Effect of mRNA secondary structure on the efficiency of translational initiation by eukaryotic ribosomes

We have used site-directed mutagenesis to determine whether the structural context surrounding the AUG triplet influences its ability to be selected as an initiation codon by the eukaryotic preinitiation complex. AUG triplets were introduced in a loop and stem structure naturally occurring at the midpoint of the 129-nucleotides-long 5'-untranslated region of the porcine proopiomelanocortin mRNA; one AUG triplet was inserted in the loop while another was inserted in the stem of the hairpin structure. The proopiomelanocortin cDNA and the mutant cDNAs were inserted downstream from the early promoter of an expression vector derived from simian virus 40 (SV40) and transfected into monkey kidney COS-1 cells. Analysis of the proopiomelanocortin-related peptides present in the culture medium 72 h after transfection revealed that both mutant cDNAs direct the synthesis of more proopiomelanocortin than the non-mutant cDNA. The increased translational efficiency observed with both mutants is probably due to the decreased secondary structures of the shortened 5'-untranslated region. In addition, comparison of the two mutants indicates that the mutant mRNA with the AUG triplet inserted in the loop region of the hairpin structure directs the synthesis of approximately 75% more proopiomelanocortin than the mutant mRNA with the AUG triplet inserted in the stem region of the same hairpin structure, supporting a role for the structural context in the efficiency of translational initiation.     

RNA secondary structure and in vitro translation efficiency

Cell-free protein synthesis is a promising technology featuring many advantages compared to in vivo expression techniques. However, most proteins are still synthesized in vivo due to relatively low protein yields commonly achieved in vitro, especially in the batch mode of reaction. In Escherichia coli S30 extract-based cell-free systems protein yields are supposed to be partially limited by a secondary structure formation of the mRNA. In this study we checked promising members of various classes of RNA chaperones and several different RNA helicases on their ability to enhance in vitro translation. The data clearly show that the addition of none of these factors provides a general solution to the problem. However, protein yields can be increased in presence of a microRNA hybridizing with the 5′ untranslated region of mRNAs, possibly by inducing structural changes improving accessibility of the Shine Dalgarno sequence for the ribosomes.     

Amplicon secondary structure prevents target hybridization to oligonucleotide microarrays

DNA microarrays that are used as end-point detectors for PCR assays are typically composed of short (15-25 mer) oligonucleotide probes bound to glass. When designing these detectors, we have frequently encountered situations where a probe would not hybridize to its complementary, terminally labeled PCR amplicon. To determine if failures could be explained by general phenomenon such as secondary structure, we designed a microarray to detect eight regions of the Escherichia coli 16S rDNA gene. We then amplified eight amplicons of different lengths using a biotin conjugated, antisense primer. Amplicons were then hybridized to the microarray and detected using a combination of signal amplification and fluorescence. In most cases, probe sequences complementary to the 5' region of the amplified products (sense orientation) did not hybridize to their respective amplicon. We tested for positional bias and showed that a biotin conjugated sense primer mirrored the same probe failures. Nick translated products, however, hybridized to all probes. Because nick translation generates many labeled fragments of random length, we concluded that this method disrupted secondary structure that otherwise prevented the amplicons from hybridizing to their respective probes. We also show that nick translation does not compromise detector sensitivity even when used with long PCR amplicons (ca. 1.5 kbp). Despite the increased cost of the nick translation, we concluded that this labeling strategy will reduce the time needed to design new assays as well as avoid possible false negatives during field applications. Alternative labeling strategies are also discussed.     

Effect of superhelical structure on the secondary structure of DNA rings

A quantity, called the linking number, is defined, which specifies the total number of twists in a circular helix. The linking number is invariant under continuous deformations of the ring and therefore enables one to calculate the influence of superhelical structures on the secondary helix of a circular molecule. The linking number can be determined by projecting the helix into a plane and counting strand crosses in the projection as described. For example, it has been shown that for each 180° twist in a left-handed superhelix, a right-handed 360° twist is removed from the secondary helix, thus allowing local unwinding.     

siRNA target site secondary structure predictions using local stable substructures

The crystal structure based model of the catalytic center of Ago2 revealed that the siRNA and the mRNA must be able to form an A-helix for correct positing of the scissile phosphate bond for cleavage in RNAi. This suggests that base pairing of the target mRNA with itself, i.e. secondary structure, must be removed before cleavage. Early on in the siRNA design, GC-rich target sites were avoided because of their potential to be involved in strong secondary structure. It is still unclear how important a factor mRNA secondary structure is in RNAi. However, it has been established that a difference in the thermostability of the ends of an siRNA duplex dictate which strand is loaded into the RNA-induced silencing complex. Here, we use a novel secondary structure prediction method and duplex-end differential calculations to investigate the importance of a secondary structure in the siRNA design. We found that the differential duplex-end stabilities alone account for functional prediction of 60% of the 80 siRNA sites examined, and that secondary structure predictions improve the prediction of site efficacy. A total of 80% of the non-functional sites can be eliminated using secondary structure predictions and duplex-end differential.     

Effect of alkylation on the secondary structure of DNA

S1 nuclease hydrolysis and bezoylated naphthoylated DEAE-cellulose (BND-cellulose) chromatography have been used to demonstrate that alkylation of DNA by dimethyl sulfate at neutral pH leads to the production of partially denatured molecules under conditions where no significant depurination occurs. DNA was alkylated with increasing concentrations of the alkylating agent, and subjected to enzymatic degradation and binding to BND cellulose. An increasing degree of DNA hydrolysis and adherence to BND cellulose was seen. On hydroxyapatite chromatography the alkylated DNA still eluted at the position of double-stranded molecules suggesting the presence of partially denatured regions. The presence of salt had a preventive effect on such denaturation.     

The effect of structure in a long target RNA on ribozyme cleavage efficiency.

Inhibition of gene expression by catalytic RNA (ribozymes) requires that ribozymes efficiently cleave specific sites within large target RNAs. However, the cleavage of long target RNAs by ribozymes is much less efficient than cleavage of short oligonucleotide substrates because of higher order structure in the long target RNA. To further study the effects of long target RNA structure on ribozyme cleavage efficiency, we determined the accessibility of seven hammerhead ribozyme cleavage sites in a target RNA that contained human immunodeficiency virus type 1 (HIV-1) vif - vpr . The base pairing-availability of individual nucleotides at each cleavage site was then assessed by chemical modification mapping. The ability of hammerhead ribozymes to cleave the long target RNA was most strongly correlated with the availability of nucleotides near the cleavage site for base pairing with the ribozyme. Moreover, the accessibility of the seven hammerhead ribozyme cleavage sites in the long target RNA varied by up to 400-fold but was directly determined by the availability of cleavage sites for base pairing with the ribozyme. It is therefore unlikely that steric interference affected hammerhead ribozyme cleavage. Chemical modification mapping of cleavage site structure may therefore provide a means to identify efficient hammerhead ribozyme cleavage sites in long target RNAs.     

Effect of lysine modification on the secondary structure of ovalbumin

In order to determine the effect of chemical modification of the -amino groups on the secondary structure of ovalbumin, we prepared six acetylated (17, 36, 54, 70, 82, and 98%) and four succinylated derivatives (25, 50, 72, and 97%) of the protein. Native ovalbumin and the acylated derivatives were homogeneous as revealed by the electrophoretic pattern. The UV-absorption and fluorescence spectra changed progressively with the extent of modification. However, circular dichroic (CD) studies indicated that acylation of 15 of the 20 lysine residues had little effect on the secondary structure of ovalbumin. Acylation of the remaining five lysine residues resulted in a fairly severe change in the secondary structure. The -helical content decreased from about 31% in the native state to 16.5% in the 97% succinylated ovalbumin and to 21.5% in the 98% acetylated derivative. A comparison of these data with the spectral and hydrodynamic data of Qasim and Salahuddin (1978) suggested that the secondary structure of ovalbumin is more resistant to acylation than is the tertiary structure and, thus, the tertiary and the secondary structures are, to some extent, mutually independent. Raising thepH to 11.2 did not alter the secondary structure of ovalbumin and increasing the ionic strength by more than 20-fold did not reverse the loss of helical structure in 97% succinylated protein. These two observations suggest that the change in secondary structure upon maximal acylation may not only involve electrostatic effects, but also certain other factors, such as steric hindrance due to the entering bulky groups.     

Improved efficiency in cryo-em secondary structure topology determination from inaccurate data

The determination of the secondary structure topology is a critical step in deriving the atomic structure from the protein density map obtained from electron cryo-microscopy technique. This step often relies on the matching of two sources of information. One source comes from the secondary structures detected from the protein density map at the medium resolution, such as 5-10 ?. The other source comes from the predicted secondary structures from the amino acid sequence. Due to the inaccuracy in either source of information, a pool of possible secondary structure positions needs to be sampled. This paper studies the question, that is, how to reduce the computation of the mapping when the inaccuracy of the secondary structure predictions is considered. We present a method that combines the concept of dynamic graph with our previous work of using constrained shortest path to identify the topology of the secondary structures. We show a reduction of 34.55% of run-time as comparison to the na?ve way of handling the inaccuracies. We also show an improved accuracy when the potential secondary structure errors are explicitly sampled verses the use of one consensus prediction. Our framework demonstrated the potential of developing computationally effective exact algorithms to identify the optimal topology of the secondary structures when the inaccuracy of the predicted data is considered.     

目标树抚育对亚热带天然次生灌丛群落结构和多样性的影响

在浙江省临安市选择典型天然次生灌丛,分别进行封禁和目标树抚育,探讨灌丛恢复为乔木林的可能性.结果表明: 4年后,与未管护(对照)相比,封禁和目标树抚育后群落平均胸径分别提高1.3和2.6倍,平均高度分别提高0.5和1.1倍;目标树抚育林木出现了对照林分没有的4.5～8.5 cm径阶和4.5～8.5 m树高阶,形成了4 m高的新林层;灌木层物种丰富度和多样性指数没有因抚育而下降;封禁管理维持了群落的树种组成,遵循原有的演替方向;目标树抚育显著改变了群落的树种组成,提高了目的树种的重要值,近期有可能恢复成为针阔混交林群落.与封禁相比,目标树抚育在优势林木胸径和高度生长、树种组成改善等方面更能达到预想的目标.在有条件经营的情况下,可以选择目标树抚育模式对天然次生灌丛进行管理,从而达到加快群落恢复演替形成乔木林的目的.     

RNAi revised - target mRNA-dependent enhancement of gene silencing

The discovery of RNA interference (RNAi) gave rise to the development of new nucleic acid-based technologies as powerful investigational tools and potential therapeutics. Mechanistic key details of RNAi in humans need to be deciphered yet, before such approaches take root in biomedicine and molecular therapy.We developed and validated an in silico-based model of siRNA-mediated RNAi in human cells in order to link in vitro-derived pre-steady state kinetic data with a quantitative and time-resolved understanding of RNAi on the cellular level. The observation that product release by Argonaute 2 is accelerated in the presence of an excess of target RNA in vitro inspired us to suggest an associative mechanism for the RNA slicer reaction where incoming target mRNAs actively promote dissociation of cleaved mRNA fragments. This novel associative model is compatible with high multiple turnover rates of RNAi-based gene silencing in living cells and accounts for target mRNA concentration-dependent enhancement of the RNAi machinery.     

Effect of riboflavin and light on the secondary structure of DNA

S1 nuclease hydrolysis and benzoylated naphthoylated DEAE cellulose (BND-cellulose) chromatography have been used to study the effect of riboflavin and visible light on DNA. Native calf thymus DNA was incubated with riboflavin in the presence of fluorescent light for various time periods and subjected to S1 nuclease hydrolysis. An increasing degree of DNA degradation was seen suggesting a destabilization of the secondary structure. A decrease in melting temperature was also observed. Incubation with riboflavin and illumination caused adherence to BND-cellulose indicating the production of single stranded regions or breaks in the native double stranded molecules. However, when incubation was done in dark and in the presence of triplet excited state quencher, potassium iodide, a reduced adherence of DNA to BND-cellulose was seen. Plasmid pBR322 DNA was also treated with riboflavin under these conditions and subjected to agarose gel electrophoresis. No degradation could be seen in dark incubated and potassium iodide treated samples. These results indicate that the adherence of DNA to BND-cellulose in dark is possibly due to the binding of aromatic residues to the resin suggesting the formation of a complex between riboflavin and DNA.     

The effects of spacer sequences on silencing efficiency of plant RNAi vectors

RNA interference (RNAi) has been used to suppress gene expression in various eukaryotic organisms. In plants, RNAi can be induced by introduction of an RNAi vector that transcribes a self-complementary hairpin RNA. Most basic RNAi constructs have an inverted repeat interrupted with a spacer sequence. To test silencing capability of RNAi constructs, we developed an in vivo assay that is based on the RNAi-mediated changes of the α-linolenic acid content in hairy roots. A tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) is the main enzyme for production of α-linolenic acid of root membrane lipids. Tobacco hairy roots transformed with the RNAi vectors against the NtFAD3 gene showed a decrease in α-linolenic acid content. The frequency of RNA silencing was more affected by spacer sequence than by spacer length, at least between 100 and 1800 bp. Since significant amounts of hairpin RNA against the NtFAD3 gene remained in the transgenic plants displaying a weak silencing phenotype, low degree of silencing was attributed to low efficiency of hairpin RNA processing mediated by Dicer-like proteins. Our results show the possibility of producing a broad range of the RNAi-induced silencing phenotypes by replacing the spacer sequence of RNAi construct.     

Effect of alkylation with streptozotocin on the secondary structure of DNA

S₁ nuclease hydrolysis and hydroxyapatite chromatography were used to study the effect of the alkylating antibiotic, streptozotocin, on the secondary structure of DNA. Native calf thymus DNA was alkylatedin vitro with increasing concentrations of streptozotocin and subjected to S 1 nuclease hydrolysis. An increasing degree of DNA degradation was seen, suggesting a destabilization of the secondary structure. Indirect evidence, deduced from alkaline hydrolysis, effect of NaCl on S₁ nuclease hydrolysis, and hydroxyapatite chromatographic analysis of alkylated DNA, suggested a significant alkylation of DNA phosphates in addition to DNA bases. Nictotinamide has been reported to alter the cytotoxic and carcinogenic effects of streptozotocin. Our experiments indicate that in the presence of nicotinamide, streptozotocin causes the formation of a greater proportion of alkylated bases in relation to alkyl phosphotriesters. This may have significance in relation to the differential cytotoxicity of streptozotocin in the absence and presence of nicotinamide.     

Single-stranded antisense siRNAs guide target RNA cleavage in RNAi

Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). Here, we describe a human biochemical system that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, we demonstrate that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with eIF2C1 and/or eIF2C2 (human GERp95) Argonaute proteins. RISC is rapidly formed in HeLa cell cytoplasmic extract supplemented with 21 nt siRNA duplexes, but also by adding single-stranded antisense RNAs, which range in size between 19 and 29 nucleotides. Single-stranded antisense siRNAs are also effectively silencing genes in HeLa cells, especially when 5'-phosphorylated, and expand the repertoire of RNA reagents suitable for gene targeting.