5' to 3' exoribonucleolytic activity is a normal component of chloroplast mRNA decay pathways

Molecular genetic studies have shown that determinants of chloroplast mRNA stability lie in both the 5' and 3' untranslated regions. While it is well-known that chloroplast mRNAs are unstable in the absence of certain nucleus-encoded factors, little is known of the decay mechanisms for chloroplast mRNA in wild-type cells. Here we used a poly(G)18 sequence, which impedes both 5'-->3' and 3'-->5' exoribonucleolytic RNA decay in vivo, to study the degradation pathway of petD mRNA in wild-type and mcd1 mutant chloroplasts of Chlamydomonas; the mcd1 mutant lacks a nucleus-encoded factor required for petD mRNA accumulation. Upon inserting poly(G) at positions -20, +25, +165 or +25/+165 relative to the mature petD 5' end, mRNAs accumulate with 5' ends corresponding to the poly(G) sequence, in addition to the normal RNA with its 5' end at +1. We interpret these results as evidence for continuous degradation of petD mRNA in wild-type cells by a 5'-->3' exoribonucleolytic activity. In the case of the -20 insertion, the accumulating RNA can be interpreted as a processing intermediate, suggesting that 5' end maturation may also involve this activity. When examined in the mcd1 mutant background, petD mRNAs with the poly(G) 5' ends, but not normal +1 ends, accumulated. However, no expression of SUIV, the petD gene product, was detected. Insertion of poly(G) at +165 in wild-type cells did not demonstrably affect SUIV accumulation, suggesting that ribosomal scanning does not occur upstream of this position. However, since neither poly(G) -20 nor +165 RNA could be translated in mcd1 cells, this raises the possibility that the MCD1 product is essential for translation.     

Synthesis and hybridization properties of oligonucleotides containing (2S,3R)-9-(2,3,4-trihydroxybutyl)adenine

The synthesis and properties of oligonucleotides (ONs) containing 9-(2,3,4-trihydroxybutyl)adenine, A(C2) and A(C3), are described. The ON containing A(C2) involves the 3'-->4' and 3-->5' phosphodiester linkages in the strand, whereas that containing A(C3) possesses the 3'-->4' and 2'-->5' phosphodiester linkages. It was found that incorporation of the analogs, A(C2) or A(C3), into ONs significantly reduces the thermal and thermodynamic stabilities of the ON/DNA duplexes, but does not largely decrease the thermal and thermodynamic stabilities of the ON/RNA duplexes as compared with the case of the ON/DNA duplexes. It was revealed that the base recognition ability of A(C2) is greater than that of A(C3) in the ON/RNA duplexes.     

Molecular dynamics simulations of DNA within a nanopore: arginine-phosphate tethering and a binding/sliding mechanism for translocation

Protein nanopores show great potential as low-cost detectors in DNA sequencing devices. To date, research has largely focused on the staphylococcal pore α-hemolysin (αHL). In the present study, we have developed simplified models of the wild-type αHL pore and various mutants in order to study the translocation dynamics of single-stranded DNA under the influence of an applied electric field. The model nanopores reflect the experimentally measured conductance values in planar lipid bilayers. We show that interactions between rings of cationic amino acids and DNA backbone phosphates result in metastable tethering of nucleic acid molecules within the pore, leading us to propose a "binding and sliding" mechanism for translocation. We also observe folding of DNA into nonlinear conformational intermediates during passage through the confined nanopore environment. Despite adopting nonlinear conformations, the DNA hexamer always exits the pore in the same orientation as it enters (3' to 5') in our simulations. The observations from our simulations help to rationalize experimentally determined trends in residual current and translocation efficiency for αHL and its mutants.     

RNA-linked nascent DNA pieces in phage T7-infected Escherichia coli. III. Detection of intact primer RNA

RNA-linked DNA fragments of T7-infected Escherichiacoli were labeled with [(32)P]orthophosphate invivo. The RNA segments of the labeled fragments were isolated by degrading the DNA portion with the 3'--> 5' exonuclease intrinsic to bacteriophage T4 DNA polymerase and fractionated according to net charge by a DEAE-Sephadex A-25 column chromatography in the presence of 7 M urea. Tri-, tetra- and pentanucleotides were obtained which have ATP residues at their 5' ends. Most of the pentanucleotides had a single deoxynucleotide at the 3' end but a minor portion was totally an oligoribonucleotide. In the light of prior results, the former is a cooligomer of an intact tetraribonucleotide primer and a monodeoxynucleotide and the latter is an intact pentaribonucleotide primer. Tri- and tetraribonucleotides with ATP at the 5' ends had no deoxynucleotide at the 3' ends, therefore it is not clear if intact triribonucleotide primers are present. The 5'-terminal dinucleotides of the tetra- and pentanucleotides were mostly pppApC and a trace amount of pppApA was present.Images     

Nonsense-mediated mRNA decay in mammalian cells involves decapping, deadenylating, and exonucleolytic activities

Nonsense-mediated mRNA decay (NMD) is a mechanism by which cells recognize and degrade mRNAs that prematurely terminate translation. To date, the polarity and enzymology of NMD in mammalian cells is unknown. We show here that downregulating the Dcp2 decapping protein or the PM/Scl100 component of the exosome (1) significantly increases the abundance of steady-state nonsense-containing but not nonsense-free mRNAs, and (2) significantly slows the decay rate of transiently induced nonsense-containing but not nonsense-free mRNA. Downregulating poly(A) ribonuclease (PARN) also increases the abundance of nonsense-containing mRNAs. Furthermore, NMD factors Upf1, Upf2, and Upf3X coimmunopurify with the decapping enzyme Dcp2, the putative 5'-->3' exonuclease Rat1, the proven 5'-->3' exonuclease Xrn1, exosomal components PM/Scl100, Rrp4, and Rrp41, and PARN. From these and other data, we conclude that NMD in mammalian cells degrades mRNAs from both 5' and 3' ends by recruiting decapping and 5'-->3' exonuclease activities as well as deadenylating and 3'-->5' exonuclease activities.     

Synthesis and properties of 3'-amino-2',4'-BNA, a bridged nucleic acid with a N3'-->P5' phosphoramidate linkage

The synthesis and properties of a bridged nucleic acid analogue containing a N3'-->P5' phosphoramidate linkage, 3'-amino-2',4'-BNA, is described. A heterodimer containing a 3'-amino-2',4'-BNA thymine monomer, and thymine and methylcytosine monomers of 3'-amino-2',4'-BNA and their 5'-phosphoramidites, were synthesized efficiently. The dimer and monomers were incorporated into oligonucleotides by conventional 3'-->5' assembly, and 5'-->3' reverse assembly phosphoramidite protocols, respectively. Compared to a natural DNA oligonucleotide, modified oligonucleotides containing the 3'-amino-2',4'-BNA residue formed highly stable duplexes and triplexes with single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded DNA (dsDNA) targets, with the average increase in melting temperature (T(m)) against ssDNA, ssRNA and dsDNA being +2.7 to +4.0 degrees C, +5.0 to +7.0 degrees C, and +5.0 to +11.0 degrees C, respectively. These increases are comparable to those observed for 2',4'-BNA-modified oligonucleotides. In addition, an oligonucleotide modified with a single 3'-amino-2',4'-BNA thymine residue showed extraordinarily high resistance to nuclease degradation, much higher than that of 2',4'-BNA and substantially higher even than that of 3'-amino-DNA and phosphorothioate oligonucleotides. The above properties indicate that 3'-amino-2',4'-BNA has significant potential for antisense and antigene applications.     

Bacillus subtilis bacteriophage SPP1 G40P helicase lacking the n-terminal domain unwinds DNA bidirectionally

Bacillus subtilis bacteriophage SPP1 G40P hexameric replicative DNA helicase unidirectionally translocates with a 5'-->3' polarity while separating the DNA strands. A G40P mutant derivative lacking the N-terminal domain (containing amino acid residues 110-442 from G40P, G40PDeltaN109) was purified and characterized. G40PDeltaN109 showed an ATPase activity that was dependent on the presence of single-stranded (ss) DNA. Unlike G40P, G40PDeltaN109 was shown to bind with similar affinity both ssDNA arms of forked structures by nuclease protection assays. In a pH-dependent manner, G40PDeltaN109 unwound a branched double-arm substrate preferentially with a 3'-->5' polarity. Our results show that the linker region and the C-terminal domain of G40P are sufficient to render an enzyme capable of encircling the ssDNA tails of the forked DNA and to unwind DNA with both 5'-->3' and 3'-->5' polarity. The presence of the N-terminal domain, which does not play an essential role in helicase action, might be required indirectly for strand discrimination and polarity of translocation.     

Syntheses of DNA duplexes containing a C-C interstrand cross-link

Short DNA duplexes that contain a N4C-ethyl-N4C interstrand cross-link were prepared on controlled pore glass supports using a DNA synthesizer. The C-C cross-link was introduced via a convertible nucleoside on the support or by using a protected C-C cross-link phosphoramidite. An orthogonal protection scheme allowed selective chain growth in either a 3'-->5' or 5'-->3' direction. The cross-linked duplexes were purified by HPLC and characterized by MALDI-TOF mass spectrometry and/or by enzymatic digestion.     

How Nanopore Translocation Experiments Can Measure RNA Unfolding

Electrokinetic translocation of biomolecules through solid-state nanopores represents a label-free single-molecule technique that may be used to measure biomolecular structure and dynamics. Recent investigations have attempted to distinguish individual transfer RNA (tRNA) species based on the associated pore translocation times, ion-current noise, and blockage currents. By manufacturing sufficiently smaller pores, each tRNA is required to undergo a deformation to translocate. Accordingly, differences in nanopore translocation times and distributions may be used to infer the mechanical properties of individual tRNA molecules. To bridge our understanding of tRNA structural dynamics and nanopore measurements, we apply molecular dynamics simulations using a simplified “structure-based” energetic model. Calculating the free-energy landscape for distinct tRNA species implicates transient unfolding of the terminal RNA helix during nanopore translocation. This provides a structural and energetic framework for interpreting current experiments, which can aid the design of methods for identifying macromolecules using nanopores.     

Ionic current blockades from DNA and RNA molecules in the alpha-hemolysin nanopore

We characterize the substate structure of current blockades produced when single-stranded polynucleotide molecules were electrophoretically driven into the alpha-hemolysin protein pore. We frequently observe substates where the ionic current is reduced by approximately 50%. Most of these substates can be associated with a molecular configuration where a polymer occupies only the vestibule region of the pore, though a few appear related to a polymer occupying only the transmembrane beta-barrel region of the pore. The duration of the vestibule configuration depends on polymer composition and on which end of the polymer, 3' or 5', subsequently threads into the narrowest constriction and initiates translocation. Below approximately 140 mV a polymer is more likely to escape from the vestibule against the applied voltage gradient, while at higher voltages a polymer is more likely to follow the voltage gradient by threading through the narrowest constriction and translocating through the pore. Increasing the applied voltage also increases the duration of the vestibule configuration. A semiquantitative model of these trends suggests that escape has stronger voltage dependence than threading, and that threading is sensitive to polymer orientation while escape is not. These results emphasize the utility of alpha-hemolysin as a model system to study biologically relevant physical and chemical processes at the single-molecule level.     

Characterization of RNA synthesized in isolated nuclei of herpes simplex virus type 1-infected KB cells.

Nuclei isolated from herpes simplex virus (HSV)-infected KB cells actively synthesize HSV RNA in vitro; the RNA can be hybridized with HSV DNA or nuclear RNA from HSV-infected cells. Nascent RNA molecules labeled in vivo with 32PO4 were elongated, utilizing the nuclear system to incorporate Hg-CTP at their 3' ends, and then isolated on an affinity column. Hybridization of isolated nascent RNA molecules showed that greater than 50% of them were HSV specific and that more than 25% were self-complementary.     

Functions of the exosome in rRNA, snoRNA and snRNA synthesis.

The yeast nuclear exosome contains multiple 3'-->5' exoribonucleases, raising the question of why so many activities are present in the complex. All components are required during the 3' processing of the 5.8S rRNA, together with the putative RNA helicase Dob1p/Mtr4p. During this processing three distinct steps can be resolved, and hand-over between different exonucleases appears to occur at least twice. 3' processing of snoRNAs (small nucleolar RNAs) that are excised from polycistronic precursors or from mRNA introns is also a multi-step process that involves the exosome, with final trimming specifically dependent on the Rrp6p component. The spliceosomal U4 snRNA (small nuclear RNA) is synthesized from a 3' extended precursor that is cleaved by Rnt1p at sites 135 and 169 nt downstream of the mature 3' end. This cleavage is followed by 3'-->5' processing of the pre-snRNA involving the exosome complex and Dob1p. The exosome, together with Rnt1p, also participates in the 3' processing of the U1 and U5 snRNAs. We conclude that the exosome is involved in the processing of many RNA substrates and that different components can have distinct functions.     

Endowing RNase H-inactive antisense with catalytic activity: 2-5A-morphants

A convergent synthetic approach was used to conjugate 2',5'-oligoadenylate (2-5A, p5'A2' [p5'A2'](n)()p5'A) to phosphorodiamidate morpholino oligomers (morphants). To provide requisite quantities of 2-5A starting material, commercially and readily available synthons for solid-phase synthesis were adapted for larger scale solution synthesis. Thus, the tetranucleotide 5'-phosphoryladenylyl(2'-->5')adenylyl(2'-->5')adenylyl(2'-->5')adenosine (p5'A2'p5'A2'](2)p5'A2', tetramer 2-5A, 9) was synthesized starting with 2',3'-O-dibenzoyl-N(6),N(6)-dibenzoyl adenosine prepared from commercially available 5'-O-(4-monomethoxytrityl) adenosine. Coupling with N(6)-benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-(tert-butyldimethylsilyl) adenosine-2'-(N,N-diisopropyl-2-cyanoethyl)phosphoramidite, followed by oxidization and deprotection, generated 5'-deprotected dimer 2-5A. Similar procedures lengthened the chain to form protected tetramer 2-5 A. The title product 9 p5'A(2'p5'A)(3) (tetramer 2-5A) was obtained through phosphorylation of the terminal 5'-hydroxy of the protected tetramer and removal of remaining protecting groups using concentrated ammonium hydroxide-ethanol (3:1, v/v) at 55 degrees C and tetrabutylammonium fluoride (TBAF) in THF at room temperature, respectively. The 2-5A-phosphorodiamidate morpholino antisense chimera 11 (2-5A-morphant) was synthesized by covalently linking an aminolinker-functionalized phosphorodiamidate morpholino oligomer with periodate oxidized 2-5A tetramer (p5'A2'[p5'A2'](2)p5'A). The resulting Schiff base was reduced with cyanoborohydride thereby transforming the ribose of the 2'-terminal nucleotide of 2-5A N-substituted morpholine. RNase L assays demonstrated that this novel 2-5A-antisense chimera had significant biological activity, thereby providing another potential tool for RNA ablation.     

Exoribonuclease R in Pseudomonas syringae is essential for growth at low temperature and plays a novel role in the 3' end processing of 16 and 5 S ribosomal RNA

The (3'-->5') exoribonuclease RNase R interacts with the endoribonuclease RNase E in the degradosome of the cold-adapted bacterium Pseudomonas syringae Lz4W. We now present evidence that the RNase R is essential for growth of the organism at low temperature (4 degrees C). Mutants of P. syringae with inactivated rnr gene (encoding RNase R) are cold-sensitive and die upon incubation at 4 degrees C, a phenotype that can be complemented by expressing RNase R in trans. Overexpressing polyribonucleotide phosphorylase in the rnr mutant does not rescue the cold sensitivity. This is different from the situation in Escherichia coli, where rnr mutants show normal growth, but pnp (encoding polyribonucleotide phosphorylase) and rnr double mutants are nonviable. Interestingly, RNase R is not cold-inducible in P. syringae. Remarkably, however, rnr mutants of P. syringae at low temperature (4 degrees C) accumulate 16 and 5 S ribosomal RNA (rRNA) that contain untrimmed extra ribonucleotide residues at the 3' ends. This suggests a novel role for RNase R in the rRNA 3' end processing. Unprocessed 16 S rRNA accumulates in the polysome population, which correlates with the inefficient protein synthesis ability of mutant. An additional role of RNase R in the turnover of transfer-messenger RNA was identified from our observation that the rnr mutant accumulates transfer-messenger RNA fragments in the bacterium at 4 degrees C. Taken together our results establish that the processive RNase R is crucial for RNA metabolism at low temperature in the cold-adapted Antarctic P. syringae.     

Light-directed 5'-->3' synthesis of complex oligonucleotide microarrays

Light-directed synthesis of high-density microarrays is currently performed in the 3'-->5' direction due to constraints in existing synthesis chemistry. This results in the probes being unavailable for many common types of enzymatic modification. Arrays that are synthesized in the 5'-->3' direction could be utilized to perform parallel genotyping and resequencing directly on the array surface, dramatically increasing the throughput and reducing the cost relative to existing techniques. In this report we demonstrate the use of photoprotected phosphoramidite monomers for light-directed array synthesis in the 5'-->3' direction, using maskless array synthesis technology. These arrays have a dynamic range of >2.5 orders of magnitude, sensitivity below 1 pM and a coefficient of variance of <10% across the array surface. Arrays containing >150,000 probe sequences were hybridized to labeled mouse cRNA producing highly concordant data (average R(2) = 0.998). We have also shown that the 3' ends of array probes are available for sequence-specific primer extension and ligation reactions.