Improved application of RNAModMapper - An RNA modification mapping software tool - For analysis of liquid chromatography tandem mass spectrometry (LC-MS/MS) data.

Research into post-transcriptional processing and modification of RNA continues to speed forward, as their ever-emerging role in the regulation of gene expression in biological systems continues to unravel. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has proven for over two decades to be a powerful ally in the elucidation of RNA modification identity and location, but the technique has not proceeded without its own unique technical challenges. The throughput of LC-MS/MS modification mapping experiments continues to be impeded by tedious and time-consuming spectral interpretation, particularly during for the analysis of complex RNA samples. RNAModMapper was recently developed as a tool to improve the interpretation and annotation of LC-MS/MS data sets from samples containing post-transcriptionally modified RNAs. Here, we delve deeper into the methodology and practice of RNAModMapper to provide greater insight into its utility, and remaining hurdles, in current RNA modification mapping experiments.

Determining degradation intermediates and the pathway of 3' to 5' degradation of histone mRNA using high-throughput sequencing.

The half-life of an mRNA is an important parameter contributing to the steady-state level of the mRNA. Rapid changes in mRNA levels can result from decreasing the half-life of an mRNA. Establishing the detailed pathway of mRNA degradation for a particular class of mRNAs requires the ability to isolate mRNA degradation intermediates. High-throughput sequencing provides a method for detecting these intermediates. Here we describe a method for determining the intermediates in 3' to 5' degradation. Characterizing these intermediates requires not only determining the precise 3' end of the molecule to a single nucleotide resolution, but also the ability to detect and characterize any untemplated nucleotides present on the intermediates. We achieve this by ligating a known sequence to all the 3' termini in the cell, and then sequence the 3' termini and the ligated linker to identify any alterations to the genomic reference sequence. We have applied this method to characterize the intermediates in histone mRNA metabolism, allowing us to deduce the pathway of 3' to 5' degradation. This method can potentially be applied to any RNA, and we discuss possible strategies for extending the method to include simultaneous determination of the 3' and 5' end of the same RNA molecule.

Ultraviolet Photodissociation and Activated Electron Photodetachment Mass Spectrometry for Top-Down Sequencing of Modified Oligoribonucleotides.

With the increased development of new RNA-based therapeutics, the need for robust analytical methods for confirming sequences and mapping modifications has accelerated. Characterizing modified ribonucleic acids using mass spectrometry is challenging because diagnostic fragmentation may be suppressed for modified nucleotides, thus hampering complete sequence coverage and the confident localization of modifications. Ultraviolet photodissociation (UVPD) has shown great potential for the characterization of nucleic acids due to extensive backbone fragmentation. Activated electron photodetachment dissociation (a-EPD) has also been used as an alternative to capitalize on the dominant charge-reduction pathway prevalent in UVPD, facilitate dissociation, and produce high abundances of fragment ions. Here, we compare higher-energy collisional activation (HCD), UVPD using 193 and 213 nm photons, and a-EPD for the top-down sequencing of modified nucleic acids, including methylated, phosphorothioate, and locked nucleic acid-modified DNA. The presence of these modifications alters the fragmentation pathways observed upon UVPD and a-EPD, and extensive backbone cleavage is observed that results in the production of fragment ions that retain the modifications and allow them to be pinpointed. LNA and 2'-O-methoxy phosphorothioate modifications caused a significant suppression of fragmentation for UVPD but not for a-EPD, whereas phosphorothioate bonds did not cause any significant suppression for either method. The incorporation of 2'-O-methyl modifications suppressed fragmentation of the antisense strand of patisiran, which resulted in some gaps in sequence coverage. However, UVPD provided the highest sequence coverage when compared to a-EPD.

Characterization and quantification of RNA post-transcriptional modifications using stable isotope labeling of RNA in conjunction with mass spectrometry analysis.

Mass spectrometry has emerged as an increasingly powerful tool for the identification and characterization of nucleic acids, in particular RNA post-transcriptional modifications. High mass accuracy instrumentation is often required to discriminate between compositional isomers of oligonucleotides. We have used stable isotope labeling ((15)N) of E. coli RNA in conjunction with mass spectrometry analysis of the combined heavy- and light-labeled RNA for the identification and quantification of oligoribonucleotides and post-transcriptional modifications. The number of nitrogen atoms in the oligoribonucleotide and fragment ions can readily be determined using this approach, enabling the discrimination between potential compositional isomers without the requirement of high mass accuracy mass spectrometers. In addition, the identification of specific fragment ions in both the unlabeled and labeled oligoribonucleotides can be used to gain further confidence in the assignment of RNA post-transcriptional modifications. Using this approach we have identified a range of post-transcriptional modifications of E. coli 16S rRNA. Furthermore, this method facilitates the rapid and accurate quantification of oligoribonucleotides, including cyclic phosphate intermediates and missed cleavages often generated from RNase digestions.

Roles for TbDSS-1 in RNA surveillance and decay of maturation by-products from the 12S rRNA locus.

The Trypanosoma brucei exoribonuclease, TbDSS-1, has been implicated in multiple aspects of mitochondrial RNA metabolism. Here, we investigate the role of TbDSS-1 in RNA processing and surveillance by analyzing 12S rRNA processing intermediates in TbDSS-1 RNAi cells. RNA fragments corresponding to leader sequence upstream of 12S rRNA accumulate upon TbDSS-1 depletion. The 5' extremity of 12S rRNA is generated by endonucleolytic cleavage, and TbDSS-1 degrades resulting upstream maturation by-products. RNAs with 5' ends at position -141 and 3' ends adjacent to the mature 5' end of 12S rRNA are common and invariably possess oligo(U) tails. 12S rRNAs with mature 3' ends and unprocessed 5' ends also accumulate in TbDSS-1 depleted cells, suggesting that these RNAs represent dead-end products normally destined for decay by TbDSS-1 in an RNA surveillance pathway. Together, these data indicate dual roles for TbDSS-1 in degradation of 12S rRNA maturation by-products and as part of a mitochondrial RNA surveillance pathway that eliminates stalled 12S processing intermediates. We further provide evidence that TbDSS-1 degrades RNAs originating upstream of the first gene on the minor strand of the mitochondrial maxicircle suggesting that TbDSS-1 also removes non-functional RNAs generated from other regions of the mitochondrial genome.

Natural occurrence of 2',5'-linked heteronucleotides in marine sponges.

2',5'-oligoadenylate synthetases (OAS) as a component of mammalian interferon-induced antiviral enzymatic system catalyze the oligomerization of cellular ATP into 2',5'-linked oligoadenylates (2-5A). Though vertebrate OASs have been characterized as 2'-nucleotidyl transferases under in vitro conditions, the natural occurrence of 2',5'-oligonucleotides other than 2-5A has never been demonstrated. Here we have demonstrated that OASs from the marine sponges Thenea muricata and Chondrilla nucula are able to catalyze in vivo synthesis of 2-5A as well as the synthesis of a series 2',5'-linked heteronucleotides which accompanied high levels of 2',5'-diadenylates. In dephosphorylated perchloric acid extracts of the sponges, these heteronucleotides were identified as A2'p5'G, A2' p5'U, A2'p5'C, G2'p5'A and G2' p5'U. The natural occurrence of 2'-adenylated NAD(+) was also detected. In vitro assays demonstrated that besides ATP, GTP was a good substrate for the sponge OAS, especially for OAS from C. nucula. Pyrimidine nucleotides UTP and CTP were also used as substrates for oligomerization, giving 2',5'-linked homo-oligomers. These data refer to the substrate specificity of sponge OASs that is remarkably different from that of vertebrate OASs. Further studies of OASs from sponges may help to elucidate evolutionary and functional aspects of OASs as proteins of the nucleotidyltransferase family.

Sequencing of single and double stranded RNA oligonucleotides by acid hydrolysis and MALDI mass spectrometry.

Treatment of RNA oligonucleotides with strong acids at pH 1-2 rapidly leads to hydrolysis of the phosphodiester bonds at the 5'-position of ribose. Analysis of the resulting degradation products by MALDI coupled to an Orbitrap high resolution mass spectrometer shows almost complete mass ladders from both sides of the nucleotides without interfering fragments from base losses or internal fragments. From the mass differences between adjacent peaks of a mass ladder, the sequence can be determined. Low cleavage efficiency at the termini leads to 2mers and 3mers which can be identified by MS/MS. In this way the complete sequences of different siRNA 21mer single and double strands could be verified. This simple and fast method can be applied for controlling sequences of synthetic oligomers, as well as for de-novo sequencing. Moreover, the method is applicable for localization and identification of RNA modifications as demonstrated using the examples of an oligonucleotide with phosphorothioate backbone and of one containing 2'-methoxy-ribose modifications.

Small ncRNA transcriptome analysis from kinetoplast mitochondria of Leishmania tarentolae.

Gene expression in mitochondria of kinetoplastid protozoa requires RNA editing, a post-transcriptional process which involves insertion or deletion of uridine residues at specific sites within mitochondrial pre-mRNAs. Sequence specificity of the RNA editing process is mediated by oligo-uridylated small, non-coding RNAs, designated as guide RNAs (gRNAs). In this study, we have analyzed the small ncRNA transcriptome from kinetoplast mitochondria of Leishmania tarentolae by generating specialized cDNA libraries encoding size-selected RNA species. Through this screen, a significant number of novel oligo-uridylated RNA species, which we have termed oU-RNAs, has been identified. Most novel oU-RNAs are present as stable RNA species in mitochondria as assessed by northern blot analysis. Thereby, novel oU-RNAs show similar expression levels and sizes as previously reported for canonical gRNAs. Several oU-RNAs are transcribed from both strands of the maxicircle and minicircles components of the mitochondrial genome, from regions where up till now no transcription has been reported. Two stable oU-RNAs exhibit an anchor sequence in antisense orientation to known gRNAs and thus might regulate editing of respective pre-mRNAs. A number of oU-RNAs map in antisense orientation to non-edited protein-coding genes suggesting that they might function by a different mechanism. In addition, our screen shows that all kinetoplast-derived RNAs are prone to some degree of uridylation.

U1 small nuclear ribonucleoprotein particle-specific proteins interact with the first and second stem-loops of U1 RNA, with the A protein binding directly to the RNA independently of the 70K and Sm proteins.

The U1 small nuclear ribonucleoprotein particle (U1 snRNP), a cofactor in pre-mRNA splicing, contains three proteins, termed 70K, A, and C, that are not present in the other spliceosome-associated snRNPs. We studied the binding of the A and C proteins to U1 RNA, using a U1 snRNP reconstitution system and an antibody-induced nuclease protection technique. Antibodies that reacted with the A and C proteins induced nuclease protection of the first two stem-loops of U1 RNA in reconstituted U1 snRNP. Detailed analysis of the antibody-induced nuclease protection patterns indicated the existence of relatively long-range protein-protein interactions in the U1 snRNP, with the 5' end of U1 RNA and its associated specific proteins interacting with proteins bound to the Sm domain near the 3' end. UV cross-linking experiments in conjunction with an A-protein-specific antibody demonstrated that the A protein bound directly to the U1 RNA rather than assembling in the U1 snRNP exclusively via protein-protein interactions. This conclusion was supported by additional experiments revealing that the A protein could bind to U1 RNA in the absence of bound 70K and Sm core proteins.

Structural requirements of adenovirus VAI RNA for its translation enhancement function.

Recently, by genetic and biochemical approaches, it has been shown that adenovirus VAI RNA is required for efficient translation of viral mRNAs at late times after infection. To understand the nucleotide sequences and the domains of the VAI RNA that are responsible for the role of VAI RNA in enhancement of translation, a mutational analysis of the VAI gene was undertaken. Deletion, substitution, and insertion mutations covering most of the nucleotide sequences of VAI RNA were introduced into the VAI gene at the plasmid level. These mutant genes were then reintroduced into the virus, and growth properties of the mutant viruses were studied. The majority of the mutants retained normal or nearly normal levels of biological function. Mutations in the region between +43 and +53 and between +107 and the 3' end of the gene resulted in a considerable loss of activity. These mutants, however, grew significantly better than did an adenovirus type 5 mutant lacking both functional VAI and VAII genes, indicating that they retain a portion of their activity. Because no one mutation was able to completely abolish the function, we suggest that the VAI RNA may have multiple functional sites for its translation modulation function. These multiple sites may be short oligonucleotide sequences that may interact with cellular or viral components or both during translation.

Synergistic effect of human immune interferon and double-stranded RNA against human colon carcinoma cells in vitro.

The cytocidal activity of human immune interferon (IFN-gamma) in combination with the synthetic double-stranded RNA, poly(I).poly(C), was investigated in human colon carcinoma cell line HT-29. Three days of treatment with IFN-gamma (10 to 25 units/ml) resulted in 30 to 40% reduction in colony formation, whereas poly(I).poly(C) (25 to 100 micrograms/ml) reduced cell viability by 10 to 20% of control. The lethal effect of the combination of IFN-gamma and poly(I).poly(C) was synergistic wherein 70 to 90% reduction in colony formation was observed. Measurements of DNA, RNA, and protein synthesis after IFN-gamma and poly(I).poly(C) treatment showed a dose-dependent reduction in all three parameters. Recombinant IFN-gamma in combination with poly(I).poly(C) exhibited a similar effect. Studies evaluating the molecular mechanism of IFN-gamma and poly(I).poly(C) toxicity indicate a lack of involvement of the double-stranded RNA-dependent (2',5')oligoadenylate-RNase L and protein kinase pathways; however, the effect appears to be related to the inhibition of ribosomal RNA transcription in this cell line.

Potentiation of the cytocidal effect of human immune interferon by different synthetic double-stranded RNAs in the refractory human colon carcinoma cell line BE.

A human cell line BE, derived from an undifferentiated carcinoma of the colon, was studied for its response to the cytocidal effects of human immune interferon (IFN-gamma) alone and in combination with various double-standard RNAs (dsRNAs). BE cells were moderately refractory to 3-day treatment with IFN-gamma (10 to 300 units/ml) where only 5 to 30% reduction in colony formation occurred. A similar exposure interval to polyriboinosinic.polyribocytidylic acid [poly(I).poly(C)] (100 micrograms/ml) had no detectable effect on colony formation. In contrast, the lethal effect of the combination of IFN-gamma and poly(I).poly(C) was synergistic and this regimen produced a 40 to 80% reduction in colony formation. The cytocidal effects of the combination of IFN-gamma with varying concentrations of the dsRNAs poly(I).poly(C), polyriboadenylic.polyribouridylic acid [poly(A).poly(U)], polyriboinosinic.polyribocytidylic acid stabilized with poly-L-lysine in carboxymethylcellulose [poly(ICLC)], and mismatched dsRNA [rIn.r(C13,U)n] were also examined. The concentration of the dsRNAs producing a 50% decrease in cell viability in combination with IFN-gamma (100 units/ml) was 6 micrograms/ml for poly(I).poly(C), 1 microgram/ml for poly(A).poly(U), 3 ng/ml for poly(ICLC), and 16 micrograms/ml for rIn.r(C13,U)n. DNA, RNA, and protein synthesis in IFN-gamma and poly(I).poly(C)-treated cells were reduced in a dose-dependent manner. However, there were no changes in either (2',5')oligoadenylate concentrations or in ribosomal RNA transcription following treatment with IFN-gamma and poly(I).poly(C). Thus, the synergism resulting from the combination of IFN-gamma and dsRNA appears to be mediated via another, as yet unknown, mechanism.

S1 nuclease as a probe of yeast ribosomal 5 S RNA conformation.

5 S RNA was isolated from Saccharomyces cerevisiae grown in the presence of 32P-phosphate and digested with nuclease S1, a single-strand specific nuclease. Two different procedures were employed to determine the sites of attack on the RNA. First, 5 S RNA was isolated from nuclease S1 digests, digested to completion with ribonuclease T1, and then 'fingerprinted' by two-dimensional electrophoresis. Quantitation of each of the characteristic RNAase T1-derived oligonucleotides was employed to determine the relative susceptibility of various regions of the molecule to nuclease S1. A second procedure to define nuclease S1-susceptible sites in the molecule employed polyacrylamide gel electrophoretic fractionation of nuclease S1 digests followed by identification of the nucleotide sequences of the released RNA fragments. Both procedures showed that the region of the molecule between residues 9 and 60 was most susceptible to nuclease S1, with preferential cleavage occurring between residues 12-25 and 50-60. These results are discussed in relation to a proposed model for the secondary structure of yeast 5 S RNA.

Hamster mitochondrial transfer RNA lacks T and the universal GUUCG sequence.

Earlier inferences (based on incorporation of radio-activity from methyl-labeled methionine) that mammalian mitochondrial tRNA lacks ribothymidine ('T') have been confirmed using [14C]uridine as precursor. Moreover, oligonucleotide fingerprint analysis of 32P-labeled samples have shown that hamster mitochondrial tRNA lacks the 'universal' pentanucleotide of conventional tRNA in which T normally occurs, CTpsiCG, in modified or unmodified form.

Primary structure of bovine liver tRNATrp.

Purified tRNATrp from bovine liver, accepting 1700 pmol tryptophan per A260nm unit, was completely digested with pancreatic ribonuclease and T1 ribonuclease. The sequences of the resulting oligonucleotides were determined and the primary structure of the tRNA was deduced. These analyses showed numerous incomplete post-transcriptional modifications, and several positions heterogenously occupied by two different nucleotides, which lead us to think that in bovine liver there exist a mixture of several tRNATrp.

The 5' ends of bacterial RNA. II. The triphosphate-terminated ends of primary gene transcripts.

Bacterial RNA, Pulse labeled with 32Pi, was digested with pancreatic RNAase. Oligonucleotides containing a triphosphate group at the 5'-hydroxyl, and therefore derived from the original beginning ends of the RNA transcripts, were purified by hydroxyapatite chromatography and analyzed by two-dimensional paper electrophoresis. A broad diversity of species was found, although the distribution among these species was not completely uniform. Possible methods of utilizing these methods in combination with in vitro synthetic techniques are discussed.

Methylated nucleosides in polyadenylate-containing yeast messenger ribonucleic acid.

Yeast messenger RNA was found to be methylated. A calculation of the specific methylation showed that the average yeast messenger RNA molecule contains only two methylated nucleosides which occur in one alkali stable oligonucleotide. Similar to other eukaryotic messengers, the 5' terminus of yeast messenger RNA is blocked by 7-methylguanosine, linked through a di- or triphosphate bridge to a ribosemethylated nucleoside. Contrary to the messengers of high eucaryotic organisms, no additional base methylated constituents were found.

Binding sites for ribosomal proteins S8 and S15 in the 16 S RNA of Escherichia coli.

A fragment of the 16 S ribosomal RNA of Escherichia coli that contains the binding sites for proteins S8 and S15 of the 30 S ribosomal subunit has been isolated and characterized. The RNA fragment, which sediments as 5 S, was partially protected from pancreatic RNAase digestion when S15 alone, or S8 and S15 together, were bound to the 16 S RNA. Purified 5 S RNA was shown to reassociate specifically with protein S15 by analysis of binding stoichiometry. Although interaction between the fragment and protein S8 alone could not be detected, the 5 S RNA selectively bound both S8 and S15 when incubated with an unfractionated mixture of 30-S subunit proteins. Nucleotide sequence analysis demonstrated that the 5 S RNA arises from the middle of the 16 S RNA molecule and encompasses approximately 150 residues from Sections C, C'1 and C'2. Section C consists of a long hairpin loop with an extensively hydrogen-bonded stem and is contiguous with Section C'1. Sections C'1 and C'2, although not contiguous, are highly complementary and it is likely that together they comprise the base-paired stem of an adjacent loop.

Protamine messenger RNA from rainbow trout testis contains the nucleotide sequence A-A-U-A-A-A in an untranslated region.

Full-length, complementary DNAs were prepared to rainbow trout protamine mRNA using reverse transcriptase and were labelled during synthesis by the replacement of dATP by [alpha32P]dATP or dTTP by [alpha32P]dTTP. The 32P-labelled protamine complementary DNAs were digested with T4 endonuclease IV. Fragments from the digests were separated in two dimensions, and those discrete fragments which could be identified from both the A-labelled and T-labelled complementary DNAs were subjected to sequence analysis. The sequences described here all arise from the non-coding region. One pentadecanucleotide contained the sequence A-A-U-A-A-A which has been reported by Proudfoot and Brownlee ((1976) Nature 263, 211-214) to occur in the noncoding regions of six other eukaryotic mRNAs.

Effects of membrane ribonuclease and 3'-nucleotidase on the digestion of polyuridylic acid by rat liver plasma membrane.

1. Fragments of isolated rat liver plasma membrane possess a ribonuclease activity which at pH 7.8 in the presence of 10 mM EDTA can digest polyuridylic acid (poly(U)) and polycytidylic acid (poly(C)) but not polyadenylic acid (poly(A)) and polyguanylic acid (poly(G)). Under these conditions, the membrane preparation does not degrade native or denatured DNA. 2. The products of the reaction with poly(U) (10 mM EDTA present) can be separated on DEAE-Sephadex into oligonucleotides of increasing chain length. Most of the products are di- to hexa-nucleotides which contain terminal 3'-phosphate groups. 3. When EDTA is not present (pH 7.8 or 8.8) the plasma membrane preparation degrades both poly(A) and poly(U). With poly(A) the product is all nucleoside while with poly(U) as substrate most of the product is nucleoside, but also some oligonucleotides are produced. 4. The ribonuclease releases acid soluble products very slowly from high concentrations of poly(U) (mg/ml). 5. Uridine trinucleotide with and without a terminal 3'-phosphate group is degraded by rat liver plasma membrane. The trinucleotide diphosphate is rapidly hydrolyzed to nucleoside while the trinucleotide itself is slowly digested and yields intermediate products, including nucleoside.