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 - Sex-lethal splicing autoregulation in vivo: interactions between SEX-LETHAL, the U1 snRNP and U2AF underlie male exon skipping. JF - Development Y1 - 2003 A1 - Nagengast, Alexis A A1 - Stitzinger, Shane M A1 - Tseng, Chin-Hsiu A1 - Mount, Stephen M A1 - Salz, Helen K KW - Alternative Splicing KW - Amino Acid Sequence KW - Animals KW - Animals, Genetically Modified KW - Drosophila melanogaster KW - Drosophila Proteins KW - Exons KW - Female KW - Gene Expression Regulation, Developmental KW - Genes, Insect KW - Homeostasis KW - Male KW - Models, Genetic KW - Molecular Sequence Data KW - Nuclear Proteins KW - Point Mutation KW - Ribonucleoprotein, U1 Small Nuclear KW - Ribonucleoproteins KW - RNA Splicing KW - RNA-Binding Proteins KW - Sequence Homology, Amino Acid KW - Sex Differentiation AB -

Alternative splicing of the Sex-lethal pre-mRNA has long served as a model example of a regulated splicing event, yet the mechanism by which the female-specific SEX-LETHAL RNA-binding protein prevents inclusion of the translation-terminating male exon is not understood. Thus far, the only general splicing factor for which there is in vivo evidence for a regulatory role in the pathway leading to male-exon skipping is sans-fille (snf), a protein component of the spliceosomal U1 and U2 snRNPs. Its role, however, has remained enigmatic because of questions about whether SNF acts as part of an intact snRNP or a free protein. We provide evidence that SEX-LETHAL interacts with SANS-FILLE in the context of the U1 snRNP, through the characterization of a point mutation that interferes with both assembly into the U1 snRNP and complex formation with SEX-LETHAL. Moreover, we find that SEX-LETHAL associates with other integral U1 snRNP components, and we provide genetic evidence to support the biological relevance of these physical interactions. Similar genetic and biochemical approaches also link SEX-LETHAL with the heterodimeric splicing factor, U2AF. These studies point specifically to a mechanism by which SEX-LETHAL represses splicing by interacting with these key splicing factors at both ends of the regulated male exon. Moreover, because U2AF and the U1 snRNP are only associated transiently with the pre-mRNA during the course of spliceosome assembly, our studies are difficult to reconcile with the current model that proposes that the SEX-LETHAL blocks splicing at the second catalytic step, and instead argue that the SEX-LETHAL protein acts after splice site recognition, but before catalysis begins.

VL - 130 CP - 3 ER - TY - JOUR T1 - Genomic sequence, splicing, and gene annotation. JF - Am J Hum Genet Y1 - 2000 A1 - Mount, S M KW - Animals KW - Consensus Sequence KW - Exons KW - genes KW - Genome KW - Genomics KW - HUMANS KW - Nucleotides KW - Regulatory Sequences, Nucleic Acid KW - RNA Splice Sites KW - RNA Splicing KW - Untranslated Regions VL - 67 CP - 4 M3 - 10.1086/303098 ER - TY - JOUR T1 - Genetic depletion reveals an essential role for an SR protein splicing factor in vertebrate cells. JF - Bioessays Y1 - 1997 A1 - Mount, S M KW - Amino Acid Sequence KW - Animals KW - Molecular Sequence Data KW - Nuclear Proteins KW - RNA Splicing KW - RNA-Binding Proteins KW - Serine-Arginine Splicing Factors KW - Vertebrates AB -

SR proteins are essential for the splicing of messenger RNA precursors in vitro, where they also alter splice site selection in a concentration-dependent manner. Although experiments involving overexpression or dominant mutations have confirmed that these proteins can influence RNA processing decisions in vivo, similar results with loss-of-function mutations have been lacking. Now, a system for genetic depletion of the chicken B cell line DT40 has revealed that the SR protein ASF/SF2 (alternative splicing factor/splicing factor 2) is essential for viability in these cells(1). This study opens the way for a complete functional dissection of this protein, and other SR proteins, in vivo.

VL - 19 CP - 3 M3 - 10.1002/bies.950190302 ER - TY - JOUR T1 - AT-AC introns: an ATtACk on dogma. JF - Science Y1 - 1996 A1 - Mount, S M KW - Animals KW - Base Composition KW - Base Sequence KW - Consensus Sequence KW - HUMANS KW - Introns KW - Molecular Sequence Data KW - Mutation KW - RNA Precursors KW - RNA Splicing KW - RNA, Small Nuclear KW - Spliceosomes VL - 271 CP - 5256 ER - TY - JOUR T1 - Genetic enhancement of RNA-processing defects by a dominant mutation in B52, the Drosophila gene for an SR protein splicing factor. JF - Mol Cell Biol Y1 - 1995 A1 - Peng, X A1 - Mount, S M KW - Alleles KW - Amino Acid Sequence KW - Animals KW - Base Sequence KW - DNA Primers KW - Drosophila melanogaster KW - Drosophila Proteins KW - Frameshift Mutation KW - Genes, Dominant KW - Genes, Insect KW - Molecular Sequence Data KW - Nuclear Proteins KW - Phosphoproteins KW - Point Mutation KW - Protein Structure, Tertiary KW - Proteins KW - RNA Splicing KW - RNA-Binding Proteins KW - Sequence Deletion KW - Sex Determination Analysis AB -

SR proteins are essential for pre-mRNA splicing in vitro, act early in the splicing pathway, and can influence alternative splice site choice. Here we describe the isolation of both dominant and loss-of-function alleles of B52, the gene for a Drosophila SR protein. The allele B52ED was identified as a dominant second-site enhancer of white-apricot (wa), a retrotransposon insertion in the second intron of the eye pigmentation gene white with a complex RNA-processing defect. B52ED also exaggerates the mutant phenotype of a distinct white allele carrying a 5' splice site mutation (wDR18), and alters the pattern of sex-specific splicing at doublesex under sensitized conditions, so that the male-specific splice is favored. In addition to being a dominant enhancer of these RNA-processing defects, B52ED is a recessive lethal allele that fails to complement other lethal alleles of B52. Comparison of B52ED with the B52+ allele from which it was derived revealed a single change in a conserved amino acid in the beta 4 strand of the first RNA-binding domain of B52, which suggests that altered RNA binding is responsible for the dominant phenotype. Reversion of the B52ED dominant allele with X rays led to the isolation of a B52 null allele. Together, these results indicate a critical role for the SR protein B52 in pre-mRNA splicing in vivo.

VL - 15 CP - 11 ER - TY - JOUR T1 - Localization of sequences required for size-specific splicing of a small Drosophila intron in vitro. JF - J Mol Biol Y1 - 1995 A1 - Guo, M A1 - Mount, S M KW - Animals KW - Base Sequence KW - Cell Line KW - DNA KW - Drosophila KW - Genes, Insect KW - HeLa Cells KW - HUMANS KW - Introns KW - Molecular Sequence Data KW - Myosin Heavy Chains KW - RNA Splicing KW - Species Specificity AB -

Many introns in Drosophila and other invertebrates are less than 80 nucleotides in length, too small to be recognized by the vertebrate splicing machinery. Comparison of nuclear splicing extracts from human HeLa and Drosophila Kc cells has revealed species-specificity, consistent with the observed size differences. Here we present additional results with the 68 nucleotide fifth intron of the Drosophila myosin heavy chain gene. As observed with the 74 nucleotide second intron of the Drosophila white gene, the wild-type myosin intron is accurately spliced in a homologous extract, and increasing the size by 16 nucleotides both eliminates splicing in the Drosophila extract and allows accurate splicing in the human extract. In contrast to previous results, however, an upstream cryptic 5' splice site is activated when the wild-type myosin intron is tested in a human HeLa cell nuclear extract, resulting in the removal of a 98 nucleotide intron. The size dependence of splicing in Drosophila extracts is also intron-specific; we noted that a naturally larger (150 nucleotide) intron from the ftz gene is efficiently spliced in Kc cell extracts that do not splice enlarged introns (of 84, 90, 150 or 350 nucleotides) derived from the 74 nucleotide white intron. Here, we have exploited that observation, using a series of hybrid introns to show that a region of 46 nucleotides at the 3' end of the white intron is sufficient to confer the species-specific size effect. At least two sequence elements within this region, yet distinct from previously described branchpoint and pyrimidine tract signals, are required for efficient splicing of small hybrid introns in vitro.

VL - 253 CP - 3 M3 - 10.1006/jmbi.1995.0564 ER - TY - JOUR T1 - Species-specific signals for the splicing of a short Drosophila intron in vitro. JF - Mol Cell Biol Y1 - 1993 A1 - Guo, M A1 - Lo, P C A1 - Mount, S M KW - Animals KW - Base Sequence KW - Cell Nucleus KW - Consensus Sequence KW - DNA KW - DNA Transposable Elements KW - Drosophila KW - Drosophila Proteins KW - Electrophoresis, Polyacrylamide Gel KW - HeLa Cells KW - HUMANS KW - Introns KW - Molecular Sequence Data KW - Mutation KW - Peptide Hydrolases KW - Proteins KW - Regulatory Sequences, Nucleic Acid KW - Retroelements KW - RNA Splicing KW - Species Specificity AB -

The effects of branchpoint sequence, the pyrimidine stretch, and intron size on the splicing efficiency of the Drosophila white gene second intron were examined in nuclear extracts from Drosophila and human cells. This 74-nucleotide intron is typical of many Drosophila introns in that it lacks a significant pyrimidine stretch and is below the minimum size required for splicing in human nuclear extracts. Alteration of sequences of adjacent to the 3' splice site to create a pyrimidine stretch was necessary for splicing in human, but not Drosophila, extracts. Increasing the size of this intron with insertions between the 5' splice site and the branchpoint greatly reduced the efficiency of splicing of introns longer than 79 nucleotides in Drosophila extracts but had an opposite effect in human extracts, in which introns longer than 78 nucleotides were spliced with much greater efficiency. The white-apricot copia insertion is immediately adjacent to the branchpoint normally used in the splicing of this intron, and a copia long terminal repeat insertion prevents splicing in Drosophila, but not human, extracts. However, a consensus branchpoint does not restore the splicing of introns containing the copia long terminal repeat, and alteration of the wild-type branchpoint sequence alone does not eliminate splicing. These results demonstrate species specificity of splicing signals, particularly pyrimidine stretch and size requirements, and raise the possibility that variant mechanisms not found in mammals may operate in the splicing of small introns in Drosophila and possibly other species.

VL - 13 CP - 2 ER - TY - JOUR T1 - Splicing signals in Drosophila: intron size, information content, and consensus sequences. JF - Nucleic Acids Res Y1 - 1992 A1 - Mount, S M A1 - Burks, C A1 - Hertz, G A1 - Stormo, G D A1 - White, O A1 - Fields, C KW - Animals KW - Base Sequence KW - Consensus Sequence KW - Databases, Factual KW - Drosophila KW - Introns KW - Molecular Sequence Data KW - RNA Splicing KW - RNA, Messenger KW - software AB -

A database of 209 Drosophila introns was extracted from Genbank (release number 64.0) and examined by a number of methods in order to characterize features that might serve as signals for messenger RNA splicing. A tight distribution of sizes was observed: while the smallest introns in the database are 51 nucleotides, more than half are less than 80 nucleotides in length, and most of these have lengths in the range of 59-67 nucleotides. Drosophila splice sites found in large and small introns differ in only minor ways from each other and from those found in vertebrate introns. However, larger introns have greater pyrimidine-richness in the region between 11 and 21 nucleotides upstream of 3' splice sites. The Drosophila branchpoint consensus matrix resembles C T A A T (in which branch formation occurs at the underlined A), and differs from the corresponding mammalian signal in the absence of G at the position immediately preceding the branchpoint. The distribution of occurrences of this sequence suggests a minimum distance between 5' splice sites and branchpoints of about 38 nucleotides, and a minimum distance between 3' splice sites and branchpoints of 15 nucleotides. The methods we have used detect no information in exon sequences other than in the few nucleotides immediately adjacent to the splice sites. However, Drosophila resembles many other species in that there is a discontinuity in A + T content between exons and introns, which are A + T rich.

VL - 20 CP - 16 ER - TY - JOUR T1 - RNA processing. Sequences that signal where to splice. JF - Nature Y1 - 1983 A1 - Mount, S M KW - Base Sequence KW - RNA Splicing KW - Saccharomyces cerevisiae VL - 304 CP - 5924 ER - TY - JOUR T1 - Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein. JF - Cell Y1 - 1983 A1 - Padgett, R A A1 - Mount, S M A1 - Steitz, J A A1 - Sharp, P A KW - Adenoviruses, Human KW - Antigens KW - Autoantigens KW - Base Sequence KW - Cell Extracts KW - HeLa Cells KW - HUMANS KW - Immune Sera KW - Nucleic Acid Precursors KW - Ribonucleoproteins KW - Ribonucleoproteins, Small Nuclear KW - RNA KW - RNA Precursors KW - RNA Splicing KW - RNA, Messenger KW - RNA, Small Cytoplasmic KW - RNA, Viral KW - Transcription, Genetic AB -

A mouse monoclonal antibody and human autoimmune sera directed against various classes of small ribonucleoprotein particles have been tested for inhibition of mRNA splicing in a soluble in vitro system. The splicing of the first and second leader exons of adenovirus late RNA was inhibited only by those sera that reacted with U1 RNP. Both U1 RNP-specific human autoimmune serum and sera directed against the Sm class of small nuclear RNPs, including a mouse monoclonal antibody, specifically inhibited splicing. Antisera specific for U2 RNP had no effect on splicing nor did antisera specific for the La or Ro class of small RNPs. These results suggest that U1 RNP is essential for the splicing of mRNA precursors.

VL - 35 CP - 1 ER - TY - JOUR T1 - The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro. JF - Cell Y1 - 1983 A1 - Mount, S M A1 - Pettersson, I A1 - Hinterberger, M A1 - Karmas, A A1 - Steitz, J A KW - Base Sequence KW - DNA-Directed RNA Polymerases KW - HUMANS KW - Nucleoproteins KW - Ribonuclease T1 KW - Ribonucleoproteins KW - Ribonucleoproteins, Small Nuclear KW - RNA KW - RNA Splicing KW - T-Phages AB -

The ability of purified U1 small nuclear RNA-protein complexes (U1 snRNPs) to bind in vitro to two RNAs transcribed from recombinant DNA clones by bacteriophage T7 RNA polymerase has been studied. A transcript which contains sequences corresponding to the small intron and flanking exons of the major mouse beta-globin gene is bound in marked preference to an RNA devoid of splice site sequences. The site of U1 snRNP binding to the globin RNA has been defined by T1 ribonuclease digestion of the RNA-U1 snRNP complex. A 15-17-nucleotide region, including the 5' splice site, remains undigested and complexed with the snRNP such that it can be co-precipitated by antibodies directed against the U1 snRNP. Partial proteinase K digestion of the U1 snRNP abolishes interaction with the globin RNA, indicating that the snRNP proteins contribute significantly to RNA binding. No RNA cleavage, splicing, or recognition of the 3' splice site by U1 snRNPs has been detected. Our results are discussed in terms of the probable role of U1 snRNPs in the messenger RNA splicing of eucaryotic cell nuclei.

VL - 33 CP - 2 ER - TY - JOUR T1 - A catalogue of splice junction sequences. JF - Nucleic Acids Res Y1 - 1982 A1 - Mount, S M KW - Animals KW - Base Sequence KW - genes KW - Genes, Viral KW - HUMANS KW - Repetitive Sequences, Nucleic Acid KW - RNA Splicing KW - Species Specificity AB -

Splice junction sequences from a large number of nuclear and viral genes encoding protein have been collected. The sequence CAAG/GTAGAGT was found to be a consensus of 139 exon-intron boundaries (or donor sequences) and (TC)nNCTAG/G was found to be a consensus of 130 intron-exon boundaries (or acceptor sequences). The possible role of splice junction sequences as signals for processing is discussed.

VL - 10 CP - 2 ER -