Failure to detect "cap" structures in mitochondrial DNA-coded poly(A)-containing RNA from HeLa cells

The structure of the 5'-termini has been investigated in mitochondrial DNA- coded poly(A)-containing RNA from HeLa cells. For this purpose, mitochondrial RNA isolated from cells labeled for 3 hours with [32P]orthophosphate in the presence of 20 µg/ml camptothecin, and selected for poly(A) content by two passages through oligo(dT)-cellulose, was digested either with the nuclease P1 or with a mixture of RNases: the digestion products were then fractionated by two-dimensional electrophoresis. No "cap" structures were detected under conditions where the presence of such structures in one out of five to ten RNA molecules would have been recognized. It is, therefore, likely that "cap" structures are completely absent in HeLa cell mitochondrial poly(A)-containing RNA

The mRNA degradation factor Xrn1 regulates transcription elongation in parallel to Ccr4

Co-transcriptional imprinting of mRNA by Rpb4 and Rpb7 subunits of RNA polymerase II (RNAPII) and by the Ccr4–Not complex conditions its posttranscriptional fate. In turn, mRNA degradation factors like Xrn1 are able to influence RNAPII-dependent transcription, making a feedback loop that contributes to mRNA homeostasis. In this work, we have used repressible yeast GAL genes to perform accurate measurements of transcription and mRNA degradation in a set of mutants. This genetic analysis uncovered a link from mRNA decay to transcription elongation. We combined this experimental approach with computational multi-agent modelling and tested different possibilities of Xrn1 and Ccr4 action in gene transcription. This double strategy brought us to conclude that both Xrn1-decaysome and Ccr4–Not regulate RNAPII elongation, and that they do it in parallel. We validated this conclusion measuring TFIIS genome-wide recruitment to elongating RNAPII. We found that xrn1Δ and ccr4Δ exhibited very different patterns of TFIIS versus RNAPII occupancy, which confirmed their distinct role in controlling transcription elongation. We also found that the relative influence of Xrn1 and Ccr4 is different in the genes encoding ribosomal proteins as compared to the rest of the genome

Fluorescence-based quantification of messenger RNA and plasmid DNA decay kinetics in extracellular biological fluids and cell extracts

Extracellular and intracellular degradation of nucleic acids remains an issue in non-viral gene therapy. Understanding biodegradation is critical for the rational design of gene therapeutics in order to maintain stability and functionality at the target site. However, there are only limited methods available that allow determining the stability of genetic materials in biological environments. In this context, the decay kinetics of fluorescently labeled plasmid DNA (pDNA) and messenger RNA (mRNA) in undiluted biological samples (i.e., human serum, human ascites, bovine vitreous) and cell extracts is studied using fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT). It is demonstrated that FCS is suitable to follow mRNA degradation, while SPT is better suited to investigate pDNA integrity. The half-life of mRNA and pDNA is approximate to 1-2 min and 1-4 h in biological samples, respectively. The resistance against biodegradation drastically improves by complexation with lipid-based carriers. Taken together, FCS and SPT are able to quantify the integrity of mRNA and pDNA, respectively, as a function of time, both in the extracellular biological fluids and cell extracts. This in turn allows to focus on the important but less understood issue of nucleic acids degradation in more detail and to rationally optimize gene delivery system as therapeutics

Protein-RNA interactions: a structural analysis

A detailed computational analysis of 32 protein-RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein-double-stranded DNA and protein-single-stranded DNA complexes. The interface properties of the protein-RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein-RNA and protein-DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein-RNA complexes, backbone contacts were more dominant in the protein-DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level

Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging

Small interfering RNA (siRNA) molecules are potent effectors of post-transcriptional gene silencing. Using noninvasive bioluminescent imaging and a mathematical model of siRNA delivery and function, the effects of target-specific and treatment-specific parameters on siRNA-mediated gene silencing are monitored in cells stably expressing the firefly luciferase protein. In vitro, luciferase protein levels recover to pre-treatment values within <1 week in rapidly dividing cell lines, but take longer than 3 weeks to return to steady-state levels in nondividing fibroblasts. Similar results are observed in vivo, with knockdown lasting ~10 days in subcutaneous tumors in A/J mice and 3–4 weeks in the nondividing hepatocytes of BALB/c mice. These data indicate that dilution due to cell division, and not intracellular siRNA half-life, governs the duration of gene silencing under these conditions. To demonstrate the practical use of the model in treatment design, model calculations are used to predict the dosing schedule required to maintain persistent silencing of target proteins with different half-lives in rapidly dividing or nondividing cells. The approach of bioluminescent imaging combined with mathematical modeling provides useful insights into siRNA function and may help expedite the translation of siRNA into clinically relevant therapeutics for disease treatment and management

Simultaneous precise editing of multiple genes in human cells

Abstract. When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results i

DNA and its counterions: A molecular dynamics study

The behaviour of mobile counterions, Na+ and K+, was analysed around a B-DNA double helix with the sequence CCATGCGCTGAC in aqueous solution during two 50 ns long molecular dynamics trajectories. The movement of both monovalent ions remains diffusive in the presence of DNA. Ions sample the complete space available during the simulation time, although individual ions sample only about one-third of the simulation box. Ions preferentially sample electronegative sites around DNA, but direct binding to DNA bases remains a rather rare event, with highest site occupancy values of &lt;13%. The location of direct binding sites depends greatly on the nature of the counterion. While Na+ binding in both grooves is strongly sequence-dependent with the preferred binding site in the minor groove, K+ mainly visits the major groove and binds close to the centre of the oligomer. The electrostatic potential of an average DNA structure therefore cannot account for the ability of a site to bind a given cation; other factors must also play a role. An extensive analysis of the influence of counterions on DNA conformation showed no evidence of minor groove narrowing upon ion binding. A significant difference between the conformations of the double helix in the different simulations can be attributed to extensive (/ transitions in the phosphate backbone during the simulation with Na+. These transitions, with lifetimes over tens of nanoseconds, however, appear to be correlated with ion binding to phosphates. The ion-specific conformational properties of DNA, hitherto largely overlooked, may play an important role in DNA recognition and binding