RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus

1988 ◽  
Vol 8 (6) ◽  
pp. 2379-2393 ◽  
Author(s):  
T H Chang ◽  
M W Clark ◽  
A J Lustig ◽  
M E Cusick ◽  
J Abelson
Keyword(s):  
Cell Nucleus ◽  
Specific Antibody ◽  
Essential Role ◽  
Mrna Splicing ◽  
Temperature Sensitive ◽  
Gene Products ◽  
Nonpermissive Temperature ◽  
Exogenous Rna

The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.

scholarly journals RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

1988 ◽  
Vol 8 (6) ◽  
pp. 2379-2393 ◽  
Author(s):  
T H Chang ◽  
M W Clark ◽  
A J Lustig ◽  
M E Cusick ◽  
J Abelson
Keyword(s):  
Cell Nucleus ◽  
Specific Antibody ◽  
Essential Role ◽  
Mrna Splicing ◽  
Temperature Sensitive ◽  
Gene Products ◽  
Nonpermissive Temperature ◽  
Exogenous Rna

The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.

scholarly journals Mutations affecting the tRNA-splicing endonuclease activity of Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 118 (4) ◽  
pp. 609-617
Author(s):  
M Winey ◽  
M R Culbertson
Keyword(s):  
Growth Defect ◽  
Temperature Sensitive ◽  
Gene Products ◽  
Endonuclease Activity ◽  
Temperature Dependent ◽  
Direct Role ◽  
Trna Splicing

Abstract Two unlinked mutations that alter the enzyme activity of tRNA-splicing endonuclease have been identified in yeast. The sen1-1 mutation, which maps on chromosome 12, causes temperature-sensitive growth, reduced in vitro endonuclease activity, and in vivo accumulation of unspliced pre-tRNAs. The sen2-1 mutation does not confer a detectable growth defect, but causes a temperature-dependent reduction of in vitro endonuclease activity. Pre-tRNAs do not accumulate in sen2-1 strains. The in vitro enzyme activities of sen1-1 and sen2-1 complement in extracts from a heterozygous diploid, but fail to complement in mixed extracts from separate sen1-1 and sen2-1 haploid strains. These results suggest a direct role for SEN gene products in the enzymatic removal of introns from tRNA that is distinct from the role of other products known to affect tRNA splicing.

scholarly journals PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle

1989 ◽  
Vol 9 (9) ◽  
pp. 3710-3719
Author(s):  
J Banroques ◽  
J N Abelson
Keyword(s):  
Essential Role ◽  
Mrna Splicing ◽  
Ribonucleoprotein Particle ◽  
Specific Antibodies ◽  
Small Nuclear Ribonucleoprotein ◽  
Assembly Pathway ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex.

The polyserine domain of the lysyl-5 hydroxylase Jmjd6 mediates subnuclear localization

2013 ◽  
Vol 453 (3) ◽  
pp. 357-370 ◽  
Author(s):  
Alexander Wolf ◽  
Monica Mantri ◽  
Astrid Heim ◽  
Udo Müller ◽  
Erika Fichter ◽  
...  
Keyword(s):  
Essential Role ◽  
Mrna Splicing ◽  
Hypoxic Stress ◽  
Subnuclear Localization ◽  
Nucleolar Proteins ◽  
Fibrillar Centre ◽  
Lysine Residues ◽  
Alternatively Spliced

Jmjd6 (jumonji-domain-containing protein 6) is an Fe(II)- and 2OG (2-oxoglutarate)-dependent oxygenase that catalyses hydroxylation of lysine residues in proteins involved in pre-mRNA splicing. Jmjd6 plays an essential role in vertebrate embryonic development and has been shown to modulate alternative splicing in response to hypoxic stress. In the present study we show that an alternatively spliced version of Jmjd6 lacking the polyS (polyserine) domain localizes to the nucleolus, predominantly in the fibrillar centre. Jmjd6 with the polyS domain deleted also interacts with nucleolar proteins. Furthermore, co-immunoprecipitation experiments and F2H (fluorescent 2-hybrid) assays demonstrate that Jmjd6 homo-oligomerization occurs in cells. In correlation with the observed variations in the subnuclear distribution of Jmjd6, the structure of Jmjd6 oligomers in vitro changes in the absence of the polyS domain, possibly reflecting the role of the polyS domain in nuclear/nucleolar shuttling of Jmjd6.

scholarly journals PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle.

1989 ◽  
Vol 9 (9) ◽  
pp. 3710-3719 ◽  
Author(s):  
J Banroques ◽  
J N Abelson
Keyword(s):  
Essential Role ◽  
Mrna Splicing ◽  
Ribonucleoprotein Particle ◽  
Specific Antibodies ◽  
Small Nuclear Ribonucleoprotein ◽  
Assembly Pathway ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex.

Cellular Samurai: katanin and the severing of microtubules

2000 ◽  
Vol 113 (16) ◽  
pp. 2821-2827 ◽  
Author(s):  
L. Quarmby
Keyword(s):  
Epithelial Cells ◽  
Essential Role ◽  
Aaa Atpase ◽  
C Elegans ◽  
Microtubule Severing ◽  
Biochemical Studies ◽  
New Evidence

Recent biochemical studies of the AAA ATPase, katanin, provide a foundation for understanding how microtubules might be severed along their length. These in vitro studies are complemented by a series of recent reports of direct in vivo observation of microtubule breakage, which indicate that the in vitro phenomenon of catalysed microtubule severing is likely to be physiological. There is also new evidence that microtubule severing by katanin is important for the production of non-centrosomal microtubules in cells such as neurons and epithelial cells. Although it has been difficult to establish the role of katanin in mitosis, new genetic evidence indicates that a katanin-like protein, MEI-1, plays an essential role in meiosis in C. elegans. Finally, new proteins involved in the severing of axonemal microtubules have been discovered in the deflagellation system of Chlamydomonas.

scholarly journals Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

2007 ◽  
Vol 18 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Yasunari Takami ◽  
Tatsuya Ono ◽  
Tatsuo Fukagawa ◽  
Kei-ichi Shibahara ◽  
Tatsuo Nakayama
Keyword(s):  
Dna Replication ◽  
Essential Role ◽  
Chromatin Assembly ◽  
Nuclear Antigen ◽  
Nucleosome Assembly ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-γ are required for cell viability. These observations highlighted the essential role of CAF-1–dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

scholarly journals Study of Staphylococcus aureusPathogenic Genes by Transfer and Expression in the Less Virulent Organism Streptococcus gordonii

2001 ◽  
Vol 69 (2) ◽  
pp. 657-664 ◽  
Author(s):  
P. Stutzmann Meier ◽  
J. M. Entenza ◽  
P. Vaudaux ◽  
P. Francioli ◽  
M. P. Glauser ◽  
...  
Keyword(s):  
Single Gene ◽  
Individual Factors ◽  
Gene Products ◽  
Streptococcus Gordonii ◽  
Pathogenic Role ◽  
Gain Of Function ◽  
Function Strategy

ABSTRACT Because Staphylococcus aureus strains contain multiple virulence factors, studying their pathogenic role by single-gene inactivation generated equivocal results. To circumvent this problem, we have expressed specific S. aureus genes in the less virulent organism Streptococcus gordonii and tested the recombinants for a gain of function both in vitro and in vivo. Clumping factor A (ClfA) and coagulase were investigated. Both gene products were expressed functionally and with similar kinetics during growth by streptococci and staphylococci. ClfA-positive S. gordoniiwas more adherent to platelet-fibrin clots mimicking cardiac vegetations in vitro and more infective in rats with experimental endocarditis (P < 0.05). Moreover, deletingclfA from clfA-positive streptococcal transformants restored both the low in vitro adherence and the low in vivo infectivity of the parent. Coagulase-positive transformants, on the other hand, were neither more adherent nor more infective than the parent. Furthermore, coagulase did not increase the pathogenicity ofclfA-positive streptococci when both clfA andcoa genes were simultaneously expressed in an artificial minioperon in streptococci. These results definitively attribute a role for ClfA, but not coagulase, in S. aureus endovascular infections. This gain-of-function strategy might help solve the role of individual factors in the complex the S. aureus-host relationship.

scholarly journals Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex

2018 ◽  
Vol 2018 ◽  
pp. 1-9
Author(s):  
Gwang Sik Kim ◽  
Young Chul Lee
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Reporter Gene Expression ◽  
Temperature Sensitive ◽  
Internal Deletion ◽  
Cell Extracts ◽  
Evolutionarily Conserved

Med6 protein (Med6p) is a hallmark component of evolutionarily conserved Mediator complexes, and the genuine role of Med6p in Mediator functions remains elusive. For the functional analysis ofSaccharomyces cerevisiaeMed6p (scMed6p), we generated a series of scMed6p mutants harboring a small internal deletion. Genetic analysis of these mutants revealed that three regions (amino acids 33–42 (Δ2), 125–134 (Δ5), and 157–166 (Δ6)) of scMed6p are required for cell viability and are located at highly conserved regions of Med6 homologs. Notably, the Med6p-Δ2 mutant was barely detectable in whole-cell extracts and purified Mediator, suggesting a loss of Mediator association and concurrent rapid degradation. Consistent with this, the recombinant forms of Med6p having these mutations partially (Δ2) restore or fail (Δ5 and Δ6) to restore in vitro transcriptional defects caused by temperature-sensitivemed6mutation. In an artificial recruitment assay, Mediator containing a LexA-fused wild-type Med6p or Med6p-Δ5 was recruited to thelexAoperator region with TBP and activated reporter gene expression. However, the recruitment of Mediator containing LexA-Med6p-Δ6 tolexAoperator region resulted in neither TBP recruitment nor reporter gene expression. This result demonstrates a pivotal role of Med6p in the postrecruitment function of Mediator, which is essential for transcriptional activation by Mediator.

scholarly journals U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction.

1991 ◽  
Vol 11 (11) ◽  
pp. 5571-5577 ◽  
Author(s):  
S L Yean ◽  
R J Lin
Keyword(s):  
Mrna Splicing ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Small Nuclear Rna ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Sensitive Allele ◽  
U4 Rna ◽  
Small Nuclear Ribonucleoproteins

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.

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