RNA Constituents Research Articles

Assembly constraints drive co-evolution among ribosomal constituents.

Ribosome biogenesis, a central and essential cellular process, occurs through sequential association and mutual co-folding of protein–RNA constituents in a well-defined assembly pathway. Here, we construct a network of co-evolving nucleotide/amino acid residues within the ribosome and demonstrate that assembly constraints are strong predictors of co-evolutionary patterns. Predictors of co-evolution include a wide spectrum of structural reconstitution events, such as cooperativity phenomenon, protein-induced rRNA reconstitutions, molecular packing of different rRNA domains, protein–rRNA recognition, etc. A correlation between folding rate of small globular proteins and their topological features is known. We have introduced an analogous topological characteristic for co-evolutionary network of ribosome, which allows us to differentiate between rRNA regions subjected to rapid reconstitutions from those hindered by kinetic traps. Furthermore, co-evolutionary patterns provide a biological basis for deleterious mutation sites and further allow prediction of potential antibiotic targeting sites. Understanding assembly pathways of multicomponent macromolecules remains a key challenge in biophysics. Our study provides a ‘proof of concept’ that directly relates co-evolution to biophysical interactions during multicomponent assembly and suggests predictive power to identify candidates for critical functional interactions as well as for assembly-blocking antibiotic target sites.

Assembly of RNPs: help needed.

Assembly of RNPs: help needed.

Fluorosomen werfen Licht auf Ribosomenproduktion

Ribosomes facilitate protein biosynthesis in all living cells. They consist of two subunits composed of protein and RNA constituents. Ribosome biogenesis is insufficiently explored due to its complexity and, at the same time, supposed to be an attractive target for new antimicrobials. For both scientific and pharmaceutical purposes the use of fluorescently labeled ribosomes (fluorosomes) could be a valuable tool.

Transformations of the macromolecular landscape at mitochondria during DNA-damage-induced apoptotic cell death.

Apoptosis is a dynamic process regulated by mitochondrion critical for cellular respiration and survival. Execution of apoptosis is mediated by multiple protein signaling events at mitochondria. Initiation and progression of apoptosis require numerous apoptogenic factors that are either released from or sequestered in mitochondria, which may transform the biomolecular makeup of the organelle. In this communication, using Raman microspectroscopy, we demonstrate that transformation in biomolecular composition of mitochondrion may be used as apoptosis marker in an individual cell. For the first time, we show that significant changes occur in the concentrations of RNA, DNA, protein, and lipid constituents of mitochondria during apoptosis. The structural analysis of proteins on mitochondria demonstrated a decrease in α-helix secondary structure content, and an increase in the levels of random coils and β-sheets on mitochondria. This may represent an additional hallmark of apoptosis. Strikingly, we observed nearly identical changes in macromolecular content of mitochondria both in the presence and absence of a key proapoptotic protein, Bax (Bcl-2-associated X protein). Increased DNA level in mitochondria corresponded with higher mitochondrial DNA (mtDNA), cellular reactive oxygen species (ROS), and mitochondrial ROS production. Upregulation of polymerase-γ (POLG), mitochondrial helicase Twinkle, and mitochondrial transcription factor A (Tfam) in response to DNA damage correlated with increased mtDNA and RNA synthesis. Elevated activity of oxidative phosphorylation complexes supports functional mitochondrial respiration during apoptosis. Thus, we define previously unknown dynamic correlation of macromolecular structure of mitochondria and apoptosis progression in the presence and absence of Bax protein. These findings open up a new approach for monitoring physiological status of cells by non invasive single-cell method.

A comparative study of ribosomal proteins: linkage between amino acid distribution and ribosomal assembly.

BackgroundAssembly of the ribosome from its protein and RNA constituents must occur quickly and efficiently in order to synthesize the proteins necessary for all cellular activity. Since the early 1960’s, certain characteristics of possible assembly pathways have been elucidated, yet the mechanisms that govern the precise recognition events remain unclear.We utilize a comparative analysis to investigate the amino acid composition of ribosomal proteins (r-proteins) with respect to their role in the assembly process. We compared small subunit (30S) r-protein sequences to those of other housekeeping proteins from 560 bacterial species and searched for correlations between r-protein amino acid content and factors such as assembly binding order, environmental growth temperature, protein size, and contact with ribosomal RNA (rRNA) in the 30S complex.ResultsWe find r-proteins have a significantly high percent of positive residues, which are highly represented at rRNA contact sites. An inverse correlation between the percent of positive residues and r-protein size was identified and is mainly due to the content of Lysine residues, rather than Arginine. Nearly all r-proteins carry a net positive charge, but no statistical correlation between the net charge and the binding order was detected. Thermophilic (high-temperature) r-proteins contain increased Arginine, Isoleucine, and Tyrosine, and decreased Serine and Threonine compared to mesophilic (lower-temperature), reflecting a known distinction between thermophiles and mesophiles, possibly to account for protein thermostability. However, this difference in amino acid content does not extend to rRNA contact sites, as the proportions of thermophilic and mesophilic contact residues are not significantly different.ConclusionsGiven the significantly higher level of positively charged residues in r-proteins and at contact sites, we conclude that ribosome assembly relies heavily on an electrostatic component of interaction. However, the binding order of r-proteins in assembly does not appear to depend on these electrostatics interactions. Additionally, because thermophiles and mesophiles exhibit significantly different amino acid compositions in their sequences but not in the identities of contact sites, we conclude that this electrostatic component of interaction is insensitive to temperature and is not the determining factor differentiating the temperature sensitivity of ribosome assembly.

37 A comparative study of ribosomal proteins: linkage between amino acid distribution and ribosomal assembly

Assembly of the ribosome from its protein and RNA constituents has been studied extensively over the past 50 years, and here we utilize a comparative analysis approach to relate the composition of ribosomal proteins (r-proteins) to their role in the assembly process. We computed the amino acid distributions for the 30S subunit r-protein sequences from 560 bacterial species and compared this composition to those of other house-keeping proteins from the same species. We found that r-proteins have a significantly higher content of positively charged residues (Lysine, K, and Arginine, R) than do nonribosomal proteins (10% for R and 11% for K in r-proteins, vs. 4.7% R and 5.9% K in non-ribosomal proteins), which is consistent with prior knowledge of net positive charges carried by r-proteins (Baker et al., 2001; Klein et al., 2004; Burton et al., 2012). Furthermore, these two residues are also highly represented at contact sites along the protein/RNA interface (contact enrichment factor (CEF) > 1). These results provide further evidence of the importance of electrostatic interactions between the positively charged proteins and negatively charged ribosomal RNA (rRNA) during ribosome assembly. Other highly represented contact residues include polar and aromatic residues, which are likely to interact with rRNA via hydrogen bonds and base stacking interactions, respectively. Interestingly, the proportion of K residues generally decreases with r-protein size, reflecting a negative correlation between protein lengths and the proportion of K (Spearman’s rank correlation, ρ = −0.802, p = 2.60e − 5). We suggest that this trend helps the smaller r-proteins, which experience higher translational entropy than large proteins, overcome the increased free energy barrier during assembly. When the r-protein sequences were categorized according to the species’ optimal growth temperature, we found that thermophiles show increased R, Isoleucine (I), and Tyrosine (Y) content, whereas mesophiles have increased proportions of Serine (S) and Threonine (T). These results reflect one typical distinction between thermophiles and mesophiles (Kumar and Nussinov 2001), yet these differences in amino acid distributions do not extend to their respective contact sites. That is, the makeup of thermophilic and mesophilic r-protein contact residues are not significantly different (p > 0.01). This indicates that, while the percent compositions of amino acids relating to qualities such as thermostability and protein folding are expected to vary with environmental temperature, the distributions of residues in contact with rRNA are comparable for all bacterial species. From this, we conclude that the electrostatic interactions that guide ribosome assembly are independent of temperature.

In vitro antifungal activity of dehydrozingerone and its fungitoxic properties.

The efficacy of Dehydrozingerone (DZ; dehydroderivative of zingerone) as an antifungal agent and its mode of action against food spoilage fungal pathogens was studied and presented. DZ is a constituent of ginger (Zingiber officinale rhizomes) and structural half analogue of curcumin. Its efficacy against Aspergillus oryzae, Aspergillus flavus, Aspergillus niger, Aspergillus ochraceus, Fusarium oxysporum and Penicillium chrysogenum was evaluated. Effect of DZ on the growth and sporulation of A. ochraceus was also studied. The fungal species were susceptible to DZ and the minimum inhibitory concentration and fungicidal concentration ranged from 755 to 911 μM and 880 to 1041 μM respectively. The mycelial and spore germination was significantly inhibited; reduction in the weight of the cell mass, carbohydrate, protein, DNA and RNA constituents in the cells isolated from cultures of A. ochraceus grown with DZ were observed. Scanning electron microscopy studies revealed morphological observations such as cell lysis, inhibition and morphological alterations in hyphae and sporulation in A. ochraceus on treatment with DZ. Current investigations revealed that DZ is a potential antifungal agent and can find application as an additive or adjuvant in food and pharmaceutical industries after appropriate toxicological studies.

Probing, by Self-Assembly, the Number of Potential Binding Sites for Minor Protein Subunits in the Procapsid of Double-Stranded RNA Bacteriophage φ6

The double-stranded RNA bacteriophage Φ6 is an extensively studied prokaryotic model system for virus assembly. There are established in vitro assembly protocols available for the Φ6 system for obtaining infectious particles from purified protein and RNA constituents. The polymerase complex is a multifunctional nanomachine that replicates, transcribes, and translocates viral RNA molecules in a highly specific manner. The complex is composed of (i) the major structural protein (P1), forming a T=1 icosahedral lattice with two protein subunits in the icosahedral asymmetric unit; (ii) the RNA-dependent RNA polymerase (P2); (iii) the hexameric packaging nucleoside triphosphatase (NTPase) (P4); and (iv) the assembly cofactor (P7). In this study, we analyzed several Φ6 virions and recombinant polymerase complexes to investigate the relative copy numbers of P2, P4, and P7, and we applied saturated concentrations of these proteins in the self-assembly system to probe their maximal numbers of binding sites in the P1 shell. Biochemical quantitation confirmed that the composition of the recombinant particles was similar to that of the virion cores. By including a high concentration of P2 or P7 in the self-assembly reaction mix, we observed that the numbers of these proteins in the resulting particles could be increased beyond those observed in the virion. Our results also suggest a previously unidentified P2-P7 dependency in the assembly reaction. Furthermore, it appeared that P4 must initially be incorporated at each, or a majority, of the 5-fold symmetry positions of the P1 shell for particle assembly. Although required for nucleation, excess P4 resulted in slower assembly kinetics.

A Computational Investigation on the Connection between Dynamics Properties of Ribosomal Proteins and Ribosome Assembly

Assembly of the ribosome from its protein and RNA constituents has been studied extensively over the past 50 years, and experimental evidence suggests that prokaryotic ribosomal proteins undergo conformational changes during assembly. However, to date, no studies have attempted to elucidate these conformational changes. The present work utilizes computational methods to analyze protein dynamics and to investigate the linkage between dynamics and binding of these proteins during the assembly of the ribosome. Ribosomal proteins are known to be positively charged and we find the percentage of positive residues in r-proteins to be about twice that of the average protein: Lys+Arg is 18.7% for E. coli and 21.2% for T. thermophilus. Also, positive residues constitute a large proportion of RNA contacting residues: 39% for E. coli and 46% for T. thermophilus. This affirms the known importance of charge-charge interactions in the assembly of the ribosome. We studied the dynamics of three primary proteins from E. coli and T. thermophilus 30S subunits that bind early in the assembly (S15, S17, and S20) with atomic molecular dynamic simulations, followed by a study of all r-proteins using elastic network models. Molecular dynamics simulations show that solvent-exposed proteins (S15 and S17) tend to adopt more stable solution conformations than an RNA-embedded protein (S20). We also find protein residues that contact the 16S rRNA are generally more mobile in comparison with the other residues. This is because there is a larger proportion of contacting residues located in flexible loop regions. By the use of elastic network models, which are computationally more efficient, we show that this trend holds for most of the 30S r-proteins.

Linkage Between Dynamics and Assembly of Ribosomal Proteins

Assembly of the ribosome from its protein and RNA constituents has been studied extensively over the past 50 years, and experimental evidence suggests that prokaryotic ribosomal proteins undergo conformational changes during assembly. However, to date, no studies have attempted to elucidate these conformational changes. The present work utilizes computational methods to analyze protein dynamics and to investigate the linkage between dynamics and binding of these proteins played during the assembly of the ribosome. We studied the dynamics of three primary proteins from E. coli and T. thermophilus 30S subunits (S15, S17, and S20) with atomic molecular dynamic simulations, followed by a study of all r-proteins using elastic network models. We find that r-proteins contain a higher than average percentage of positive residues (Lys + Arg is 18.7% for E. coli and 21.2% for T. thermophilus). Also, positive residues constitute a large proportion of RNA contacting residues (39% for E. coli and 46% for T. thermophilus). This indicates the importance of charge-charge interactions in the assembly of the ribosome. Molecular Dynamics simulations show that solvent-exposed proteins (S15 and S17) tend to adopt more stable solution conformations than an RNA-embedded protein (S20). We also find protein residues that contact the 16S rRNA are generally more mobile in comparison with the other residues. This is because there is a larger proportion of contacting residues located in flexible loop regions. Using elastic network models, which are computationally more efficient, we show that this trend holds for most of the 30S r-proteins.

Increasing surface enhanced Raman spectroscopy effect of RNA and DNA components by changing the pH of silver colloidal suspensions

This work focused on establishing the parameters for enhancing the Raman signals of DNA and RNA constituents: nitrogenous bases, nucleosides and nucleotides, using metallic nanoparticles as surface enhanced Raman scattering substrates. Silver nanospheres were synthesized using sodium borohydride as a reducing agent and sodium citrate as a capping agent. The prepared nanoparticles had a surface plasmon band at ∼384 nm and an average size of 12 ± 3 nm. The nanoparticles’ surface charge was manipulated by changing the pH of the Ag colloidal suspensions in the range of 1–13. Low concentrations as 0.7 μM were detected under the experimental conditions. The optimum pH values were: 7 for adenine, 9 for AMP, 5 for adenosine, 7 for dAMP and 11 for deoxyadenosine.

The post-transcriptional steps of eukaryotic ribosome biogenesis.

One of the most important tasks of any cell is to synthesize ribosomes. In eukaryotes, this process occurs sequentially in the nucleolus, the nucleoplasm and the cytoplasm. It involves the transcription and processing of pre-ribosomal RNAs, their proper folding and assembly with ribosomal proteins and the transport of the resulting pre-ribosomal particles to the cytoplasm where final maturation events occur. In addition to the protein and RNA constituents of the mature cytoplasmic ribosomes, this intricate process requires the intervention of numerous protein and small RNA trans-acting factors. These transiently interact with pre-ribosomal particles at various stages of their maturation. Most of the constituents of pre-ribosomal particles have probably now been identified and research in the field is starting to unravel the timing of their intervention and their precise mode of action. Moreover, quality control mechanisms are being discovered that monitor ribosome synthesis and degrade the RNA components of defective pre-ribosomal particles.

General, rapid, and transcription-dependent fragmentation of nucleolar antigens in S. cerevisiae mRNA export mutants

In the yeast Saccharomyces cerevisiae, mutation of some effectors of mRNA nuclear export leads to the rapid accumulation of HSP104 RNA in transcription site-associated foci. We have screened the S. cerevisiae complement of viable gene deletion mutants for their inability to export HSP104 RNA. The 15 strains identified comprise deletions of components of the THO, Thp1p/Sac3p, and nuclear pore complexes. In all three mutant classes, retained RNA overlaps the HSP104 transcription site. Thus, an early block to HSP104 RNA export is general. Incubation of the identified deletion strains, as well as seven additional mutants, under conditions where mRNA export is blocked results in rapid dissipation of nucleolar protein and RNA constituents. Time course experiments show that dissipation of nucleolar antigens succeeds mRNA retention and is reversed when the load of nuclear mRNA ceases. Consistent with a causal role of excess nuclear mRNA, nucleolar morphology in an mRNA export mutant environment remains intact when transcription by RNA polymerase II is inhibited.

Excitation and ionic fragmentation of gas-phase biomolecules using electrons and synchrotron radiation

An experimental study of the electronic excitation and ionic dissociation of two important classes of biomolecules–natural products (biogenic volatile organic compounds, VOCs, and volatile components of essential oils) and DNA and RNA constituents (aminoacids and bases) is here exemplified with recent results on the fragmentation of thymine and isoprene as induced by synchrotron radiation and fast electrons. Fragmentation of the thymine molecule was seen to dramatically increase as the photon energy increased from 21 to 300 eV and 450 eV. At the highest photon energy, simply and doubly charged N and O atoms were observed. The parent ion (m/z = 126) could be observed at all photon energies. The fragmentation pattern observed in the 1.0 keV electron impact mass spectrum of thymine resembled more closely the fragmentation observed with 21 eV photons. In isoprene, the dominant fragments observed at 21 eV and 310 eV photon energy as well as in the 1.0 keV electron impact mass spectrum were C5H7+(m/z = 67), C4H5+(m/z = 53), C3H3+(m/z = 39) and C2H3+(m/z = 27). Previously unreported fragments, namely H+, C+, CH+, CH2+, and CH3+ were observed at the high photon energies and at the electron impact mass spectrum.

An early evolutionary origin for the minor spliceosome

The minor spliceosome is a ribonucleoprotein complex that catalyses the removal of an atypical class of spliceosomal introns (U12-type) from eukaryotic messenger RNAs. It was first identified and characterized in animals, where it was found to contain several unique RNA constituents that share structural similarity with and seem to be functionally analogous to the small nuclear RNAs (snRNAs) contained in the major spliceosome. Subsequently, minor spliceosomal components and U12-type introns have been found in plants but not in fungi. Unlike that of the major spliceosome, which arose early in the eukaryotic lineage, the evolutionary history of the minor spliceosome is unclear because there is evidence of it in so few organisms. Here we report the identification of homologues of minor-spliceosome-specific proteins and snRNAs, and U12-type introns, in distantly related eukaryotic microbes (protists) and in a fungus (Rhizopus oryzae). Cumulatively, our results indicate that the minor spliceosome had an early origin: several of its characteristic constituents are present in representative organisms from all eukaryotic supergroups for which there is any substantial genome sequence information. In addition, our results reveal marked evolutionary conservation of functionally important sequence elements contained within U12-type introns and snRNAs.

HPLC analysis of plant DNA methylation: a study of critical methodological factors

HPLC analysis of nucleosides is important for determining total DNA methylation in plants and can be used to help characterise epigenetic changes during stress, growth and development. This is of particular interest for in vitro plant cultures as they are highly susceptible to genetic change. HPLC methodologies have been optimised for mammalian and microbial DNA, but not for plants. This study examines critical methodological factors in the HPLC analysis of plant DNA methylation using in vitro cultures of Ribes ciliatum. HPLC revealed that complete removal of RNA from plant DNA extractions is difficult using RNase (A and T1) digestions and LiCl precipitation. This suggests that base analysis should be avoided when using these RNA removal techniques, as bases from residual RNA fragments will inflate peak areas for DNA-derived bases. Nucleoside or nucleotide analysis is therefore recommended as a more suitable option as RNA and DNA constituents can be readily separated. DNA digestion was also a critical factor as methylation was under-estimated following incomplete nuclease digestion and over-estimated following incomplete phosphatase digestion. The units of enzyme required for complete DNA digestion was optimised and found to be 20–200 times less for nuclease P1 and 15 times less for alkaline phosphatase as compared with previous protocols. Digestion performance was conveniently monitored using marker peaks that indicate incomplete digestion products. This study identifies critical components of HPLC analysis and offers a comprehensive guide for the stringent analysis of DNA methylation in plants.

Induction of RNA interference in Caenorhabditis elegans by RNAs derived from plants exhibiting post-transcriptional gene silencing.

The term 'gene silencing' refers to transcriptional and post-transcriptional control of gene expression. Related processes are found across kingdoms in plants and animals. We intended to test whether particular RNA constituents of a silenced plant can induce silencing in an animal. We generated Nicotiana benthamiana lines that expressed green fluorescent protein (GFP) from a transgene. Plants in which GFP expression was spontaneously silenced showed siRNAs characteristic of post-transcriptional gene silencing (PTGS). RNA extracts prepared from silenced plants were injected into a GFP-expressing strain of Caenorhabditis elegans, where they induced RNA interference (RNAi). Extracts from non-silenced plants were inactive. This directly demonstrates a relationship and a mechanistic link between PTGS and RNAi. Controls confirmed that the silencing agent was an RNA. Size fractionation on denaturing gels revealed that an RNA of approximately 85 nt was most active in inducing silencing in the worm. Northern blot analysis of the region in question did not detect a prominent GFP-specific RNA of sense or antisense polarity, indicating that the RNA species which induced silencing was present only in low concentration or did not hybridize due to formation of an intramolecular double strand. In view of its high activity, it is possible that this agent is responsible for the systemic spread of silencing in plants and it might represent the aberrant RNA, a previously postulated inducer of silencing.

Analysis of HCV‐Ig Isotype Complexes by a Novel Immuno‐Capture RT–PCR Method

HCV can be present in the circulating blood either as a free virus or as a virion-immunoglobulin (Ig) complex. All isotypes of Igs may form the virus complexes, but it remains unclear what specific role of each Ig plays in the clearance of HCV. In the present study, we have combined immuno-capture and RT-PCR, and developed a quick double-specificity method for detecting and distinguishing different HCV-Ig complexes. We compared our new method, the immuno-capture RT-PCR (iRT-PCR), with the conventional RT-PCR (cRT-PCR) for the sensitivity of detecting HCV in 35 clinically diagnosed patients with HCV infection. The results showed that 31 patients were detected to be positive by using iRT-PCR, whereas 16 patients were positive with the use of cRT-PCR. HCV-IgM, HCV-IgG, HCV-IgA could separately be detected by iRT-PCR and their positive rates were 66.7%, 51.0%, 62.7%, respectively. HCV bound to antibody was a common feature of hepatitis C (HC) and 86.3% of patients were positive at least by one of the HCV-Ig tests. The patterns of HCV RNA constituents varied according to disease categories. In summary, iRT-PCR is a valuable method for analysis of the composition of the immune complexes, which may provide new and valuable insights into HCV pathogenesis.

Self-Assembly of a Viral Molecular Machine from Purified Protein and RNA Constituents

We present the assembly of the polymerase complex (procapsid) of a dsRNA virus from purified recombinant proteins. This molecular machine packages and replicates viral ssRNA genomic precursors in vitro. After addition of an external protein shell, these in vitro self-assembled viral core particles can penetrate the host plasma membrane and initiate a productive infection. Thus, a viral procapsid has been assembled and rendered infectious using purified components. Using this system, we have studied the mechanism of assembly of the common dsRNA virus shell and the incorporation of a symmetry mismatch within an icosahedral capsid. Our work demonstrates that this molecular machine, self-assembled under defined conditions in vitro, can function in its natural environment, the cell cytoplasm.

Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1

The interdependency of steps in the processing of pre-rRNA in Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes. To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins. When incubated with protein extract, the spacer formed a specific large RNP. This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresis. Modification exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses confirmed an interaction with this sequence and indicated that this domain is critical to the efficient maturation of the precursor RNA. The protein constituents, purified by affinity chromatography using the ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicated at least 20 protein components ranging in size from 20–200 kDa. Peptide mapping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA binding proteins which included four different RNA-binding motifs. Another protein was putatively identified as a pseudouridylate synthase. Additional RNA constituents were not detected. The significance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed.