A two-dimensional thin layer chromatographic procedure for the sequential analysis of oligonucleotides employing tritium post-labeling.

Two dimensional PEI-cellulose thin layer chromatography can resolve sequentially degraded oligonucleotide fragments of tRNA. This technique entails the sequential degradation of the oligonucleotide with snake venom phosphodiesterase in the presence of bacterial alkaline phosphatase, and periodate oxidation followed by tritiated sodium borohydride reduction of the 3' terminal nucleoside. Subsequently the tritiated oligonucleotide fragments were resolved by two dimensional PEI-cellulose TLC. The results of these experiments indicate that, in some cases, the complete nucleotide sequence of a large oligonucleotide fragment may be determined by interpretation of the observed mobility shifts, thereby eliminiating the need for additional analysis of the oligonucleotide. In addition, the use of two-dimensional rather than one-dimensional resolution of the tritium labeled fragments allows for a complete separation of any interfering background spots from the sequentially degraded oligonucleotides. This procedure was applied to the complete nucleotide sequence analysis of several ribonuclease T1Val and ribonuclease A digestion products from human placenta tRNA.     

Mature methyl-deficient tRNA isolated from a mammary adenocarcinoma

A transplantable rat tumor, mammary adenocarcinoma 13762, accumulates tRNA which can be methylated in vitro by mammalian tRNA (adenine-1) methyltransferase. This unusual ability of the tumor RNA to serve as substrate for a homologous tRNA methylating enzyme is correlated with unusually low levels of the A₅₈-specific adenine-1 methyltransferase. The nature of the methyl-accepting RNA has been examined by separating tumor tRNA on two-dimensional polyacrylamide gels. Comparisons of ethidium bromide-stained gels of tumor vs. liver tRNA show no significant quantitative differences and no accumulation of novel tRNAs or precursor tRNAs in adenocarcinoma RNA. Two-dimensional separations of tumor RNA after in vitro [¹⁴C]methylation using purified adenine-1 methyltransferase indicate that about 25% of the tRNA species are strongly methyl-accepting RNAs. Identification of six of the tRNAs separated on two-dimensional gels has been carried out by hybridization of cloned tRNA genes to Northern blots. Three of these, tRNA^Lys3, tRNA^Gln and tRNA^Met_i, are among the adenocarcinoma methyl-accepting RNAs. The other three RNAs, all of which are leucine-specific tRNAs, show no methyl-accepting properties. Our results suggest that low levels of a tRNA methyltransferase in the adenocarcinoma cause selected species of tRNA to escape the normal A₅₈ methylation, resulting in the appearance of several mature tRNAs which are deficient in 1-methyladenine. The methyl-accepting tRNAs from the tumor appear as ethidium bromide-stained spots of similar intensity to those seen for RNA from rat liver; therefore, methyladenine deficiency does not seem to impair processing of these tRNAs.     

Relative quantitation of transfer RNAs using liquid chromatography mass spectrometry and signature digestion products

Transfer ribonucleic acids (tRNAs) are challenging to identify and quantify from unseparated mixtures. Our lab previously developed the signature digestion approach for identifying tRNAs without specific separation. Here we describe the combination of relative quantification via enzyme-mediated isotope labeling with this signature digestion approach for the relative quantification of tRNAs. These quantitative signature digestion products were characterized using liquid chromatography mass spectrometry (LC-MS), and we find that up to 5-fold changes in tRNA abundance can be quantified from sub-microgram amounts of total tRNA. Quantitative tRNA signature digestion products must (i) incorporate an isotopic label during enzymatic digestion; (ii) have no m/z interferences from other signature digestion products in the sample and (iii) yield a linear response during LC-MS analysis. Under these experimental conditions, the RNase T1, A and U2 signature digestion products that potentially could be used for the relative quantification of Escherichia coli tRNAs were identified, and the linearity and sequence identify of RNase T1 signature digestion products were experimentally confirmed. These RNase T1 quantitative signature digestion products were then used in proof-of-principle experiments to quantify changes arising due to different culturing media to 17 tRNA families. This method enables new experiments where information regarding tRNA identity and changes in abundance are desired.     

Micro thin-layer techniques for rapid sequence analysis of P-labeled RNA: Double digestion and pancreatic ribonuclease analyses

Double digestion of oligonucleotides obtained from ribonuclease T₁ or pancreatic ribonuclease A fingerprints results in the following series of products: (Ap)_0-nCp, (Ap)_0-nUp, and (Ap)_0-nGp. A new micromethod is described for the rapid analysis of these digests. The procedure consists of two-dimensional chromatography on a small PEI thin-layer plate. In the first dimension, the oligonucleotides are separated independent of their Ap content into three groups, which represent the Cp-, Gp-, and Up- 3′-terminal oligonucleotide series, respectively. In the second dimension, the products are fractionated according to their chain length. This method, which allows direct identification of even the longer Ap tracts in a double digest of an oligonucleotide or an RNA chain, is very rapid and highly sensitive and can be applied to the simultaneous analysis of a large number of samples in a single run. The procedure has also been adapted to the analysis of pancreatic ribonuclease A digests of small RNA fragments.     

Structural comparison of human, bovine, rat, and Walker 256 carcinosarcoma asparaginyl-tRNA

The complete nucleotide sequences of human placenta, human liver, and bovine liver tRNAAsn have been determined. A comparison of these tRNA structures with the previously reported nucleotide sequences of rat liver and Walker 256 carcinosarcoma tRNAAns reveals that the primary nucleotide sequences of the major species of mammalian cytoplasmic tRNAasn are conserved in higher eucaryotes. The complete nucleotide sequence of these tRNAs is: pG-U-C-U-C-U-G-U-m1G-m2G-C-G-C-A-A-D-C-G-G-D-X-A-G-C-G-C-m2(2)G-psi-psi-C-G-G-C-U-Q(G)-U-U-t6A-A-C-C-G-A-A-A-G-m7G-D-U-G-G-U-G-G-Z-psi-C-G-m1A-G-C-C-C-A-C-C-C-A-G-G-G-A-C-G-C-C-AOH where X is 3-(3-amino-3-carboxyl-n-propyl)uridine, Q is 7-(4,5-cis-dihydroxyl-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanosine, Z is an unknown modified nucleotide, and Q(G) represents the replacement of Q nucleoside by G nucleoside in Walker 256 carcinosarcoma tRNAAsn. These primary structures were determined by combined use of the 3H- and 32P-post-labeling techniques. Sequences were compared by tritium nucleoside trialcohol analysis, completed RNAase T1 digestion followed by 3H-labeled fingerprinting on polyethylenimine-impregnated cellulose by two-dimensional thin-layer chromatography (TLC), and polyacrylamide gel electrophoresis of either 5'-32P- and/or 3'-[32P]pCp-labeled tRNA after partial ribonuclease digestions.     

The 5'-termini of the oligonucleotides from ribonuclease T 1 digested E. coli ribosomes

Oligonucleotides remaining in the 70s $\text{Escherichia}\text{coli}$ ribosomal particles after varying degrees of digestion with ribonuclease T₁ were phosphorylated with polynucleotide kinase in the presence of γ-labeled³²P-ATP. The resulting radioactively labeled RNA molecules were further digested with pancreatic ribonuclease and analyzed by a two-dimensional finger-printing technique. The numbers of labeled oligonucleotides were proportional to the duration of T₁ digestion; most of these oligonucleotides yielded ¹pAp and/or ¹pCp as their 5′-end groups upon alkaline hydrolysis.     

The nucleotide sequences of some large ribonuclease T1 products from bacteriophage R17 ribonucleic acid

A method of `fingerprinting' high-molecular-weight <sup>32</sup>P-labelled RNA species, using a two-dimensional thin-layer-chromatographic separation of ribonuclease T<sub>1</sub> digestion products, has been applied to RNA from the Escherichia coli bacteriophage R17. The `fingerprinting' technique, besides giving a unique pattern that can be used as a characterization of the RNA, has made it possible to isolate a number of the larger oligonucleotides and to determine their nucleotide sequences.     

The use of ribonuclease U2 in RNA sequence determination

The catalog of oligomers produced by ribonuclease T₁ digestion ofEscherichi coli 16S ribosomal RNA has been determined by a new method that involves the use of ribonuclease U₂ fromUstilago sphaerogena. The sequences for the larger T₁ oligomers (8 or more bases) determined in this way differ in more than 50 % of the cases from those reported previously (determined by other methods).     

E. coli tRNAPhe modified at the 3-(3-amino-3-carboxypropyl)uridine with a photoaffinity label is fully functional for aminoacylation and for ribosomal interaction

E. coli tRNA^Phe was modified at its 3-(3-amino-3-carboxypropyl)uridine residue with the N-hydroxysuccinimide ester of N-4-azido-2-nitrophenyl)glycine. Exclusive modification of this base was shown by two-dimensional TLC analysis of the T₁ oligonucleotide and nucleoside products of nuclease digestion. The fully modified tRNA could be aminoacylated to the same level as control tRNA. The aminoacylated tRNA was as active as control tRNA in non-enzymatic binding to the P site of ribosomes, and in EFTu-dependent binding to the rirobosomal A site. The functional activity of this photolabile modified tRNA allows it to be used to probe the A and P binding sites on ribosomes and on other proteins that interact with tRNA. Crosslinking to the ribosomal P site has been shown.     

Gross map location of Escherichia coli transfer RNA genes.

Chromosomal locations of Escherichia coli genes specifying more than 20 different transfer RNA species were determined by utilizing two different methods. One was based upon gene dosage effects caused by F′ factors. In 15 different F′ strains and their corresponding F^? strains, relative contents of individual tRNAs were measured after separating the tRNAs by two-dimensional polyacrylamide gel electrophoresis. Approximate doubling of the content of particular tRNA was found in individual F′ strains, as showing gross map location of the tRNA gene. The other method was based on the amplified synthesis of tRNAs occurring after prophage induction of λ lysogens. Synthesis of individual tRNAs was measured after the induction of λ phages integrated at five different bacterial sites. Characteristic overproduction of different tRNAs was observed in individual prophage strains. This finding also gave approximate map locations of tRNA genes close to the prophage sites. The mapping data obtained by the two methods were consistent with each other and also with the reported positions in the cases where previously mapped. On the basis of map location of the tRNA_f1^Met gene newly determined, the λ-transducing phage carrying the tRNA_f1^Met gene was found.     

Incorporation of cytokinin N-benzyladenine into tobacco callus transfer ribonucleic Acid and ribosomal ribonucleic Acid preparations

The incorporation of the cytokinin N⁶-benzyladenine into tobacco (Nicotiana tabacum) callus tRNA and rRNA preparations isolated from tissue grown on medium containing either N⁶-benzyladenine-8-¹⁴C or N⁶-benzyladenine-8-¹⁴C: benzene-³H(G) has been examined. N⁶-benzyladenine was incorporated into both the tRNA and rRNA preparations as the intact base. Over 90% of the radioactive N⁶-benzyladenosine recovered from the RNA preparations was associated with the rRNA. Purification of the crude rRNA by either MAK chromatography or Sephadex G-200 gel filtration had no effect on the N⁶-benzyladenosine content of the RNA preparation. The distribution of N⁶-benzyladenosine moieties in tobacco callus tRNA fractionated by BD-cellulose chromatography did not correspond to the distribution of ribosylzeatin activity. N⁶-benzyladenosine was released from the rRNA preparation by treatment with venom phosphodiesterase and phosphatase, ribonuclease T₂ and phosphatase, or ribonuclease T₂ and a 3′-nucleotidase. N⁶-benzyladenosine was not released from the RNA preparation by treatment with either ribonuclease T₂ or phosphatase alone or by successive treatment with ribonuclease T₂ and a 5′-nucleotidase. Brief treatment of the rRNA preparation with ribonuclease T₁ and pancreatic ribonuclease converted the N⁶-benzyladenosine moieties into an ethyl alcohol soluble form. On the basis of these and earlier results, the N⁶-benzyladenosine recovered from the tobacco callus RNA preparations appears to be present as a constituent of RNA and not as a nonpolynucleotide contaminant.     

A double-labeling procedure for sequence analysis of picomole amounts of nonradioactive RNA fragments.

A double-labeling procedure for sequence analysis of nonradioactive polyribonucleotides is detailed, which is based on controlled endonucleolytic degradation of 3'-terminally (3H)-labeled oligonucleotide-(3') dialcohols and 5"-terminal analysis of the partial (3H)-labeled fragments following their separation according to chain length by polyethyleneimine- (PEI-)cellulose TLC and detection by fluorography. Undesired nonradioactive partial digestion products are eliminated by periodate oxidation. The 5'-termini are assayed by enzymic incorporation of (32p)-label into the isolated fragments, enzymic release of (32p)-labeled nucleoside-(5') monophosphates, two-dimensional PEI-cellulose chromatography, and autoradiography. Using this procedure, as little as 0.1 - 0.3 A260 unit of tRNA is needed to sequence all fragments in complete ribonuclease T1 and A digests, whereas radioactive derivative methods previously described by us1-4 required 4 - 6 A260 units.     

The occurrence and distribution of poly(a) ribonucleic Acid in soybean

The occurrence and distribution of poly(A) sequences in the RNA of soybean (Glycine max var. Wayne) have been studied. Only one of the two species of AMP-rich RNA contains poly(A). D-RNA does not contain detectable poly(A) sequences. The TB-RNA is the poly(A) RNA in this system. At least a part (up to 50% or more) of the mRNA in polyribosomes contains a poly(A) sequence. The poly(A) RNA is heterodisperse in size but has a mean size of approximately 18S (2,000 nucleotides) in urea and formamide gels. The poly(A) fragment resulting from ribonuclease A and T₁ digestion migrates as a broad band overlapping the 4 to 5.8S regions of the gels with a mean size of somewhat greater than 5S. No evidence was found for the occurrence of a discrete oligo(A) fragment in the poly(A) RNA; however, oligonucleotides which migrate faster than the poly(A) fraction were observed in preparations which were not bound to oligo(dT) cellulose prior to electrophoresis. This oligonucleotide region was enriched in AMP (up to about 65%) as would be expected after ribonuclease A and T₁ digestion.     

Human Tyrosine tRNA Is Also Internally Cleavable by E. coli Ribonuclease P RNA Ribozyme in Vitro

Transfer RNA is an essential molecule for biological system, and each tRNA molecule commonly has a cloverleaf structure. Previously, we experimentally showed that some Drosophila tRNA (tRNA^Ala, tRNA^His, and tRNA_i ^Met) molecules fit to form another, non-cloverleaf, structure in which the 3'-half of the tRNA molecules forms an alternative hairpin, and that the tRNA molecules are internally cleaved by the catalytic RNA of bacterial ribonuclease P (RNase P). Until now, the hyperprocessing reaction of tRNA has only been reported with Drosophila tRNAs. This time, we applied the hyperprocessing reaction to one of human tRNAs, human tyrosine tRNA, and we showed that this tRNA was also hyperprocessed by E. coli RNase P RNA. This tRNA is the first example for hyperprocessed non-Drosophila tRNAs. The results suggest that the hyperprocessing reaction can be a useful tool to detect destablized tRNA molecules from any species.     

Queuine is incorporated into brain transfer RNA

Pig brain tRNA was assayed for the presence of queuosine in the first position of the anticodon for each of the Q-family of tRNAs (aspartyl, asparaginyl, histidyl and tyrosyl). The brain tRNA was aminoacylated with each of the four amino acids and the aminoacylated tRNA's analyzed by RPC-5 chromatography. The results of this study show that for all four tRNAs of the family, queuine is substituted for guanine in virtually 100% of the anticodons. Therefore, it can be concluded that queuine is able to cross the blood-brain barrier and that brain contains quanine-queuine tRNA transglycosylase, the enzyme responsible for the excision of guanine from the orginal transcipts of these tRNAs and insertion of queuine. The determination of whether the tRNA contained queuine was made from the elution profile of the RPC-5 chromatrograms and the results confirmed by a change in the RPC-5 elution profile when the tRNAs were reacted with BrCN or NaIO₄.     

Glycine and asparagme tRNA sequences from the archaebacteriuin,Methanobacterium thermoautotrophicum

Two tRNA sequences from Methanobacterium thermoautotrophium are reported. Both tRNA^Gly_GCC and tRNA_NUU^Asn, the first tRNA sequences from methanogens, were determined by partial hydrolyses (both chemical and enzymatic) and analyzed by gel electrophoresis. The two tRNAs contain the unusual T-loop modifications, Cm and m¹I, which are present in other archaebacterial tRNAs. Finally the presence of an unknown modification in the D-loop has been inferred by a large jump in the sequence ladder. These tRNAs are approximately equidistant from eubacterial or eukaryotic tRNAs.     

A heterogeneous tRNA granule structure exhibiting rapid,bi-directional neuritic transport

mRNA translation is regulated by diverse mechanisms that converge at the initiation and elongation steps to determine the rate, profile, and localization of proteins synthesized. A consistently relevant feature of these mechanisms is the spatial re-distribution of translation machinery, a process of particular importance in neural cells. This process has, however, been largely overlooked with respect to its potential role in regulating the local concentration of cytoplasmic tRNAs, even as a multitude of data suggest that spatial regulation of the tRNA pool may help explain the remarkably high rates of peptide elongation. Here, we report that Cy3/Cy5-labeled bulk tRNAs transfected into neural cells distribute into granule-like structures – “tRNA granules” – that exhibit dynamic mixing of tRNAs between granules and rapid, bi-directional vectorial movement within neurites. Imaging of endogenous tRNA_gly and tRNA_lys by fluorescent in situ hybridization revealed a similar granular distribution of tRNAs in somata and neurites; this distribution was highly overlapping with granules imaged by introduction of exogenous Cy5-tRNA_thr and Cy3-tRNA_val. A subset of tRNA granules located in the cell body, neurite branch points and growth cones displayed fluorescence resonance energy transfer (FRET) between Cy3 and Cy5-labeled tRNAs indicative of translation, and co-localization with elongation machinery. A population of smaller, rapidly trafficked granules in neurites lacked FRET and showed poor colocalization with translation initiation and elongation factors, suggesting that they are a translationally inactive tRNA transport particle. Our data suggest that tRNAs are packaged into granules that are rapidly transported to loci where translation is needed, where they may greatly increase the local concentration of tRNAs in support of efficient elongation. The potential implications of this newly described structure for channeling of elongation, local translation, and diseases associated with altered tRNA levels or function are discussed.