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The two similarly expressed genes encoding U3 snRNA in Schizosaccharomyces pombe lack introns.

Both genes encoding U3 small nuclear RNA (snRNA) from the budding yeast Saccharomyces cerevisiae were recently shown to be interrupted by introns of the type removed by the pre-mRNA splicing machinery. We previously described one of the two U3 genes from the fission yeast Schizosaccharomyces pombe. In the present work, the second S. pombe U3 coding sequence was identified, and direct RNA sequence analysis was used to show that neither the U3A nor the U3B gene from this organism contains an intervening sequence. Our data also demonstrate that, as expected, the two RNAs exhibit great primary- and secondary-structure conservation. These similarities are not likely to be the result of a recent gene duplication or conversion event, because the DNA sequences flanking the U3A and U3B genes have diverged substantially. A notable exception is a 19-bp block, centered 36 nucleotides upstream from the transcriptional start site, in which the two loci match in 15 positions; this motif may represent an RNA polymerase II upstream regulatory element, because related sequences are found preceding fission yeast U1, U2, U4, and U5 snRNA genes. The significance of a short conserved sequence just downstream of the U3A and U3B genes is unknown, as it is not found 3' to other snRNA coding sequences in S. pombe. The 5' one-third of U3B RNA can be folded into a dual hairpin structure, as we previously proposed for Schizosaccharomyces pombe U3A and for other lower eukaryotic U3 homologues. Quantitation of fission yeast U3A and U3B indicates that, in contrast to snR17A and B in Saccharomyces cerevisiae, these RNAs accumulate to similar levels.

Base Sequence↗

Degradation of ribosomal RNA during Plasmodium falciparum gametocytogenesis.

The life cycle of Plasmodium falciparum is characterized by complex regulatory changes that allow adaptation of the parasites to different environmental conditions, which are especially pronounced during transmission between the mammalian host and the insect vector. Previous studies have shown that P. falciparum uses three types of ribosomal RNAs (rRNA A, S1, and S2 types) at different stages of its life cycle. We used Oxford Nanopore Technologies direct RNA sequencing to investigate the dynamics of rRNA usage throughout the parasite's intraerythrocytic development, as well as in salivary gland sporozoites. Our study revealed a preponderance of A-type rRNAs during the intraerythrocytic cycle and gametocytogenesis, while S-type rRNAs slowly increase in abundance in mosquito stages starting 3 days post-infection. Salivary gland sporozoites showed an even proportion of all rRNA types. By examining the length distributions of rRNA molecules, we detected an extensive and specific degradation of rRNAs during gametocytogenesis, starting in stage II gametocytes and continuing until the final stages of gametocyte development. We hypothesize that rRNA degradation may be linked to the global translational repression and metabolic quiescence described in stage V gametocytes, similar to mechanisms observed in bacterial and eukaryotic stress responses.IMPORTANCEOur study uses Oxford Nanopore direct RNA sequencing of tightly synchronized blood-stage Plasmodium falciparum parasites to investigate the expression of ribosomal RNAs during asexual and sexual development. P. falciparum utilizes distinct types of rRNA during its development. However, due to the challenges of differentiating these highly similar molecules, their regulation and the mechanism underlying the switch between rRNA types remain unclear. We observe significant rRNA degradation in mature gametocytes, leading us to propose that this potentially leads to a reduced number of functional ribosomes when parasites become quiescent and translation is repressed.

Plasmodium falciparum↗

Nucleotide sequence of the 5'- and 3'- domains for rabbit 18S ribosomal RNA.

By direct RNA sequence analysis we have determined the primary structures of both the 5' and 3' domains for rabbit 18S ribosomal RNA. Purified 18S rRNA was labeled in vitro at either its 5' or 3' terminus with 32P, base-specifically fragmented enzymatically and chemically, and the resulting fragments electrophoretically fractionated by size in adjacent lanes of 140 cm long polyacrylamide sequencing gels run in 90% formamide. A phylogenetic comparison of both the mammalian 5' proximal 400 residues and the 3' distal 301 nucleotides with the previously determined yeast and Xenopus laevis 18S rRNA sequence shows extensive conservation interspersed with tracts having little homology. Clusters of G + C rich sequences are present within the mammalian 5' domain which are entirely absent in both the Xenopus laevis and yeast 18S rRNAs. Most base differences and insertions within the mammalian 18S rRNA when compared with yeast or Xenopus rRNA result in an increase in the G + C content of these regions. We have found nucleotide sequence analysis of the ribosomal RNA directly permits detection of both cistron heterogeneities and mapping of many of the modified bases.

Animals↗

Nucleotide sequence of barley yellow mosaic virus RNA 1: a close evolutionary relationship with potyviruses.

The complete nucleotide sequence of barley yellow mosaic virus (BaYMV) RNA 1 was obtained by analysis of overlapping cDNA clones and by direct RNA sequencing. The sequence is 7632 nucleotides in length, excluding a 3' poly(A) tail. The first AUG codon at nucleotide 172 appeared to be the initiator for a single long open reading frame encoding a protein of 2410 amino acids with an Mr of 270,755. Amino acid sequence comparisons revealed that the BaYMV 270K protein contains three regions upstream of the C-terminal capsid protein which share significant homologies with the cytoplasmic inclusion and two nuclear inclusion proteins of potyviruses thus indicating their similarities in genetic organization. However, the apparent low levels of homology in the corresponding proteins of BaYMV and potyviruses are in contrast with the high conservation among potyviruses. Moreover, our data indicate that BaYMV RNA 1 has no counterpart to the two cistrons located in the 5'-terminal region of the potyvirus genome. Although the data suggest a close evolutionary relationship between BaYMV and potyviruses, the striking differences set BaYMV apart from potyviruses.

Amino Acid Sequence↗

Distant strains of the fish rhabdovirus VHSV maintain a sixth functional cistron which codes for a nonstructural protein of unknown function.

We used direct RNA sequencing to determine the genomic organization of the region downstream from the G gene of viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus. This region contains a gene coding for a protein, identified as nonvirion protein (NV), and the gene coding for the RNA polymerase (L). Thus, VHSV genome organization was confirmed to be 3'-N-P-M-G-NV-L-5'. In both a virulent European (07-71) and an avirulent North American (Makah) strain, the NV gene is transcribed into a small mRNA that codes for a protein of 122 amino acids. It has no significant sequence similarity with the infectious hematopoietic necrosis virus NV protein nor with any other known protein. We expressed the NV protein as a fusion protein with the glutathione S-transferase of Schistosoma japonicum and used the purified fusion protein to immunize rabbits. The rabbit antiserum precipitated from infected cell extracts--and not from noninfected cells or purified virions--a protein of 14 kDa, well in accordance with the expected NV gene product size. The prediction that the NV protein is a nonstructural protein is supported by its absence from mature virions although it is present in infected cells.

Amino Acid Sequence↗

Long-read transcriptomics corrects Trichomonas vaginalis intron annotations and refines transcript-end features.

BACKGROUND: Trichomonas vaginalis causes the most prevalent non-viral sexually transmitted infection worldwide. Despite its large genome (181.5 Mb; 36,310 predicted protein-coding genes in NYU_TvagG3_2), intron annotations remain limited and inconsistently validated. A recent short-read RNA-seq study reported 63 putative active introns, but short reads can misassign splice boundaries and cannot resolve complete transcript structures. METHODS: We integrated Oxford Nanopore direct RNA sequencing (DRS), ONT cDNA long-read sequencing, and Illumina RNA-seq to refine intron annotations, transcript-end features, and UTR boundaries in T. vaginalis. Candidate introns were validated by targeted PCR and Sanger sequencing, and representative splicing events were further assessed using public SRA datasets. RESULTS: Starting from 31 historically annotated introns, motif-guided long-read screening and orthogonal validation identified 17 additional validated introns, increasing the curated set to 48 confirmed introns. Among these 17 events, three were previously unrecognized in the current NYU_TvagG3_2 reference annotation. We also corrected five reported loci, including two false-positive introns, two splice-coordinate misannotations, and one gene-sequence error. DRS further supported transcript termination site mapping, UAAA polyadenylation-signal profiling relative to poly(A) addition sites, and single-molecule poly(A)-tail estimation. StringTie mixed-mode assemblies provided updated UTR boundaries for intron-bearing transcripts and transcripts without curated introns. CONCLUSIONS: This study provides a rigorously validated, long-read-refined resource of intron annotations, UTR boundaries, and UAAA-guided transcript-end features for T. vaginalis, together with a reproducible workflow for non-model protists. These refinements improve the current reference annotation and support future studies of functional genomics, parasite biology, pathogenesis, and diagnostic development.

Trichomonas vaginalis↗

Analysis of genetic information of an insect picorna-like virus, infectious flacherie virus of silkworm: evidence for evolutionary relationships among insect, mammalian and plant picorna(-like) viruses.

We synthesized the cDNAs of an insect picornavirus, infectious flacherie virus of silkworm (IFV), genomic RNA and inserted it into a bacterial plasmid (pUC119). The 9,650 nucleotides (nts) sequence except for the poly(A) tail was obtained from the cloned cDNAs, and the sequence integrity was confirmed by primer extension and direct RNA sequencing. The sequence has a large open reading frame (ORF) of 9,255 nts (3,085 codons) flanked by the short 5' non-coding region (156 nts) and by the rather long 3' non-coding (239 nts). The structural proteins VP3, 4, 1 and 2 were located at the N-terminus of the polyprotein in this order and were preceded by a tentative small peptide. Computer analysis identified the sequences similar to the consensus sequences of 2C (helicase?), 3C (protease), and 3D (RNA polymerase) conserved among mammalian and plant picorna(-like) viruses. In addition, the predicted genome organization of IFV was quite similar to those of picornaviruses. Further analyses of the characteristics of the genome structure and a tentative phylogenetic tree constructed on the basis of the amino acid sequence similarity emphasized the evolutionary relationships among the insect and plant viruses.

Adenoviridae↗

Localization of extensive deletions in the structural genes of two neurotropic variants of murine coronavirus JHM.

The intracellular RNA of two neurotropic variants of the JHM strain of mouse hepatitis virus (MHV) independently isolated from the brain and spinal cord of an infected Wistar Furth rat were compared with that of the parental virus. The mRNAs corresponding to the genes encoding the peplomer (S) and the hemagglutinin-esterase (HE) proteins of the variant viruses were found to be smaller in size. The possible sequence changes were studied by oligonucleotide fingerprinting and direct RNA sequencing. Both variants have a large deletion of 246 amino acids in the carboxy-terminal end of the HE protein. However, this truncated protein was not detected in the infected cells, suggesting either a translational regulation or rapid degradation of the truncated protein in these cells. The variant virus isolated from the spinal cord has a second deletion of 147 amino acids in the amino-terminal half of the S protein. This deletion site corresponds to a hypervariable region where deletions have been frequently noted among MHV variants with different biological properties. These findings suggest that the changes in pathogenic properties of the two neural isolates are associated with drastic alterations of the viral structural glycoproteins.

Base Sequence↗

Complete nucleotide sequence and genomic organization of a primate calicivirus, Pan-1.

The primate calicivirus, Pan-1, was originally isolated from several primate species. It displayed typical calicivirus morphology by electron microscopy. We determined the genomic sequence of Pan-1 by cDNA cloning and direct RNA sequencing. Pan-1 shares a similar genomic organization and a high degree of sequence identity with feline caliciviruses. The Pan-1 genome contains 8,304 nucleotides, plus a poly-A tail, and is longer than any other calicivirus strains with a completely known sequence. The extra sequences of Pan-1 include a unique 424-nucleotide sequence at the 5' end of ORF1, additional amino acids at the N-terminus of the capsid, and a longer 3' UTR.

Animals↗

The yeast Hansenula wingei U3 snoRNA gene contains an intron and its coding sequence co-evolved with the 5' ETS region of the pre-ribosomal RNA.

The 5' external transcribed spacer (ETS) region of the pre-rRNA in Saccharomyces cerevisiae contains a sequence with 10 bp of perfect complementarity to the U3 snoRNA. Base pairing between these sequences has been shown to be required for 18S rRNA synthesis, although interaction over the full 10 bp of complementarity is not required. We have identified the homologous sequence in the 5' ETS from the evolutionarily distant yeast Hansenula wingei; unexpectedly, this shows two sequence changes in the region predicted to base pair to U3. By PCR amplification and direct RNA sequencing, a single type of U3 snoRNA coding sequence was identified in H. wingei. As in the S. cerevisiae U3 snoRNA genes, it is interrupted by an intron with features characteristic of introns spliced in a spliceosome. Consequently, this unusual property is not restricted to the yeast genus Saccharomyces. The introns of the H. wingei and S. cerevisiae U3 genes show strong differences in length and sequence, but are located at the same position in the U3 sequence, immediately upstream of the phylogenetically conserved Box A region. The 3' domains of the H. wingei and S. cerevisiae U3 snoRNAs diverge strongly in primary sequence, but have very similar predicted secondary structures. The 5' domains, expected to play a direct role in pre-ribosomal RNA maturation, are more conserved. The sequence predicted to base pair to the pre-rRNA contains two nucleotide substitutions in H. wingei that restore 10 bp of perfect complementarity to the 5' ETS. This is a strong phylogenetic evidence for the importance of the U3/pre-rRNA interaction.

Base Sequence↗

Initiation, pausing, and termination of transcription in the threonine operon regulatory region of Escherichia coli.

The 6 S leader RNA transcript from the Escherichia coli threonine operon controlling region was synthesized in vitro using purified RNA polymerase and restriction fragment DNA templates. The terminated leader transcript was analyzed by RNase T1 digestion followed by electrophoresis on 20% polyacrylamide, 8 M urea gels. Oligonucleotides of 7, 8, 13, 15, and 35 bases in length were detected and correlated with the known DNA sequence. The kinetics of RNase T1 digestion indicated that the RNA forms extensive secondary structure, especially at the 3'-terminus of the transcript. The sites of transcription initiation were determined by labeling the 5'-end of the transcript with [gamma-32P]ATP or -GTP followed by direct RNA sequencing. The DNA sequence preceding the initiation site shows homology with the equivalent regions of other bacterial and bacteriophage promoters. The transcription termination sites were determined by mapping of the RNase T1 oligonucleotides arising from the 3'-terminus of the transcript. Comparison of the mobilities of the 3'-oligonucleotides with the mobilities of standards on 20% polyacrylamide, 8 M urea gels indicated that the RNA contains a heterogeneous 3'-terminus. The two predominant oligonucleotides were CU7 and CU8. The 3'-terminus of the transcript also contains a region of dyad symmetry immediately preceding a stretch of uridine residues, characteristic of other rho-independent transcripts. In addition, kinetic studies indicated that RNA polymerase pauses approximately 50 base pairs upstream from the site of termination. The pause site appears to be immediately distal to another region of dyad symmetry.

Amino Acid Sequence↗

Sequences at four termini of the giardiavirus double-stranded RNA.

The linear double-stranded RNA (dsRNA) genome of giardiavirus (GLV) was estimated to be 6100 nucleotides (nt) [Wang et al., Proc. Natl. Acad. Sci. USA 90 (1993) 8585-8599]. As the 5'- and 3'-untranslated regions (UTR) of viral genomes are known to contain critical information for viral replication, we reexamine the sequences at all four termini of GLV dsRNA by (i) direct RNA sequencing with RT, (ii) tailing GLV dsRNA with UTP or CTP in addition to ATP in 3' rapid amplification of cDNA ends (RACE) and (iii) adding poly(dG) to the products of primer extension in 5'-RACE. The results confirmed the reported sequence for the 5'-terminus of the GLV sense strand RNA, but uncovered an additional 177 nt at the 3'-terminus. The new study also showed conclusively that there are no protruding overhangs at either terminus of GLV dsRNA.

Base Sequence↗

Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli.

The complete nucleotide sequence of the 16S RNA gene from the rrnB cistron of Escherichia coli has been determined by using three rapid DNA sequencing methods. Nearly all of the structure has been confirmed by two to six independent sequence determinations on both DNA strands. The length of the 16S rRNA chain inferred from the DNA sequence is 1541 nucleotides, in close agreement with previous estimates. We note discrepancies between this sequence and the most recent version of it reported from direct RNA sequencing [Ehresmann, C., Stiegler, P., Carbon, P. & Ebel, J.P. (1977) FEBS Lett. 84, 337-341]. A few of these may be explained by heterogeneity among 16S rRNA sequences from different cistrons. No nucleotide sequences were found in the 16S rRNA gene that cannot be reconciled with RNase digestion products of mature 16S rRNA.

Base Sequence↗

The 5'-terminal sequence of VSV(NJ) (Ogden): is the interaction of the NS protein with the NS binding site responsible for heterotypic interference activity?

The 5'-terminal sequence of VSV(NJ) (Ogden) and VSV(NJ) (Hazelhurst) was compared in an attempt to understand why the defective interfering particle, DI-LT, heterotypically interferes with VSV(NJ) (Ogden) but not with VSV(NJ) (Hazelhurst). The 5'-terminal sequence of VSV(NJ) (Ogden) genomic RNA was determined by direct RNA sequencing and by DNA sequencing of cDNA clones of the 3'-terminal sequence of VSV(NJ) (Ogden) DI particle genome. Primer extension analysis of the 5'-terminus of VSV(NJ) (Ogden) standard genomic RNA confirmed these data. Within the last 47 nucleotides, equivalent to the negative-strand leader RNA, the only nucleotide changes between VSV(NJ) (Ogden) and VSV(NJ) (Hazelhurst) occur between nucleotides 19 and 26, representing part of the putative NS binding region described by Isaac and Keene (J. Virol. 43, 241-249 (1982] for VSV(IND) DI particles. The spacer (S) region, located between the polyadenylation signal of the L gene and the 47th nucleotide of the leader RNA, contains more differences. The polyadenylation signal of the L gene is fully conserved, but the remainder of the L gene region (177 nucleotides) has highly diverged between VSV(NJ) (Ogden) and VSV(NJ) (Hazelhurst). The changes in the NS binding region of the negative-strand leader RNA provide further evidence for the divergent evolution of VSV(NJ) (Ogden) and VSV(NJ) (Hazelhurst). The NS binding region has been implicated as a crucial site for the initiation of RNA transcription and replication. The interaction of the NS protein with this site may determine the ability of DI particles to interfere heterotypically.

Base Sequence↗

Organization and expression of 5S rRNA genes in the parasitic nematode, Brugia malayi.

DNA sequence analysis of genes encoding 5S rRNA in the human parasitic nematode Brugia malayi (B. malayi) indicates a surprising degree of heterogeneity. This variation in coding sequence is not accompanied by corresponding heterogeneity in flanking regions which are highly conserved. Six out of eight potential 5S coding regions differed; of these sequence variants, two were abundant in the B. malayi genome. Direct RNA sequence analysis indicated that one of these abundant variants accounts for most if not all of expressed 5S RNA at two stages of development.

Animals↗

The maize stripe virus major noncapsid protein messenger RNA transcripts contain heterogeneous leader sequences at their 5' termini.

Primer extension analyses and a PCR-based cloning strategy were used to identify and characterize 5' nucleotide sequences on the maize stripe virus (MStV) RNA4 mRNA transcripts encoding the major noncapsid protein (NCP). Direct RNA sequence analysis by primer extension showed that the NCP mRNA transcripts had 10-15 nucleotides beyond the 5' terminus of the MStV RNA4 nucleotide sequence. MStV genomic RNAs isolated from ribonucleoprotein particles (RNPs) lacked the additional 5' nucleotides. cDNA clones representing the 5' region of the mRNA transcripts were constructed, and the nucleotide sequences of the 5' regions were determined for 16 clones. Each was found to have a distinct 10-15 nucleotide sequence immediately 5' of the MStV RNA4 sequence. Eleven of 16 clones had the correct MStV RNA4 5' nucleotide sequence, while five showed minor variations at or near the 5' most MStV RNA4 nucleotide. These characteristics show strong similarities to other viral mRNA transcripts which are synthesized by cap snatching.

Base Sequence↗

A critical appraisal of base-resolution m6A profiling techniques.

N6-methyladenosine (m6A) is the most prevalent internal modification in eukaryotic mRNA, influencing RNA fate and gene regulation. Early antibody-based approaches enabled transcriptome-wide profiling but lacked resolution and quantitative accuracy. Newer approaches now achieve base-resolution m6A detection using improved crosslinking, chemical or enzymatic conversion, and single-molecule sequencing. Antibody-free methods provide quantitative stoichiometry from minimal input, while nanopore direct RNA sequencing offers real-time, single-molecule readouts across entire transcriptomes. Collectively, these methods form a versatile toolkit that integrates global mapping with precise site-level analysis, advancing knowledge of context-dependent m6A regulation in physiology and disease. This review compares their principles, strengths, and limitations to guide method selection and highlight how next-generation epitranscriptomic tools are paving the way for clinical and therapeutic applications.

Humans↗

Cloning and expression of a cDNA copy of the viral K28 killer toxin gene in yeast.

The killer toxin K28, secreted by certain killer strains of the yeast Saccharomyces cerevisiae is genetically encoded by a 1.9 kb double-stranded RNA, M-dsRNA (M28), that is present within the cell as a cytoplasmically inherited virus-like particle (VLP). For stable maintenance and replication, M28-VLPs depend on a second dsRNA virus (LA), which has been shown to encode the major capsid protein (cap) and a capsid-polymerase fusion protein (cap-pol) that provides the toxin-coding M-satellites with their transcription and replicase functions. K28 toxin-coding M28-VLPs were isolated, purified and used in vitro for the synthesis of the single-stranded M28 transcript, which was shown to be of plus strand polarity and to bind to oligo(dT)-cellulose, indicating that M28(+)ssRNA contains an internal A-rich tract. Strand separation of the 1.9 kb M28-dsRNA and direct RNA sequencing of its 3' ends was performed in order to obtain specific DNA oligonucleotides that could be used as primers for cDNA synthesis. The nucleotide sequence of the toxin-coding M28-cDNA identified a single open reading frame (ORF) coding for a polypeptide of 345 amino acids, which contained two potential Kex2p/Kex1p processing sites and three potential sites for protein N-glycosylation. The toxin-coding cDNA was cloned and expressed in sensitive non-killer strains under the control of the yeast PGK promoter. Upon transformation, this construct conferred the complete K28 phenotype, demonstrating that both toxin and immunity determinants are contained within the cloned cDNA. In vitro translational analysis of the M28(+)ssRNA in vitro transcript identified the primary gene product of M28 as a K28 preprotoxin of 38 kDa (M-p38).

Amino Acid Sequence↗