Endogenous RNA cleavages at the ribosomal SRL site likely reflect miRNA (miR) mediated translational suppression

We previously suggested a mechanism whereby the RNA induced silencing complex (RISC) brings about a specific cleavage at the sarcin–ricin loop (SRL) of 28S ribosomal RNA thereby eliciting translational suppression. Here we experimentally show that endogenous cleavages take place at the SRL site, in both mammalian cells and in Caenorhabditis elegans. Furthermore we demonstrate that bulged and looped-out residues present in the imperfect miRNA–[mRNA target site] duplexes, are complementary to the SRL site. These results support, and are compatible with, our described mechanism whereby microRNAs mediate cleavage of the highly conserved 28S rRNA sarcin/ricin loop leading to translational suppression.     

Recombinant Fusion Proteins for the Industrial Production of Disulfide Bridge Containing Peptides: Purification, Oxidation without Concatamer Formation, and Selective Cleavage

We report the biotechnical production of peptides of approximately 35–50 amino acids in length containing one intramolecular disulfide bridge, using a recombinant fusion tail approach. This method fills the technological gap when either (a) chemical synthesis fails due to known problematic peptide sequences or (b) if simple recombinant expression is unsuccessful due to degradation. The fusion tail described here serves several purposes: (i) it enables high expression levels inEscherichia colito be achieved; (ii) it renders the fusion protein fairly soluble; (iii) it contains a histidine affinity tag for easy purification on Ni-chelate resins, which also serves as a catalyst for the oxygen-dependent formation of the disulfide bridge; and (iv) it suppresses the formation of concatamers during the oxidation process through steric hindrance. The purified fusion protein is then immobilized on a reversed phase column for two purposes: (i) chemical cleavage of the fusion tail by cyanogen bromide and (ii) subsequent purification of the peptide. A very hydrophilic fusion partner is required so that immobilization on the reversed phase column always occurs due to the peptide. Sensitive hydrophobic residues are thereby protected from the cleavage reagent while the cleaved hydrophilic fusion tail is easily separated from the hydrophobic peptide. The method is exemplified by eight peptides representing an immunodominant epitope of the human immunodeficiency virus, but may be useful for a significant variety of similar peptides.     

An enzyme in the test tube,and a transcription factor in the cell: Moonlighting proteins and cellular factors that affect their behavior

In the cell, expression levels, allosteric modulators, post‐translational modifications, sequestration, and other factors can affect the level of protein function. For moonlighting proteins, cellular factors like these can also affect the kind of protein function. This minireview discusses examples of moonlighting proteins that illustrate how a single protein can have different functions in different cell types, in different intracellular locations, or under varying cellular conditions. This variability in the kind of protein activity, added to the variability in the amount of protein activity, contributes to the difficulty in predicting the behavior of proteins in the cell.     

In silico determination of intracellular glycosylation and phosphorylation sites in human selectins: implications for biological function

Post-translational modifications provide the proteins with the possibility to perform functions in addition to those determined by their primary sequence. However, analysis of multifunctional protein structures in the environment of cells and body fluids is made especially difficult by the presence of other interacting proteins. Bioinformatics tools are therefore helpful to predict protein multifunctionality through the identification of serine and threonine residues wherein the hydroxyl group is likely to become modified by phosphorylation or glycosylation. Moreover, serines and threonines where both modifications are likely to occur can also be predicted (YinYang sites), to suggest further functional versatility. Structural modifications of hydroxyl groups of P-, E-, and L-selectins have been predicted and possible functions resulting from such modifications are proposed. Functional changes of the three selectins are based on the assumption that transitory and reversible protein modifications by phosphate and O-GlcNAc cause specific conformational changes and generate binding sites for other proteins. The computer-assisted prediction of glycosylation and phosphorylation sites in selectins should be helpful to assess the contribution of dynamic protein modifications in selectin-mediated inflammatory responses and cell-cell adhesion processes that are difficult to determine experimentally.     

The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans

Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex--or miRNA-induced silencing complex (miRISC)--can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression.     

Assembly of Saccharomyces cerevisiae 60S ribosomal subunits: role of factors required for 27S pre-rRNA processing

The precise functions of most of the ～200 assembly factors and 79 ribosomal proteins required to construct yeast ribosomes in vivo remain largely unexplored. To better understand the roles of these proteins and the mechanisms driving ribosome biogenesis, we examined in detail one step in 60S ribosomal subunit assembly-processing of 27SA(3) pre-rRNA. Six of seven assembly factors required for this step (A(3) factors) are mutually interdependent for association with preribosomes. These A(3) factors are required to recruit Rrp17, one of three exonucleases required for this processing step. In the absence of A(3) factors, four ribosomal proteins adjacent to each other, rpL17, rpL26, rpL35, and rpL37, fail to assemble, and preribosomes are turned over by Rat1. We conclude that formation of a neighbourhood in preribosomes containing the A(3) factors establishes and maintains stability of functional preribosomes containing 27S pre-rRNAs. In the absence of these assembly factors, at least one exonuclease can switch from processing to turnover of pre-rRNA.     

Phosphorylation and glycosylation interplay: protein modifications at hydroxy amino acids and prediction of signaling functions of the human beta3 integrin family

Protein functions are determined by their three-dimensional structures and the folded 3-D structure is in turn governed by the primary structure and post-translational modifications the protein undergoes during synthesis and transport. Defining protein functions in vivo in the cellular and extracellular environments is made very difficult in the presence of other molecules. However, the modifications taking place during and after protein folding are determined by the modification potential of amino acids and not by the primary structure or sequence. These post-translational modifications, like phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc) modifications, are dynamic and result in temporary conformational changes that regulate many functions of the protein. Computer-assisted studies can help determining protein functions by assessing the modification potentials of a given protein. Integrins are important membrane receptors involved in bi-directional (outside-in and inside-out) signaling events. The beta3 integrin family, including, alpha(IIb)beta3 and alpha(v)beta3, has been studied for its role in platelet aggregation during clot formation and clot retraction based on hydroxyl group modification by phosphate and GlcNAc on Ser, Thr, or Tyr and their interplay on Ser and Thr in the cytoplasmic domain of the beta3 subunit. An antagonistic role of phosphate and GlcNAc interplay at Thr758 for controlling both inside-out and outside-in signaling events is proposed. Additionally, interplay of GlcNAc and phosphate at Ser752 has been proposed to control activation and inactivation of integrin-associated Src kinases. This study describes the multifunctional behavior of integrins based on their modification potential at hydroxyl groups of amino acids as a source of interplay.     

RNAi and microRNA: breakthrough technologies for the improvement of plant nutritional value and metabolic engineering

This article raises the complex issue of improving plant nutritional value through metabolic engineering and the potential of using RNAi and micro RNA technologies to overcome this complexity, focusing on a few key examples. It also highlights current knowledge of RNAi and microRNA functions and discusses recent progress in the development of new RNAi vectors and their applications. RNA interference (RNAi) and microRNA (miRNA) are recent breakthrough discoveries in the life sciences recognized by the 2006 Nobel Prize in Physiology or Medicine. The importance of these discoveries relates not only to elucidating the fundamental regulatory aspects of gene expression, but also to the tremendous potential of their applications in plants and animals. Here, we review recent applications of RNAi and microRNA for improving the nutritional value of plants, discuss applications of metabolomics technologies in genetic engineering, and provide an update on the related RNAi and microRNA technologies.     

Searching for Local Similarities between HIV-1 and Human Proteins. Application to Vaccines

A method for identification of fragments with high local similarity to human proteins within potentially immunopathogenic regions of HIV-1 proteins was developed. The method is based on the use of an original matrix of antigenic similarity of amino acids. The regions whose fragments are frequent in human proteins, and regions exhibiting high similarity to the proteins responsible for important physiological functions, were identified in HIV-1 proteins. A possibility of participation of such regions in immunopathogenesis of HIV infection either through induction of cross-reacting effectors of the immune system or through molecular mimicry of physiologically important human proteins, leading to alteration of homeostasis of the organism, is discussed. Most of the regions identified in HIV-1 proteins contain either T-cell (CD8⁺ CTL or CD4⁺ Th) or B-cell epitopes, or both of them simultaneously. The criteria for the design of safe polyepitopic antiviral vaccines that would allow exclusion of epitopes with (immuno)pathogenic potential are discussed. According to these criteria, polyepitopic immunogens should be free of the virus protein regions whose fragments are frequent in human proteins, as well as of regions exhibiting pronounced local similarity to proteins that provide for important physiological functions.     

A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA

Processing of primary microRNA (pri-miRNA) stem–loops by the Drosha–DGCR8 complex is the initial step in miRNA maturation and crucial for miRNA function. Nonetheless, the underlying mechanism that determines the Drosha cleavage site of pri-miRNAs has remained unclear. Two prevalent but seemingly conflicting models propose that Drosha–DGCR8 anchors to and directs cleavage a fixed distance from either the basal single-stranded (ssRNA) or the terminal loop. However, recent studies suggest that the basal ssRNA and/or the terminal loop may influence the Drosha cleavage site dependent upon the sequence/structure of individual pri-miRNAs. Here, using a panel of closely related pri-miRNA variants, we further examine the role of pri-miRNA structures on Drosha cleavage site selection in cells. Our data reveal that both the basal ssRNA and terminal loop influence the Drosha cleavage site within three pri-miRNAs, the Simian Virus 40 (SV40) pri-miRNA, pri-miR-30a, and pri-miR-16. In addition to the flanking ssRNA regions, we show that an internal loop within the SV40 pri-miRNA stem strongly influences Drosha cleavage position and efficiency. We further demonstrate that the positions of the internal loop, basal ssRNA, and the terminal loop of the SV40 pri-miRNA cooperatively coordinate Drosha cleavage position and efficiency. Based on these observations, we propose that the pri-miRNA stem, defined by internal and flanking structural elements, guides the binding position of Drosha–DGCR8, which consequently determines the cleavage site. This study provides mechanistic insight into pri-miRNA processing in cells that has numerous biological implications and will assist in refining Drosha-dependent shRNA design.     

Disulfide bonds and disorder in granulin‐3: An unusual handshake between structural stability and plasticity

Granulins (GRNs) are a family of small (～6 kDa) proteins generated by the proteolytic processing of their precursor, progranulin (PGRN), in many cell types. Both PGRN and GRNs are implicated in a plethora of biological functions, often in opposing roles to each other. Lately, GRNs have generated significant attention due to their implicated roles in neurodegenerative disorders. Despite their physiological and pathological significance, the structure‐function relationships of GRNs are poorly defined. GRNs contain 12 conserved cysteines forming six intramolecular disulfide bonds, making them rather exceptional, even among a few proteins with high disulfide bond density. Solution NMR investigations in the past have revealed a unique structure containing putative interdigitated disulfide bonds for several GRNs, but GRN‐3 was unsolvable due to its heterogeneity and disorder. In our previous report, we showed that abrogation of disulfide bonds in GRN‐3 renders the protein completely disordered (Ghag et al., Prot Eng Des Sel 2016). In this study, we report the cellular expression and biophysical analysis of fully oxidized, native GRN‐3. Our results indicate that both E. coli and human embryonic kidney (HEK) cells do not exclusively make GRN‐3 with homogenous disulfide bonds, likely due to the high cysteine density within the protein. Biophysical analysis suggests that GRN‐3 structure is dominated by irregular loops held together only by disulfide bonds, which induced remarkable thermal stability to the protein despite the lack of regular secondary structure. This unusual handshake between disulfide bonds and disorder within GRN‐3 could suggest a unique adaptation of intrinsically disordered proteins towards structural stability.     

Towards understanding the crosstalk between protein post‐translational modifications: Homo‐ and heterotypic PTM pair distances on protein surfaces are not random

Post‐translational modifications (PTMs) represent an important regulatory layer influencing the structure and function of proteins. With broader availability of experimental information on the occurrences of different PTM types, the investigation of a potential “crosstalk” between different PTM types and combinatorial effects have moved into the research focus. Hypothesizing that relevant interferences between different PTM types and sites may become apparent when investigating their mutual physical distances, we performed a systematic survey of pairwise homo‐ and heterotypic distances of seven frequent PTM types considering their sequence and spatial distances in resolved protein structures. We found that actual PTM site distance distributions differ from random distributions with most PTM type pairs exhibiting larger than expected distances with the exception of homotypic phosphorylation site distances and distances between phosphorylation and ubiquitination sites that were found to be closer than expected by chance. Random reference distributions considering canonical acceptor amino acid residues only were found to be shifted to larger distances compared to distances between any amino acid residue type indicating an underlying tendency of PTM‐amenable residue types to be further apart than randomly expected. Distance distributions based on sequence separations were found largely consistent with their spatial counterparts suggesting a primary role of sequence‐based pairwise PTM‐location encoding rather than folding‐mediated effects. Our analysis provides a systematic and comprehensive overview of the characteristics of pairwise PTM site distances on proteins and reveals that, predominantly, PTM sites tend to avoid close proximity with the potential implication that an independent attachment or removal of PTMs remains possible. Proteins 2016; 85:78–92. © 2016 Wiley Periodicals, Inc.     

Similarities between a predicted secondary structure for the M1 RNA ribozyme and the tRNA binding center of 16 S rRNA from E. coli

We propose a new model for the secondary structure of the M1 RNA component of E. coli RNase P which is based on significant sequence homologies with parts of the E. coli 16 S rRNA. A large domain of the new model resembles closely the secondary structure of the tRNA binding center of 16 S rRNA. We suggest that this domain of M1 RNA when functioning as a ribozyme binds the mature part of the precursor tRNA.     

Feathers with Ocular Architecture: Implications for Functional and Evolutionary Similarities of Visual Signals and Receptors

Studies of visual receptors typically assume that only functionally similar structures are relevant to the evolution of complex eyes. This approach ignores growing evidence that different functional classes of organs often share structural and developmental patterns that pertain to biological sameness (deep homology). However, the potential relevance of non-receptor structures to eye evolution remains largely unexplored. An “ocular” feather color mechanism is described whose structural and optical features resemble those of chambered, image-forming eyes to a remarkable degree. These similarities include a laterally expanded, domed light receiving surface similar to that of an eye, an encapsulated spongy tissue mass whose coherent light scattering properties in the human-visible (destructive) and ultraviolet (constructive) wavelength ranges resemble those of cornea and lens, intervening spaces such as those with humors, and a laminar pigmented shelf whose structure and optics resemble a mirrored tapetum lucidum found behind many retinas. Fourier analysis and optical principles indicate that ocular structures adhere to the same light-handling properties regardless of higher function (receptor or signal). The extent to which chambered eyes and ocular feathers have evolved independently is surprisingly equivocal. On the one hand, broad differences in the location, composition, and development of chambered eyes and ocular feather signals suggest convergent evolution on an ocular organization. However, some level of evolutionary parallelism (generative homology) between chambered eyes and ocular feathers is implicated by similarities in constructional materials, tissue development, and signal transduction cascades. Structural, optical, and developmental similarities also occur between more primitive eyes and the colored dermal papillae responsible for avian skin ornamentation. Functional constraints on light-handling requirements, coupled with developmental constraints in high-stress environments on the body surface, may enhance the similar evolutionary outcomes in the different functional setting. Regardless of the mechanistic details, repeated evolution of eye-like structures in different functional settings reveals a biological potential to produce such organs that is much greater than would be inferred from a survey of receptor structures alone.     

Direct incorporation and extension of a fluorescent nucleotide through rolling circle DNA amplification for the detection of microRNA 24-3P

We designed and synthesized several fluorescent nucleotides from thiophene, anthracene and pyrene, which have different sizes, and screened their incorporation and extension capability during the rolling circle amplification of DNA. The thiophene-based fluorescent nucleotide (dUthioTP) could highly incorporate and extended into the rolling circle DNA product, while other fluorescent nucleotides (dUanthTP, and dUpyrTP) could not. This dUthioTP fluorescent nucleotide could be used for the detection of miRNA 24-3P, which is related PRRSV. This direct labeling system during rolling circle DNA amplification exhibited an increased fluorescence signal showing gel formation for the detection of miRNA 24-3P. This direct labeling system is a very simple and cost-efficient method for the detection miRNA 24-3P and also exhibited highly sensitive and selective detection properties.