TY - Generic T1 - Genomic analysis of sequence-dependent DNA curvature in Leishmania. Y1 - 2013 A1 - Smircich, Pablo A1 - Forteza, Diego A1 - El-Sayed, Najib M A1 - Garat, Beatriz KW - Chromosome mapping KW - Comparative Genomic Hybridization KW - Computational Biology KW - DNA, Protozoan KW - Genome, Protozoan KW - Genomics KW - HUMANS KW - Leishmania KW - Nucleic Acid Conformation AB -

Leishmania major is a flagellated protozoan parasite of medical importance. Like other members of the Trypanosomatidae family, it possesses unique mechanisms of gene expression such as constitutive polycistronic transcription of directional gene clusters, gene amplification, mRNA trans-splicing, and extensive editing of mitochondrial transcripts. The molecular signals underlying most of these processes remain under investigation. In order to investigate the role of DNA secondary structure signals in gene expression, we carried out a genome-wide in silico analysis of the intrinsic DNA curvature. The L. major genome revealed a lower frequency of high intrinsic curvature regions as well as inter- and intra- chromosomal distribution heterogeneity, when compared to prokaryotic and eukaryotic organisms. Using a novel method aimed at detecting region-integrated intrinsic curvature (RIIC), high DNA curvature was found to be associated with regions implicated in transcription initiation. Those include divergent strand-switch regions between directional gene clusters and regions linked to markers of active transcription initiation such as acetylated H3 histone, TRF4 and SNAP50. These findings suggest a role for DNA curvature in transcription initiation in Leishmania supporting the relevance of DNA secondary structures signals.

JA - PLoS One VL - 8 CP - 4 M3 - 10.1371/journal.pone.0063068 ER - TY - JOUR T1 - Spliceosomal small nuclear RNA genes in 11 insect genomes. JF - RNA Y1 - 2007 A1 - Mount, Stephen M A1 - Gotea, Valer A1 - Lin, Chiao-Feng A1 - Hernandez, Kristina A1 - Makalowski, Wojciech KW - Animals KW - Base Sequence KW - Bees KW - Computational Biology KW - Diptera KW - Evolution, Molecular KW - Genes, Insect KW - Genome, Insect KW - Molecular Sequence Data KW - Nucleic Acid Conformation KW - Phylogeny KW - Promoter Regions, Genetic KW - RNA Splicing KW - RNA, Small Nuclear KW - Sequence Analysis, RNA KW - Spliceosomes AB -

The removal of introns from the primary transcripts of protein-coding genes is accomplished by the spliceosome, a large macromolecular complex of which small nuclear RNAs (snRNAs) are crucial components. Following the recent sequencing of the honeybee (Apis mellifera) genome, we used various computational methods, ranging from sequence similarity search to RNA secondary structure prediction, to search for putative snRNA genes (including their promoters) and to examine their pattern of conservation among 11 available insect genomes (A. mellifera, Tribolium castaneum, Bombyx mori, Anopheles gambiae, Aedes aegypti, and six Drosophila species). We identified candidates for all nine spliceosomal snRNA genes in all the analyzed genomes. All the species contain a similar number of snRNA genes, with the exception of A. aegypti, whose genome contains more U1, U2, and U5 genes, and A. mellifera, whose genome contains fewer U2 and U5 genes. We found that snRNA genes are generally more closely related to homologs within the same genus than to those in other genera. Promoter regions for all spliceosomal snRNA genes within each insect species share similar sequence motifs that are likely to correspond to the PSEA (proximal sequence element A), the binding site for snRNA activating protein complex, but these promoter elements vary in sequence among the five insect families surveyed here. In contrast to the other insect species investigated, Dipteran genomes are characterized by a rapid evolution (or loss) of components of the U12 spliceosome and a striking loss of U12-type introns.

VL - 13 CP - 1 M3 - 10.1261/rna.259207 ER - TY - JOUR T1 - Suppressor U1 snRNAs in Drosophila. JF - Genetics Y1 - 1994 A1 - Lo, P C A1 - Roy, D A1 - Mount, S M KW - Alternative Splicing KW - Animals KW - Base Sequence KW - Cell Line KW - Cell Nucleus KW - DNA Primers KW - Drosophila melanogaster KW - Female KW - Genes, Suppressor KW - Genetic Variation KW - GENOTYPE KW - Introns KW - Male KW - Molecular Sequence Data KW - Mutagenesis, Site-Directed KW - Nucleic Acid Conformation KW - Oligodeoxyribonucleotides KW - PHENOTYPE KW - Recombinant Proteins KW - Ribonucleoprotein, U1 Small Nuclear KW - RNA, Small Nuclear KW - Transfection KW - Transformation, Genetic AB -

Although the role of U1 small nuclear RNAs (snRNAs) in 5' splice site recognition is well established, suppressor U1 snRNAs active in intact multicellular animals have been lacking. Here we describe suppression of a 5' splice site mutation in the Drosophila melanogaster white gene (wDR18) by compensatory changes in U1 snRNA. Mutation of positions -1 and +6 of the 5' splice site of the second intron (ACG[GTGAGT to ACC]GTGAGC) results in the accumulation of RNA retaining this 74-nucleotide intron in both transfected cells and transgenic flies. U1-3G, a suppressor U1 snRNA which restores base-pairing at position +6 of the mutant intron, increases the ratio of spliced to unspliced wDR18 RNA up to fivefold in transfected Schneider cells and increases eye pigmentation in wDR18 flies. U1-9G, which targets position -1, suppresses wDR18 in transfected cells less well. U1-3G,9G has the same effect as U1-3G although it accumulates to lower levels. Suppression of wDR18 has revealed that the U1b embryonic variant (G134 to U) is active in Schneider cells and pupal eye discs. However, the combination of 9G with 134U leads to reduced accumulation of both U1b-9G and U1b-3G,9G, possibly because nucleotides 9 and 134 both participate in a potential long-range intramolecular base-pairing interaction. High levels of functional U1-3G suppressor reduce both viability and fertility in transformed flies. These results show that, despite the difficulties inherent in stably altering splice site selection in multicellular organisms, it is possible to obtain suppressor U1 snRNAs in flies.

VL - 138 CP - 2 ER - TY - JOUR T1 - Drosophila melanogaster genes for U1 snRNA variants and their expression during development. JF - Nucleic Acids Res Y1 - 1990 A1 - Lo, P C A1 - Mount, S M KW - Animals KW - Base Sequence KW - Blotting, Southern KW - Cloning, Molecular KW - Drosophila melanogaster KW - Gene Expression Regulation KW - genes KW - Genetic Variation KW - Molecular Sequence Data KW - Nucleic Acid Conformation KW - Pseudogenes KW - Restriction Mapping KW - RNA, Small Nuclear AB -

We have cloned and characterized a complete set of seven U1-related sequences from Drosophila melanogaster. These sequences are located at the three cytogenetic loci 21D, 82E, and 95C. Three of these sequences have been previously studied: one U1 gene at 21D which encodes the prototype U1 sequence (U1a), one U1 gene at 82E which encodes a U1 variant with a single nucleotide substitution (U1b), and a pseudogene at 82E. The four previously uncharacterized genes are another U1b gene at 82E, two additional U1a genes at 95C, and a U1 gene at 95C which encodes a new variant (U1c) with a distinct single nucleotide change relative to U1a. Three blocks of 5' flanking sequence similarity are common to all six full length genes. Using specific primer extension assays, we have observed that the U1b RNA is expressed in Drosophila Kc cells and is associated with snRNP proteins, suggesting that the U1b-containing snRNP particles are able to participate in the process of pre-mRNA splicing. We have also examined the expression throughout Drosophila development of the two U1 variants relative to the prototype sequence. The U1c variant is undetectable by our methods, while the U1b variant exhibits a primarily embryonic pattern reminiscent of the expression of certain U1 variants in sea urchin, Xenopus, and mouse.

VL - 18 CP - 23 ER - TY - JOUR T1 - Pseudogenes for human small nuclear RNA U3 appear to arise by integration of self-primed reverse transcripts of the RNA into new chromosomal sites. JF - Cell Y1 - 1983 A1 - Bernstein, L B A1 - Mount, S M A1 - Weiner, A M KW - Animals KW - Base Sequence KW - DNA KW - genes KW - HUMANS KW - Nucleic Acid Conformation KW - Rats KW - Recombination, Genetic KW - Repetitive Sequences, Nucleic Acid KW - RNA KW - RNA, Small Nuclear KW - RNA-Directed DNA Polymerase KW - Templates, Genetic KW - Transcription, Genetic AB -

We find that both human and rat U3 snRNA can function as self-priming templates for AMV reverse transcriptase in vitro. The 74 base cDNA is primed by the 3' end of intact U3 snRNA, and spans the characteristically truncated 69 or 70 base U3 sequence found in four different human U3 pseudogenes. The ability of human and rat U3 snRNA to self-prime is consistent with a U3 secondary structure model derived by a comparison between rat U3 snRNA and the homologous D2 snRNA from Dictyostelium discoideum. We propose that U3 pseudogenes are generated in vivo by integration of a self-primed cDNA copy of U3 snRNA at new chromosomal sites. We also consider the possibility that the same cDNA mediates gene conversion at the 5' end of bona fide U3 genes where, over the entire region spanned by the U3 cDNA, the two rat U3 sequence variants U3A and U3B are identical.

VL - 32 CP - 2 ER - TY - JOUR T1 - Small ribonucleoproteins from eukaryotes: structures and roles in RNA biogenesis. JF - Cold Spring Harb Symp Quant Biol Y1 - 1983 A1 - Steitz, J A A1 - Wolin, S L A1 - Rinke, J A1 - Pettersson, I A1 - Mount, S M A1 - Lerner, E A A1 - Hinterberger, M A1 - Gottlieb, E KW - Animals KW - Base Sequence KW - HeLa Cells KW - HUMANS KW - Mice KW - Molecular Weight KW - Nucleic Acid Conformation KW - Nucleic Acid Hybridization KW - Nucleoproteins KW - Ribonucleoproteins KW - Ribonucleoproteins, Small Nuclear KW - RNA Polymerase III KW - Transcription, Genetic VL - 47 Pt 2 ER - TY - JOUR T1 - Sequence of U1 RNA from Drosophila melanogaster: implications for U1 secondary structure and possible involvement in splicing. JF - Nucleic Acids Res Y1 - 1981 A1 - Mount, S M A1 - Steitz, J A KW - Animals KW - Antibodies KW - Autoimmune Diseases KW - Base Sequence KW - Cells, Cultured KW - Cloning, Molecular KW - Drosophila melanogaster KW - HUMANS KW - Lupus Erythematosus, Systemic KW - Nucleic Acid Conformation KW - Nucleic Acid Hybridization KW - Ribonuclease T1 KW - Ribonucleoproteins KW - RNA KW - RNA, Small Nuclear AB -

U1 RNA from cultured Drosophila melanogaster cells (Kc) was identified by its ability to be recognized, as an RNP, by anti-(U1)RNP antibodies from human lupus patients. Its sequence was deduced largely from direct analysis of the RNA molecule and then confirmed by DNA sequence determinations on a genomic clone isolated by hybridization to Drosophila U1 RNA. The Drosophila U1 RNA sequence exhibits 72% agreement with human U1 RNA. Nucleotides 3-11, which are complementary to the entire consensus sequence for donor (5') splice junctions in hnRNA, and to part of the acceptor (3') consensus, are exactly conserved. However, nucleotides 14-21, postulated to interact only with acceptor junctions, differ. Comparison of the Drosophila U1 sequence with vertebrate U1 sequences allows a particular secondary structure model to be preferred over others. These results are consistent with the hypothesis that U1 snRNPs are involved in splicing, but suggest specific modifications of the model detailing molecular interactions between U1 RNA and hnRNA during the splicing reaction.

VL - 9 CP - 23 ER -