Effect of small molecules on cell reprogramming.

The essential idea of regenerative medicine is to fix or replace tissues or organs with alive and patient-specific implants. Pluripotent stem cells are able to indefinitely self-renew and differentiate into all cell types of the body which makes them a potent substantial player in regenerative medicine. The easily accessible source of induced pluripotent stem cells may allow obtaining and cultivating tissues in vitro. Reprogramming refers to regression of mature cells to its initial pluripotent state. One of the approaches affecting pluripotency is the usage of low molecular mass compounds that can modulate enzymes and receptors leading to the formation of pluripotent stem cells (iPSCs). It would be great to assess the general character of such compounds and reveal their new derivatives or modifications to increase the cell reprogramming efficiency. Many improvements in the methods of pluripotency induction have been made by various groups in order to limit the immunogenicity and tumorigenesis, increase the efficiency and accelerate the kinetics. Understanding the epigenetic changes during the cellular reprogramming process will extend the comprehension of stem cell biology and lead to potential therapeutic approaches. There are compounds which have been already proven to be or for now only putative inducers of the pluripotent state that may substitute for the classic reprogramming factors (Oct3/4, Sox2, Klf4, c-Myc) in order to improve the time and efficiency of pluripotency induction. The effect of small molecules on gene expression is dosage-dependent and their application concentration needs to be strictly determined. In this review we analysed the role of small molecules in modulations leading to pluripotency induction, thereby contributing to our understanding of stem cell biology and uncovering the major mechanisms involved in that process.

The mistletoe lectin I--phloretamide structure reveals a new function of plant lectins.

The X-ray structure at 2.7A resolution of the complex between the European mistletoe lectin I (Viscum album, ML-I) and the plant growth hormone, 3-(p-hydroxyphenyl)-propionic acid amide (phloretamide, PA) from xylem sap has revealed the binding of PA at the so far undescribed hydrophobic cavity located between the two subunits of this ribosome-inhibiting protein. No such cavity is observed in related lectins. The binding of PA is achieved through interactions with the non-conserved residues Val228A, Leu230A, Arg388B, and the C-terminal Pro510B. It is conceivable that binding of PA to ML-I is part of a defence mechanism of the parasite against the host, whereby the parasite prevents the growth hormone of the host from interfering with its own regulatory system. The specific binding of PA to ML-I indicates that heterodimeric RIPs are multifunctional proteins whose functions in the cell have not yet been fully recognized and analyzed.

Regulatory RNAs in mammals.

Recent years have brought a dramatic change in our understanding of the role of ribonucleic acids (RNAs) within the cell. In addition to the already well-known classes of RNAs that take part in the transmission of genetic information from DNA to proteins, a new highly heterogeneous group of RNA molecules has emerged. The regulatory nonprotein-coding RNAs (npcRNAs) have been shown to be involved in modulation of gene expression on both the transcriptional and post-transcriptional level. They participate in mechanisms of chromatin modification, regulation of transcription factor activity, and influencing mRNA stability, processing, and translation. npcRNAs are key factors in genetic imprinting, dosage compensation of X-chromosome-linked genes, and many processes of differentiation and development.

Canine 5S rRNA: nucleotide sequence and chromosomal assignment of its gene cluster in four canid species.

The purpose of this study was to determine the nucleotide sequence of canine 5S rRNA and use this information to develop a molecular probe to assign the gene locus to chromosomes of the dog and three other related canid species using fluorescence in situ hybridization. The nucleotide sequence of canine liver 5S rRNA is 120 base pairs long and identical to the 5S rRNA nucleotide sequence of all other mammalian species investigated so far. A single 5S rRNA gene cluster was localized pericentromerically on chromosomes of four canid species: dog 4q1.3, red fox 4q1.3, blue fox 3q1.3 and Chinese raccoon dog 8q1.3. Chromosome arms carrying the 5S rRNA gene cluster showed striking similarities in their QFQ banding patterns, suggesting high conservation of these chromosome arms among the four species studied. The chromosomal assignments of 5S rRNA genes are among the first gene mapping results for the blue fox and the Chinese raccoon dog, and are in accordance with published data on comparative chromosome maps from human, dog, red fox, blue fox and raccoon dogs.

Methionyl-tRNA synthetase.

Methionyl-tRNA synthetase (MetRS) belongs to the family of 20 enzymes essential for protein biosynthesis. It links covalently methionine with its cognate tRNA. Crystal structures solved for bacterial MetRSs have given a number of interesting insights into enzyme architecture and methionylation catalysis. A comparison of sequences of MetRSs belonging to all kingdoms of life, as well as numerous biochemical and genetic studies have revealed the presence of various additional domains appended to the catalytic core of synthetase. They are responsible for interactions with tRNA and proteins. Tertiary structure of C-terminal tRNA-binding appendices can be deduced from those determined for their homologues: tRNA binding protein 111 and endothelial monocyte-activating polypeptide II. Contacts between MetRS and other proteins could be mediated not only by noncatalytic peptides but also by structural elements present in the catalytic core, e.g. Arg-Gly-Asp (RGD) motifs. Additional activities involve MetRS in the maintenance of translational fidelity and in coordination of ribosome biogenesis with protein synthesis.

Regulatory RNAs.

In addition to mRNA, rRNA and tRNA, which play central roles within cells, there are a number of regulatory, non-coding RNAs (ncRNAs). Of varying lengths, ncRNAs have no long open reading frame. While not encoding proteins, they may act as riboregulators, and their main function is posttranscriptional regulation of gene expression. Many ncRNAs have been identified and characterized both in prokaryotes and eukaryotes, and are involved in the specific recognition of cellular nucleic acid targets through complementary base pairing, controlling cell growth and differentiation. Some are associated with the abnormalities in imprinted inheritance that occur in several well-known developmental and neurobehavioral disorders. Other ncRNAs accomplish regulation by modulating the activity of proteins. Several rRNAs are able to sustain enzymatic reactions implicated in the translation process including synthesis of peptide bonds within the ribosome. The different roles played by widely distributed RNAs acting in diverse ways, suggest the flexibility and versality of these molecules in regulatory processes. This review summarizes the available biochemical and structural data on known regulatory RNAs.

Structure and functions of 5S rRNA.

The ribosome is a macromolecular assembly that is responsible for protein biosynthesis in all organisms. It is composed of two-subunit, ribonucleoprotein particles that translate the genetic material into an encoded polypeptides. The small subunit is the site of codon-anticodon interaction between the messenger RNA (mRNA) and transfer RNA (tRNA) substrates, and the large subunit catalyses peptide bond formation. The peptidyltransferase activity is fulfilled by 23S rRNA, which means that ribosome is a ribozyme. 5S rRNA is a conserved component of the large ribosomal subunit that is thought to enhance protein synthesis by stabilizing ribosome structure. This paper shortly summarises new results obtained on the structure and function of 5S rRNA.

The specific hydrolysis of HIV-1 TAR RNA element with the anti-TAR hammerhead ribozyme: structural and functional implications.

The main transcriptional regulator of the human immunodeficiency virus is the Tat protein, which recognises and binds to a fragment RNA at the 5' end of viral mRNA, named transactivation response element (TAR) RNA. Extensive mutagenesis studies have shown that a region of TAR RNA important for Tat binding involves a set of nucleotides surrounding a characteristic UCU nucleotide bulge. The specific Tat-TAR complex formation enhances the rate of transcription elongation but inhibition of that interaction prevents the human immunodeficiency virus type 1 (HIV-1) replication. If so, a possibility of virus inactivation would be a site specific degradation of the TAR RNA element. To break down and inactivate TAR RNA, we designated the anti-hammerhead (HH) ribozyme to cleave nucleosides within the bulge. We showed for the first time the new type of the AUC hammerhead ribozyme, which hydrolyses specifically the TAR RNA element at C8 nucleotide in the bulge (C24 in the standard TAR RNA numbering). The cleavage reaction has broad magnesium requirements. Mn and particularly Ca are less efficient. Argininamide interferes with the cleavage of TAR RNA induced by the ribozyme. These results have two implications; (i) structural, where the HIV-1 TAR RNA element in solution occurs in equilibrium of only two forms, one of which, a double stranded RNA, meets structural requirements for ribozyme pairing and cleavage, and (ii) functional, the HH ribozyme can be explored for an inactivation of HIV-1 through the TAR RNA element deintegration.

The non-coding RNAs as riboregulators.

The non-coding RNAs database (http://biobases.ibch.poznan.pl/ncRNA/) contains currently available data on RNAs, which do not have long open reading frames and act as riboregulators. Non-coding RNAs are involved in the specific recognition of cellular nucleic acid targets through complementary base pairing to control cell growth and differentiation. Some of them are connected with several well known developmental and neuro-behavioral disorders. We have divided them into four groups. This paper is a short introduction to the database and presents its latest, updated edition.

Aminoacyl-tRNA synthetases database.

Aminoacyl-tRNA synthetases (AARSs) are at the center of the question of the origin of life. They constitute a family of enzymes integrating the two levels of cellular organization: nucleic acids and proteins. AARSs arose early in evolution and are believed to be a group of ancient proteins. They are responsible for attaching amino acid residues to their cognate tRNA molecules, which is the first step in the protein synthesis. The role they play in a living cell is essential for the precise deciphering of the genetic code. The analysis of AARSs evolutionary history was not possible for a long time due to a lack of a sufficiently large number of their amino acid sequences. The emerging picture of synthetases' evolution is a result of recent achievements in genomics [Woese,C., Olsen,G.J., Ibba,M. and Söll,D. (2000) Microbiol. Mol. Biol. Rev., 64, 202-236]. In this paper we present a short introduction to the AARSs database. The updated database contains 1047 AARS primary structures from archaebacteria, eubacteria, mitochondria, chloroplasts and eukaryotic cells. It is the compilation of amino acid sequences of all AARSs known to date, which are available as separate entries via the WWW at http://biobases.ibch.poznan.pl/aars/.

Structural changes of tRNA and 5S rRNA induced with magnesium and visualized with synchrotron mediated hydroxyl radical cleavage.

The structure of native yeast tRNA(Phe) and wheat germ ribosomal 5S RNA induced by different magnesium ion concentrations was studied in solution with a synchrotron mediated hydroxyl radical RNA cleavage reaction. We showed that very small amounts of Mg+2 can induce significant changes in the hydroxyl radical cleavage pattern of tRNA(Phe). It also turned out that a reactivity of tRNAz(Phe) towards *OH coincides with the strong metal binding sites. Because of the Mg ions are heavily hydrated one can suggest the strong correlation of the observed nucleosides reactivity in vicinity of Mg2+ binding sites with availability of water molecules as a source of hydroxyl radical. On the other hand the structure of wheat germ 5S rRNA is less sensitive to the hydroxyl radical reaction than tRNA(Phe) although some changes are visible at 4 mM Mg ions. It is probably due to the lack of strong Mg+2 binding sites in that molecule. The reactivity of nucleotides in loops C and D of 5S rRNA is not effected, what suggests their flexibility or involvement in higher order structure formation. There is different effect of magnesium on tRNA and 5S rRNA folding. We found that nucleotides forming strong binding sites for magnesium are very sensitive to X-ray generated hydroxyl radical and can be mapped with *OH. The results show, that guanine nucleotides are preferentially hydrated. X-ray footprinting mediated hydroxyl radical RNA cleavage is a very powerful method and has been applied to studies of stable RNAs for the first time.

Identification of 6-furfuryladenine (kinetin) in human urine.

In contrast to the current view of kinetin (K, N(6)-furfuryladenine) as an unnatural and synthetic cytokinin, recently it has been identified in plant DNA and plant extract. Here we describe identification of K in human urine using chromatography/mass-spectrometry analysis for the first time. The amount of kinetin in urine taken from unhealthy patients lung carcinoma was established to be 0.5 ng in 20 ml and a 100-fold reduced amount in healthy subjects. Since this rare base is a potential source of structural constrains it has to be removed from DNA by enzymatic DNA-repair reactions. It seems that the presence of kinetin in human is linked to oxidative damage processes.

A recurrent general RNA binding domain appended to plant methionyl-tRNA synthetase acts as a cis-acting cofactor for aminoacylation.

The cDNA encoding rice methionyl-tRNA synthetase was isolated. The protein exhibited a C-terminal polypeptide appended to a classical MetRS domain. This supplementary domain is related to endothelial monocyte activating polypeptide II (EMAPII), a cytokine produced in mammals after cleavage of p43, a component of the multisynthetase complex. It is also related to Arc1p and Trbp111, two tRNA binding proteins. We expressed rice MetRS and a derivative with a deletion of its EMAPII-like domain. Band-shift analysis showed that this extra-domain provides MetRS with non-specific tRNA binding properties. The EMAPII-like domain contributed a 10-fold decrease in K:(M) for tRNA in the aminoacylation reaction catalyzed by the native enzyme, as compared with the C-terminally truncated MetRS. Consequently, the EMAPII domain provides MetRS with a better catalytic efficiency at the free tRNA concentration prevailing in vivo. This domain binds the acceptor minihelix of tRNA(Met) and facilitates its aminoacylation. These results suggest that the EMAPII module could be a relic of an ancient tRNA binding domain that was incorporated into primordial synthetases for aminoacylation of RNA minihelices taken as the ancestor of modern tRNA.

Minireview: analysis of rape seed napin structure and potential roles of the storage protein.

Structural and functional data on 2S albumins and particularly rape seed napins are reviewed and, based on the coordinates of the three-dimensional structure of napin-like albumin BnIb, are used to model different rape napins. Surprisingly, the modeled napins, despite great sequence homology, differ in tertiary arrangements of the polypeptide chains. It is proposed that these differences in 3D structures of the analyzed rape napins may reflect their functions, which may cover many other potential beneficial purposes besides simple storage.

Specific induction of Z-DNA conformation by a nuclear localization signal peptide of lupin glutaminyl tRNA synthetase.

Recently we have sequenced cDNA of plant glutaminyl-tRNA synthetase (GlnRS) from Lupinus luteus. At the N terminal part the protein contains a lysine rich polypeptide (KPKKKKEK), which is identical to a nuclear localization signal (NLS). In this paper we showed that two synthetic peptides (20 and 8 amino acids long), which were derived from lupin GlnRS containing the NLS sequence interact with DNA, but one of them (8aa long) changing its conformation from the B to the Z form. This observation clearly suggests that the presence of the NLS polypeptide in a leader sequence of GlnRS is required not only for protein transport into nucleus but also for regulation of a gene expression. This is the first report suggesting a role of the NLS signal peptide in structural changes of DNA.

An analysis of G-U base pair occurrence in eukaryotic 5S rRNAs.

The structure-function relationship in RNA molecules is a key to understanding of the expression of genetic information. Various types of RNA play crucial roles at almost every step of protein biosynthesis. In recent years, it has been shown that one of the most important structural elements in RNA is a wobble pair G-U. In this paper, we present for the first time an analysis of the distribution of G-U pairs in eukaryotic 5S ribosomal RNAs. Interestingly, the G-U pair in 5S rRNA species is predominantly found in two intrahelical regions of the stems I and V and at the junction of helix IV and loop A. The distribution of G-U pairs and the nature of adjacent bases suggests their possible role as a recognition site in interactions with other components of protein biosynthesis machinery.

Lessons from sequenced genomes. Overlapping genes in Methanococcus jannaschii?

This paper describes our finding on overlapping genes in Methanococcus jannaschii genome. We found that one of the open reading frames (ORFs) within the M. jannaschii genome contains the nucleotide sequence of tRNA(Ser), which raises a serious question of the correctness of the initiation codon assignment for that ORF. We suggest that there are two other possible AUG initiation codons downstream from the TTG triplet, which was initially considered as a translation start site. Only one of the AUG triplets is preceded by the Shine-Dalgarno sequence that seems to be required for binding the ribosome and initiation of translation.

5S ribosomal RNA.

Ribosomes have been visualized in electron micrographs in 1943 but 5S rRNA was discovered 20 years later. The next four decades witnessed big advances in our understanding of the ribosome using biochemical, genetic and low resolution structural approaches. During those times many experimental data accumulates also on 5S rRNA, but its precise function remains unknown. To understand the role of this RNA in ribosome a high-resolution structure is urgently needed. Because the ribosome is a dynamic machine, details on the interaction of 5S rRNA with proteins within entire ribosome are required. Big progress in the structural analysis of ribosome will stimulate further understanding of 5S rRNA.