEGF IRES-mediated initiation of translation requires the modulated interaction of trans-acting factors. To identify trans-acting factors that control VEGF mRNA translation under hypoxic conditions we established an in vitro translation system based on human adenocarcinoma cells (MCF-7). Cytoplasmic extracts of MCF-7 cells grown under hypoxia (1% oxygen) recapitulate VEGF IRES-mediated reporter mRNA translation. Employing the VEGF mRNA 5′-UTR and 3′-UTR in an RNA affinity approach we isolated interacting proteins from translational active MCF-7 extract prepared from cells grown under normoxia or hypoxia. Interestingly, mass spectrometry analysis identified the DEAD-box RNA helicase 6 (DDX6) that interacts with the VEGF mRNA 5′-UTR. Recombinant DDX6 inhibits VEGF IRES-mediated translation in normoxic MCF-7 extract. Under hypoxia the level of DDX6 declines, and its interaction with VEGF mRNA is diminished in vivo. Depletion of DDX6 by RNAi further promotes VEGF expression in MCF-7 cells. Increased secretion of VEGF from DDX6 knockdown cells positively affects vascular tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Our results indicate that the decrease of DDX6 under hypoxia contributes to the activation of VEGF expression and promotes its proangiogenic function.