Enzymatic synthesis of tRNA fragments

In the present paper the results of enzymatic synthesis of yeast tRNA1Val fragments have been summarized. It is shown that complex use of nucleolytic enzymes is a convenient and effective method of synthesis of the defined sequence oligoribonucleotides. The consecutive use of different nucleolytic enzymes (ribonucleases with different substrate specificity and polynucleotide phosphorylase) and RNA ligase has permitted to obtain various fragments (or their analogs) of T psi-loop, D-arm, anticodon arm and acceptor stem. Some fragments containing modified nucleosides such as tetranucleotide GpDpCpGp (fragment 15-18), octanucleotide GpUpCpUpApGpDpC (analog of fragment 10-17), nonanucleotide GpTpUpCpGpApUpCpC (analog of T psi-loop), decanucleotide psi pCpUpGpCpUpUpIpApC (analog of fragment 27-36), hexanucleotide CpApCpGpCpA (fragment 36-41) and others were synthesized.     

tRNA fragments in human health and disease

Transfer RNA (tRNA) is traditionally considered to be an adaptor molecule that helps ribosomes to decode messenger RNA (mRNA) and synthesize protein. Recent studies have demonstrated that tRNAs also serve as a major source of small non-coding RNAs that possess distinct and varied functions. These tRNA fragments are heterogeneous in size, nucleotide composition, biogenesis and function. Here we describe multiple roles that tRNA fragments play in cell physiology and discuss their relevance to human health and disease.     

Angiogenin-induced tRNA fragments inhibit translation initiation

Angiogenin is a stress-activated ribonuclease that cleaves tRNA within anticodon loops to produce tRNA-derived stress-induced fragments (tiRNAs). Transfection of natural or synthetic tiRNAs inhibits protein synthesis and triggers the phospho-eIF2α-independent assembly of stress granules (SGs), essential components of the stress response program. We show that selected tiRNAs inhibit protein synthesis by displacing eIF4G/eIF4A from uncapped > capped RNAs. tiRNAs also displace eIF4F, but not eIF4E:4EBP1, from isolated m(7)G cap. We identify a terminal oligoguanine motif that is required to displace the eIF4F complex, inhibit translation, and?induce SG assembly. We show that the tiRNA-associated translational silencer YB-1 contributes to angiogenin-, tiRNA-, and oxidative stress-induced translational repression. Our data reveal some of the mechanisms by which stress-induced tRNA cleavage inhibits protein synthesis and activates a cytoprotective stress response program.     

Hallmarks for senescence in carcinogenesis: novel signaling players

Cellular senescence is a potent anti-cancer mechanism controlled by tumor suppressor genes, particularly p53 and pRb, which is characterized by the irreversible loss of proliferation. Senescence induced by DNA damage, oncogenic stimulation, or excessive mitogenic input, serves as a barrier that counteracts cancer progression. Emerging evidence in cellular and in in vivo models revealed the involvement of additional signaling players in senescence, including PML, CK2, Bcl-2, PI3K effectors such as Rheb, Rho small GTPases, and cytokines. Recent studies have also implicated protein kinase C (PKC) isozymes as modulators of senescence phenotypes and showed that phorbol esters, widely used PKC activators, can induce senescence in a number of cancer cells. These novel findings suggest a complex array of cross-talks between senescence pathways and may have significant implications in cancer therapy.     

RNA fragments mimicking tRNA analogs interact with cytochrome c

In times, when drug seeking assays focus on the natural molecular triggers and their analogs, a deeper insight into molecular mechanisms governing the initial step of intrinsic apoptosis (cytochrome c release) is essential to suppress the immortality of pathologically changed cells. In this study, we examined RNA molecules mimicking mitochondrial tRNAs interacting with cytochrome c and possibly affecting its cellular function. tRNA analogs were designed and synthesized prior to the conformational analysis and gel assays clearly stating the nucleic acid–protein complex formation. The circular dichroism spectroscopic (CD) and microscale thermophoresis examination revealed the structural and conformational differences between four tRNA analogs in their interactions with cytochrome c. Obtained CD spectra and gel studies resulted in the complex ratio estimation and conclusion that not only the complex formation may be preferential towards specific tRNAs present in the cell, but nucleobase modifications are not essential for such interaction.     

Differentiating analogous tRNA methyltransferases by fragments of the methyl donor

Bacterial TrmD and eukaryotic-archaeal Trm5 form a pair of analogous tRNA methyltransferase that catalyze methyl transfer from S-adenosyl methionine (AdoMet) to N(1) of G37, using catalytic motifs that share no sequence or structural homology. Here we show that natural and synthetic analogs of AdoMet are unable to distinguish TrmD from Trm5. Instead, fragments of AdoMet, adenosine and methionine, are selectively inhibitory of TrmD rather than Trm5. Detailed structural information of the two enzymes in complex with adenosine reveals how Trm5 escapes targeting by adopting an altered structure, whereas TrmD is trapped by targeting due to its rigid structure that stably accommodates the fragment. Free energy analysis exposes energetic disparities between the two enzymes in how they approach the binding of AdoMet versus fragments and provides insights into the design of inhibitors selective for TrmD.     

Analysis of genomic tRNA revealed presence of novel genomic features in cyanobacterial tRNA

Transfer RNAs (tRNA) are important molecules that involved in protein translation machinery and acts as a bridge between the ribosome and codon of the mRNA. The study of tRNA is evolving considerably in the fields of bacteria, plants, and animals. However, detailed genomic study of the cyanobacterial tRNA is lacking. Therefore, we conducted a study of cyanobacterial tRNA from 61 species. Analysis revealed that; cyanobacteria contain thirty-six to seventy-eight tRNA gens per genome that encodes for 20 tRNA isotypes. The number of iso-acceptors (anti-codons) ranged from thirty-two to forty-three per genome. tRNA^Ile with anti-codon AAU, GAU, and UAU was reported to be absent from the genome of Gleocapsa PCC 73,106 and Xenococcus sp. PCC 7305. Instead, they were contained anti-codon CAU that is common to tRNA^Met and tRNA^Ile as well. The iso-acceptors ACA (tRNA^Cys), ACC (tRNA^Gly), AGA, ACU (tRNA^Ser), AAA (tRNA^Phe), AGG (tRNA^Pro), AAC (tRNA^Val), GCG (tRNA^Arg), AUG (tRNA^His), and AUC (tRNA^Asp) were absent from the genome of cyanobacterial lineages studied so far. A few of the cyanobacterial species encode suppressor tRNAs, whereas none of the species were found to encode a selenocysteine iso-acceptor. Cyanobacterial species encode a few putative novel tRNAs whose functions are yet to be elucidated.     

Mitochondrial DNA from lupine: restriction analysis and cloning of fragments coding for tRNA

Mitochondrial DNA (mtDNA) was isolated from lupine. Restriction analysis was used to estimate its size, which is about 180 kb. A BamHI bank of this mtDNA was constructed using plasmids pBR322 and pBR327 as vectors. Eight clones containing plasmids hybridizing to mitochondrial tRNA (mttRNA) were isolated. Restriction maps of these plasmids were determined. Six of these plasmids hybridized to unique fragments and two to two fragments of very similar size, all obtained by BamHI cleavage of mtDNA.     

Peroxisomes as novel players in cell calcium homeostasis

Ca2+ concentration in peroxisomal matrix ([Ca2+](perox)) has been monitored dynamically in mammalian cells expressing variants of Ca2+-sensitive aequorin specifically targeted to peroxisomes. Upon stimulation with agonists that induce Ca2+ release from intracellular stores, peroxisomes transiently take up Ca2+ reaching peak values in the lumen as high as 50-100 microm, depending on cell types. Also in resting cells, peroxisomes sustain a Ca2+ gradient, [Ca2+](perox) being approximately 20-fold higher than [Ca2+] in the cytosol ([Ca2+](cyt)). The properties of Ca2+ traffic across the peroxisomal membrane are different from those reported for other subcellular organelles. The sensitivity of peroxisomal Ca2+ uptake to agents dissipating H+ and Na+ gradients unravels the existence of a complex bioenergetic framework including V-ATPase, Ca2+/H+, and Ca2+/Na+ activities whose components are yet to be identified at a molecular level. The different [Ca2+](perox) of resting and stimulated cells suggest that Ca2+ could play an important role in the regulation of peroxisomal metabolism.     

Glycosyltransferases and cell wall biosynthesis: novel players and insights

Plants need an enormous biosynthetic machinery to synthesize the complex polysaccharides that are present in the plant cell wall. The isolation, characterization and mapping of wall mutants, together with biochemical approaches, have led to significant advances in our understanding of both wall polysaccharide synthesis at a molecular level and the function of polysaccharides in plant growth and development. Moreover, potential regulation mechanisms and associated protein factors are emerging from recent data.     

Reconstitution of chemically synthesized ribooligonucleotides with naturally occurring tRNA fragments.

Chemically synthesized yeast tRNA terminal fragments were reconstituted with natural tRNA fragments which were obtained by partial digestion with RNase T1. The synthetic 3'-nonanucleotide (I) accepted alanine (3% with respect to the intact tRNA) when combined with a 4-fold excess of the natural 5'-quarter and the chemically synthesized hexanucleotide (II) stimulated the aminoacylation of the natural 3'-half molecule.