Plant pre-m RNA splicing and splicing components

1993 ◽  
Vol 342 (1301) ◽  
pp. 217-224 ◽  
Keyword(s):  
Transgenic Plants ◽  
Rna Splicing ◽  
Specific Protein ◽  
Messenger Rnas ◽  
Rna Helicases ◽  
Gene Encoding ◽  
Small Nuclear Ribonucleoprotein ◽  
Genes Encoding ◽  
Snrnp Protein

Pre-mRNA splicing or the removal of introns from precursor messenger RNAs depends on the accurate recognition of intron sequences by the plant splicing machinery. The major components of this machinery are small nuclear ribonucleoprotein protein particles (snRNPs) which consist of snRNAs and snRNP proteins. We have analysed various aspects of intron sequence and structure in relation to splice site selection and splicing efficiency and we have cloned snRNA genes and a gene encoding the snRNP protein, U2B". In the absence of an in vitro splicing system for plants, transient expression in protoplasts and stable plant transform ations have been used to analyse splicing of intron constructs. We aim to address the function of the UsnRNP-specific protein, U2B", via the production of transgenic plants expressing antisense U2B" transcripts and epitope-tagged U2B" protein. In addition, we have cloned genes encoding other proteins which potentially interact with RNA, such as RNA helicases, and strategies involving transgenic plants are being developed to analyse their function.

Transgene expression and agronomic improvement of rice

1993 ◽  
Vol 342 (1301) ◽  
pp. 197-201 ◽  
Keyword(s):  
Transgenic Plants ◽  
Triosephosphate Isomerase ◽  
Viral Infections ◽  
Specific Protein ◽  
Coding Region ◽  
Gene Encoding ◽  
Gus Reporter ◽  
Genes Encoding ◽  
Flanking Region ◽  
Viral Sequences

A reliable system for transformation and regeneration of rice protoplasts yielding fertile transgenic plants has been established. After co-electroporation of DNAs encoding a selectable marker and the gene of interest, protoplasts are regenerated to yield fertile plants. To date more than 70 different genes of interest have been successfully introduced and their patterns of expression are being studied. As in the case of dicot plants transformed by the Ti-plasm id vector approach, integration and expression appear to be stable in the transgenic monocots over several generations. Detailed com parative studies on gene expression in rice are underway using promoters for triosephosphate isomerase, a ubiquitously expressed gene encoding a cytosolic enzyme vital in the glycolytic cycle, two genes encoding members of the cyclophilin family, peptidyl-prolyl cis-trans -isomerases that are abundant in meristematic regions and are thought to participate in the correct folding of nascent proteins, and a gene encoding a tissue (root)- specific protein. Initial analyses suggest that the spatial expression of these genes in transgenic plants, using GUS reporter constructs, appears to be very sensitive to the nature of the 3' flanking region present in the gene construct. Constructs containing a coding region for arcelin, a bean seed protein with putative anti-insecticidal properties, and others containing viral sequences that may provide novel approaches for protection against tungro and other viral infections have been introduced into rice plants.

scholarly journals Interaction between the Negative Regulator of Splicing Element and a 3′ Splice Site: Requirement for U1 Small Nuclear Ribonucleoprotein and the 3′ Splice Site Branch Point/Pyrimidine Tract

1999 ◽  
Vol 73 (3) ◽  
pp. 2394-2400 ◽  
Author(s):  
Craig R. Cook ◽  
Mark T. McNally
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Rna Splicing ◽  
Nuclear Extract ◽  
Negative Regulator ◽  
Rous Sarcoma ◽  
Small Nuclear Ribonucleoprotein ◽  
A Minor

ABSTRACT The negative regulator of splicing (NRS) from Rous sarcoma virus suppresses viral RNA splicing and is one of several ciselements that account for the accumulation of large amounts of unspliced RNA for use as gag-pol mRNA and progeny virion genomic RNA. The NRS can also inhibit splicing of heterologous introns in vivo and in vitro. Previous data showed that the splicing factors SF2/ASF and U1, U2, and U11 small nuclear ribonucleoproteins (snRNPs) bind the NRS, and a correlation was established between SF2/ASF and U11 binding and activity, suggesting that these factors are important for function. These observations, and the finding that a large spliceosome-like complex (NRS-C) assembles on NRS RNA in nuclear extract, led to the proposal that the NRS is recognized as a minor-class 5′ splice site. One model to explain NRS splicing inhibition holds that the NRS interacts nonproductively with and sequesters U2-dependent 3′ splice sites. In this study, we provide evidence that the NRS interacts with an adenovirus 3′ splice site. The interaction was dependent on the integrity of the branch point and pyrimidine tract of the 3′ splice site, and it was sensitive to a mutation that was previously shown to abolish U11 snRNP binding and NRS function. However, further mutational analyses of NRS sequences have identified a U1 binding site that overlaps the U11 site, and the interaction with the 3′ splice site correlated with U1, not U11, binding. These results show that the NRS can interact with a 3′ splice site and suggest that U1 is of primary importance for NRS splicing inhibition.

m3G cap hypermethylation of U1 small nuclear ribonucleoprotein (snRNP) in vitro: evidence that the U1 small nuclear RNA-(guanosine-N2)-methyltransferase is a non-snRNP cytoplasmic protein that requires a binding site on the Sm core domain

1994 ◽  
Vol 14 (6) ◽  
pp. 4160-4172
Author(s):  
G Plessel ◽  
U Fischer ◽  
R Lührmann
Keyword(s):  
Nuclear Transport ◽  
Iodoacetic Acid ◽  
Small Nuclear Rna ◽  
Vitro Assay ◽  
Core Domain ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrnp ◽  
Snrnp Protein ◽  
Localization Signal

The RNA components of small nuclear ribonucleoproteins (U snRNPs) possess a characteristic 5'-terminal trimethylguanosine cap structure (m3G cap). This cap is an important component of the nuclear localization signal of U snRNPs. It arises by hypermethylation of a cotranscriptionally added m7G cap. Here we describe an in vitro assay for the hypermethylation, which employs U snRNP particles reconstituted in vitro from purified components and subsequent analysis by m3G cap-specific immunoprecipitation. Complementation studies in vitro revealed that both cytosol and S-adenosylmethionine are required for the hypermethylation of an m7G-capped U1 snRNP reconstituted in vitro, indicating that the U1 snRNA-(guanosine-N2)-methyltransferase is a trans-active non-snRNP protein. Chemical modification revealed one cytoplasmic component required for hypermethylation and one located on the snRNP: these components have different patterns of sensitivity to modification by N-ethylmaleimide and iodoacetic acid (IAA). In the presence of cytosol and S-adenosylmethionine, an intact Sm core domain is a necessary and sufficient substrate for cap hypermethylation. These data, together with our observation that isolated native U1 snRNPs but not naked U1 RNA inhibit the trimethylation of in vitro-reconstituted U1 snRNP, indicate that the Sm core binds the methyltransferase specifically. Moreover, isolated native U2 snRNP also inhibits trimethylation of U1 snRNP, suggesting that other Sm-class U snRNPs might share the same methyltransferase. IAA modification of m7G-capped U1 snRNPs inhibited hypermethylation when they were microinjected into Xenopus oocytes and consequently also inhibited nuclear import. In contrast, modification with IAA of m3G-capped U1 snRNPs reconstituted in vitro did not interfere with their nuclear transport in oocytes. These data suggest that m3G cap formation and nuclear transport of U1 snRNPs are mediated by distinct factors, which require distinct binding sites on the Sm core of U1 snRNP.

scholarly journals The yeast PRP6 gene encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein, and the PRP9 gene encodes a protein required for U2 snRNP binding.

1990 ◽  
Vol 10 (12) ◽  
pp. 6417-6425 ◽  
Author(s):  
N Abovich ◽  
P Legrain ◽  
M Rosbash
Keyword(s):  
Temperature Sensitive ◽  
Ribonucleoprotein Particle ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Snrnp Protein ◽  
Yeast Genes ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

PRP6 and PRP9 are two yeast genes involved in pre-mRNA splicing. Incubation at 37 degrees C of strains that carry temperature-sensitive mutations at these loci inhibits splicing, and in vivo experiments suggested that they might be involved in commitment complex formation (P. Legrain and M. Rosbash, Cell 57:573-583, 1989). To examine the specific role that the PRP6 and PRP9 products may play in splicing or pre-mRNA transport to the cytoplasm, we have characterized in vitro splicing and spliceosome assembly in extracts derived from prp6 and prp9 mutant strains. We have also characterized RNAs that are specifically immunoprecipitated with the PRP6 and PRP9 proteins. Both approaches indicate that PRP6 encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein and that the PRP9 protein is required for a stable U2 snRNP-substrate interaction. The results are discussed with reference to the previously observed in vivo phenotypes of these mutants.

scholarly journals Comprehensive database and evolutionary dynamics of U12-type introns

2020 ◽  
Author(s):  
Devlin C Moyer ◽  
Graham E Larue ◽  
Courtney E Hershberger ◽  
Scott W Roy ◽  
Richard A Padgett
Keyword(s):  
Rna Splicing ◽  
Evolutionary Dynamics ◽  
Messenger Rnas ◽  
Spliceosomal Introns ◽  
Conversion Model ◽  
Nuclear Maturation ◽  
Small Nuclear Ribonucleoprotein ◽  
Associated Proteins ◽  
Non Coding Rnas ◽  
Nuclear Ribonucleoprotein

Abstract During nuclear maturation of most eukaryotic pre-messenger RNAs and long non-coding RNAs, introns are removed through the process of RNA splicing. Different classes of introns are excised by the U2-type or the U12-type spliceosomes, large complexes of small nuclear ribonucleoprotein particles and associated proteins. We created intronIC, a program for assigning intron class to all introns in a given genome, and used it on 24 eukaryotic genomes to create the Intron Annotation and Orthology Database (IAOD). We then used the data in the IAOD to revisit several hypotheses concerning the evolution of the two classes of spliceosomal introns, finding support for the class conversion model explaining the low abundance of U12-type introns in modern genomes.

scholarly journals m3G cap hypermethylation of U1 small nuclear ribonucleoprotein (snRNP) in vitro: evidence that the U1 small nuclear RNA-(guanosine-N2)-methyltransferase is a non-snRNP cytoplasmic protein that requires a binding site on the Sm core domain.

1994 ◽  
Vol 14 (6) ◽  
pp. 4160-4172 ◽  
Author(s):  
G Plessel ◽  
U Fischer ◽  
R Lührmann
Keyword(s):  
Nuclear Transport ◽  
Iodoacetic Acid ◽  
Small Nuclear Rna ◽  
Vitro Assay ◽  
Core Domain ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrnp ◽  
Snrnp Protein ◽  
Localization Signal

The RNA components of small nuclear ribonucleoproteins (U snRNPs) possess a characteristic 5'-terminal trimethylguanosine cap structure (m3G cap). This cap is an important component of the nuclear localization signal of U snRNPs. It arises by hypermethylation of a cotranscriptionally added m7G cap. Here we describe an in vitro assay for the hypermethylation, which employs U snRNP particles reconstituted in vitro from purified components and subsequent analysis by m3G cap-specific immunoprecipitation. Complementation studies in vitro revealed that both cytosol and S-adenosylmethionine are required for the hypermethylation of an m7G-capped U1 snRNP reconstituted in vitro, indicating that the U1 snRNA-(guanosine-N2)-methyltransferase is a trans-active non-snRNP protein. Chemical modification revealed one cytoplasmic component required for hypermethylation and one located on the snRNP: these components have different patterns of sensitivity to modification by N-ethylmaleimide and iodoacetic acid (IAA). In the presence of cytosol and S-adenosylmethionine, an intact Sm core domain is a necessary and sufficient substrate for cap hypermethylation. These data, together with our observation that isolated native U1 snRNPs but not naked U1 RNA inhibit the trimethylation of in vitro-reconstituted U1 snRNP, indicate that the Sm core binds the methyltransferase specifically. Moreover, isolated native U2 snRNP also inhibits trimethylation of U1 snRNP, suggesting that other Sm-class U snRNPs might share the same methyltransferase. IAA modification of m7G-capped U1 snRNPs inhibited hypermethylation when they were microinjected into Xenopus oocytes and consequently also inhibited nuclear import. In contrast, modification with IAA of m3G-capped U1 snRNPs reconstituted in vitro did not interfere with their nuclear transport in oocytes. These data suggest that m3G cap formation and nuclear transport of U1 snRNPs are mediated by distinct factors, which require distinct binding sites on the Sm core of U1 snRNP.

scholarly journals Casein Kinase 1 Delta Regulates the Pace of the Mammalian Circadian Clock

2009 ◽  
Vol 29 (14) ◽  
pp. 3853-3866 ◽  
Author(s):  
Jean-Pierre Etchegaray ◽  
Kazuhiko K. Machida ◽  
Elizabeth Noton ◽  
Cara M. Constance ◽  
Robert Dallmann ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Perinatal Period ◽  
Casein Kinase ◽  
Gene Encoding ◽  
Exon 2 ◽  
Casein Kinase 1 ◽  
Functional Differences ◽  
Clock Proteins ◽  
Genes Encoding

ABSTRACT Both casein kinase 1 delta (CK1δ) and epsilon (CK1ε) phosphorylate core clock proteins of the mammalian circadian oscillator. To assess the roles of CK1δ and CK1ε in the circadian clock mechanism, we generated mice in which the genes encoding these proteins (Csnk1d and Csnk1e, respectively) could be disrupted using the Cre-loxP system. Cre-mediated excision of the floxed exon 2 from Csnk1d led to in-frame splicing and production of a deletion mutant protein (CK1δΔ2). This product is nonfunctional. Mice homozygous for the allele lacking exon 2 die in the perinatal period, so we generated mice with liver-specific disruption of CK1δ. In livers from these mice, daytime levels of nuclear PER proteins, and PER-CRY-CLOCK complexes were elevated. In vitro, the half-life of PER2 was increased by ∼20%, and the period of PER2::luciferase bioluminescence rhythms was 2 h longer than in controls. Fibroblast cultures from CK1δ-deficient embryos also had long-period rhythms. In contrast, disruption of the gene encoding CK1ε did not alter these circadian endpoints. These results reveal important functional differences between CK1δ and CK1ε: CK1δ plays an unexpectedly important role in maintaining the 24-h circadian cycle length.

scholarly journals Regulation of Alternative Splicing by the ATP-Dependent DEAD-Box RNA Helicase p72

2002 ◽  
Vol 22 (16) ◽  
pp. 5698-5707 ◽  
Author(s):  
Arnd Hönig ◽  
Didier Auboeuf ◽  
Marjorie M. Parker ◽  
Bert W. O'Malley ◽  
Susan M. Berget
Keyword(s):  
Alternative Splicing ◽  
Rna Splicing ◽  
Globin Gene ◽  
Rna Helicase ◽  
Close Relative ◽  
Rna Helicases ◽  
Enhancer Element ◽  
Dead Box ◽  
Precursor Rna

ABSTRACT Although a number of ATP-dependent RNA helicases are important for constitutive RNA splicing, no helicases have been implicated in alternative RNA splicing. Here, we show that the abundant DEAD-box RNA helicase p72, but not its close relative p68, affects the splicing of alternative exons containing AC-rich exon enhancer elements. The effect of p72 was tested by using mini-genes that undergo different types of alternative splicing. When the concentration of p72 was increased in transient transfections, the inclusion of enhancer-containing CD44 alternative exons v4 and v5 increased using a mini-gene that contained these exons and their flanking introns inserted into a β-globin gene. Other types of alternative splicing were not impacted by altering p72 concentrations. Mutation of the p72 helicase ATP-binding site or deletion of the carboxy-terminal region of the protein reduced the ability of the transfected protein to affect CD44 variable exon splicing. Use of in vitro extracts overexpressing p72 indicated that p72 becomes associated with complexes containing precursor RNA. Helicases have been implicated both in altering RNA-RNA interactions and in remodeling RNA-protein complexes. CD44 exon v4 contains a potential internal secondary structure element that base pairs the 5′ splice site with a region inside the exon located between enhancer elements. Mutations that destroyed this complementarity modestly increased inclusion in the absence of p72 but still responded to increasing p72 concentration like the wild-type exon, suggesting that p72 might have effects on protein-RNA interactions. In agreement with this hypothesis, p72 was not able to restore the inclusion of an exon mutated for its major enhancer element. Our results suggest that RNA helicases may be important alternative splicing regulatory factors.

scholarly journals The RNA helicase Dhx15 mediates Wnt-induced antimicrobial protein expression in Paneth cells

2021 ◽  
Vol 118 (4) ◽  
pp. e2017432118
Author(s):  
Yalong Wang ◽  
Kaixin He ◽  
Baifa Sheng ◽  
Xuqiu Lei ◽  
Wanyin Tao ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Intestinal Inflammation ◽  
Paneth Cell ◽  
Enteric Bacteria ◽  
Paneth Cells ◽  
Antimicrobial Protein ◽  
Rna Helicases

RNA helicases play roles in various essential biological processes such as RNA splicing and editing. Recent in vitro studies show that RNA helicases are involved in immune responses toward viruses, serving as viral RNA sensors or immune signaling adaptors. However, there is still a lack of in vivo data to support the tissue- or cell-specific function of RNA helicases owing to the lethality of mice with complete knockout of RNA helicases; further, there is a lack of evidence about the antibacterial role of helicases. Here, we investigated the in vivo role of Dhx15 in intestinal antibacterial responses by generating mice that were intestinal epithelial cell (IEC)-specific deficient for Dhx15 (Dhx15 f/f Villin1-cre, Dhx15ΔIEC). These mice are susceptible to infection with enteric bacteria Citrobacter rodentium (C. rod), owing to impaired α-defensin production by Paneth cells. Moreover, mice with Paneth cell-specific depletion of Dhx15 (Dhx15 f/f Defensinα6-cre, Dhx15ΔPaneth) are more susceptible to DSS (dextran sodium sulfate)-induced colitis, which phenocopy Dhx15ΔIEC mice, due to the dysbiosis of the intestinal microbiota. In humans, reduced protein levels of Dhx15 are found in ulcerative colitis (UC) patients. Taken together, our findings identify a key regulator of Wnt-induced α-defensins in Paneth cells and offer insights into its role in the antimicrobial response as well as intestinal inflammation.

scholarly journals U2AF1 mutations alter splice site recognition in hematological malignancies

2013 ◽  
Author(s):  
Janine O Ilagan ◽  
Aravind Ramakrishnan ◽  
Brian Hayes ◽  
Michele E Murphy ◽  
Ahmad S Zebari ◽  
...  
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Hematological Malignancies ◽  
Normal Function ◽  
Site Preference ◽  
Sequencing Studies ◽  
Genes Encoding ◽  
Quantitative Changes ◽  
Site Recognition

Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 3' splice site recognition factor U2AF1 alter its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 3' splice site motif in patients, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allele-specific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1's zinc finger domains.

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