scholarly journals Characterization of the intron-encoded U19 RNA, a new mammalian small nucleolar RNA that is not associated with fibrillarin.

1996 ◽  
Vol 16 (4) ◽  
pp. 1391-1400 ◽  
Author(s):  
T Kiss ◽  
M L Bortolin ◽  
W Filipowicz
Keyword(s):  
Hela Cell ◽  
Yeast Cells ◽  
Sequence Motifs ◽  
Cell Extracts ◽  
5.8S Rrna ◽  
Precursor Rna ◽  
Hydroxyl Termini ◽  
Nucleolar Rna ◽  
Nucleolar Rnas

We have characterized a new member (U19) of a group of mammalian small nuclear RNAs that are not precipitable with antibodies against fibrillarin, a conserved nucleolar protein associated with most of the small nucleolar RNAs characterized to date. Human U19 RNA is 200 nucleotides long and possesses 5'-monophosphate and 3'-hydroxyl termini. It lacks functional boxes C and D, sequence motifs required for fibrillarin binding in many other snoRNAs. Human and mouse RNA are 86% homologous and can be folded into similar secondary structures, a finding supported by the results of nuclease probing of the RNA. In the human genome, U19 RNA is encoded in the intron of an as yet not fully characterized gene and could be faithfully processed from a longer precursor RNA in HeLa cell extracts. During fractionation of HeLa cell nucleolar extracts on glycerol gradients, U19 RNA was associated with higher-order structures of approximately 65S, cosedimenting with complexes containing 7-2/MRP RNA, a conserved nucleolar RNA shown to be involved in 5.8S rRNA processing in yeast cells.

scholarly journals Processing of the Precursors to Small Nucleolar RNAs and rRNAs Requires Common Components

1998 ◽  
Vol 18 (3) ◽  
pp. 1181-1189 ◽  
Author(s):  
Elisabeth Petfalski ◽  
Thomas Dandekar ◽  
Yves Henry ◽  
David Tollervey
Keyword(s):  
Small Nucleolar Rnas ◽  
Rrna Processing ◽  
5.8S Rrna ◽  
Genes Encoding ◽  
Sequences Analysis ◽  
Upstream Processing ◽  
Exonuclease Digestion ◽  
Nucleolar Rna ◽  
Nucleolar Rnas ◽  
Common Components

ABSTRACT The genes encoding the small nucleolar RNA (snoRNA) species snR190 and U14 are located close together in the genome of Saccharomyces cerevisiae. Here we report that these two snoRNAs are synthesized by processing of a larger common transcript. In strains mutant for two 5′→3′ exonucleases, Xrn1p and Rat1p, families of 5′-extended forms of snR190 and U14 accumulate; these have 5′ extensions of up to 42 and 55 nucleotides, respectively. We conclude that the 5′ ends of both snR190 and U14 are generated by exonuclease digestion from upstream processing sites. In contrast to snR190 and U14, the snoRNAs U18 and U24 are excised from the introns of pre-mRNAs which encode proteins in their exonic sequences. Analysis of RNA extracted from a dbr1-Δ strain, which lacks intron lariat-debranching activity, shows that U24 can be synthesized only from the debranched lariat. In contrast, a substantial level of U18 can be synthesized in the absence of debranching activity. The 5′ ends of these snoRNAs are also generated by Xrn1p and Rat1p. The same exonucleases are responsible for the degradation of several excised fragments of the pre-rRNA spacer regions, in addition to generating the 5′ end of the 5.8S rRNA. Processing of the pre-rRNA and both intronic and polycistronic snoRNAs therefore involves common components.

Characterization of cleavage and polyadenylation specificity factor and cloning of its 100-kilodalton subunit

1994 ◽  
Vol 14 (12) ◽  
pp. 8183-8190
Author(s):  
A Jenny ◽  
H P Hauri ◽  
W Keller
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Tryptic Digest ◽  
Cell Extracts ◽  
C Terminus ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Precursor Rna ◽  
Novel Protein

During the formation of the 3' ends of mRNA, the cleavage and polyadenylation specificity factor (CPSF) is required for 3' cleavage of the transcript as well as for subsequent polyadenylation. Using peptide sequences from a tryptic digest, we have cloned the 100-kDa subunit of CPSF. This subunit is a novel protein showing no homology to any known polypeptide in databases. Polyclonal antibodies against the C terminus of the protein inhibit the polyadenylation reaction. Polyclonal and monoclonal antibodies were used to characterize the composition of CPSF. Immunoprecipitations of CPSF from HeLa cell extracts and from labeled chromatographic fractions show the coprecipitation of all four subunits of 160, 100, 73, and 30 kDa. Proteins of 160 and 30 kDa that are specifically cross-linked to precursor RNA by UV irradiation were identified as CPSF subunits by immunoprecipitation. Immunofluorescent detection of CPSF in HeLa cells localized it in the nucleoplasm, excluding cytoplasm and nucleolar structures.

scholarly journals Characterization of cleavage and polyadenylation specificity factor and cloning of its 100-kilodalton subunit.

1994 ◽  
Vol 14 (12) ◽  
pp. 8183-8190 ◽  
Author(s):  
A Jenny ◽  
H P Hauri ◽  
W Keller
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Tryptic Digest ◽  
Cell Extracts ◽  
C Terminus ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Precursor Rna ◽  
Novel Protein

During the formation of the 3' ends of mRNA, the cleavage and polyadenylation specificity factor (CPSF) is required for 3' cleavage of the transcript as well as for subsequent polyadenylation. Using peptide sequences from a tryptic digest, we have cloned the 100-kDa subunit of CPSF. This subunit is a novel protein showing no homology to any known polypeptide in databases. Polyclonal antibodies against the C terminus of the protein inhibit the polyadenylation reaction. Polyclonal and monoclonal antibodies were used to characterize the composition of CPSF. Immunoprecipitations of CPSF from HeLa cell extracts and from labeled chromatographic fractions show the coprecipitation of all four subunits of 160, 100, 73, and 30 kDa. Proteins of 160 and 30 kDa that are specifically cross-linked to precursor RNA by UV irradiation were identified as CPSF subunits by immunoprecipitation. Immunofluorescent detection of CPSF in HeLa cells localized it in the nucleoplasm, excluding cytoplasm and nucleolar structures.

scholarly journals Ribosomal RNA 2'-O-methylations regulate translation by impacting ribosome dynamics

2021 ◽  
Author(s):  
Sohail Khoshnevis ◽  
R. Elizabeth Dreggors-Walker ◽  
Virginie Marchand ◽  
Yuri Motorin ◽  
Homa Ghalei
Keyword(s):  
Methylation Pattern ◽  
Yeast Cells ◽  
Start Codon ◽  
Ribosomal Subunits ◽  
Ribonucleoprotein Complexes ◽  
Cellular Translation ◽  
Domains Of Life ◽  
Codon Selection ◽  
Nucleolar Rnas

Protein synthesis by ribosomes is critically important for gene expression in all cells. The ribosomal RNAs (rRNAs) are marked by numerous chemical modifications. An abundant group of rRNA modifications, present in all domains of life, is 2'-O-methylation guided by box C/D small nucleolar RNAs (snoRNAs) which are part of small ribonucleoprotein complexes (snoRNPs). Although 2'-O-methylations are required for proper production of ribosomes, the mechanisms by which these modifications contribute to translation have remained elusive. Here, we show that a change in box C/D snoRNP biogenesis in actively growing yeast cells results in the production of hypo 2'-O-methylated ribosomes with distinct translational properties. Using RiboMeth-Seq for the quantitative analysis of 2'-O methylations, we identify site-specific perturbations of the rRNA 2'-O-methylation pattern and uncover sites that are not required for ribosome production under normal conditions. Characterization of the hypo 2'-O-methylated ribosomes reveals significant translational fidelity defects including frameshifting and near-cognate start codon selection. Using rRNA structural probing, we show that hypo 2'-O-methylation affects the inherent dynamics of the ribosomal subunits and impacts the binding of translation factor eIF1 thereby causing translational defects. Our data reveal an unforeseen spectrum of 2'-O-methylation heterogeneity in yeast rRNA and suggest a significant role for rRNA 2'-O-methylation in regulating cellular translation by controlling ribosome dynamics and ligand binding.

scholarly journals Nuclear Retention Elements of U3 Small Nucleolar RNA

1999 ◽  
Vol 19 (12) ◽  
pp. 8412-8421 ◽  
Author(s):  
Wayne Speckmann ◽  
Aarthi Narayanan ◽  
Rebecca Terns ◽  
Michael P. Terns
Keyword(s):  
Nucleolar Localization ◽  
Sequence Motifs ◽  
Nuclear Retention ◽  
Conserved Sequence ◽  
Coiled Bodies ◽  
Conserved Sequence Motifs ◽  
Site Of Action ◽  
Sequence Requirements ◽  
Nucleolar Rna ◽  
Nucleolar Rnas

ABSTRACT The processing and methylation of precursor rRNA is mediated by the box C/D small nucleolar RNAs (snoRNAs). These snoRNAs differ from most cellular RNAs in that they are not exported to the cytoplasm. Instead, these RNAs are actively retained in the nucleus where they assemble with proteins into mature small nucleolar ribonucleoprotein particles and are targeted to their intranuclear site of action, the nucleolus. In this study, we have identified the cis-acting sequences responsible for the nuclear retention of U3 box C/D snoRNA by analyzing the nucleocytoplasmic distributions of an extensive panel of U3 RNA variants after injection of the RNAs into Xenopus oocyte nuclei. Our data indicate the importance of two conserved sequence motifs in retaining U3 RNA in the nucleus. The first motif is comprised of the conserved box C′ and box D sequences that characterize the box C/D family. The second motif contains conserved box sequences B and C. Either motif is sufficient for nuclear retention, but disruption of both motifs leads to mislocalization of the RNAs to the cytoplasm. Variant RNAs that are not retained also lack 5′ cap hypermethylation and fail to associate with fibrillarin. Furthermore, our results indicate that nuclear retention of U3 RNA does not simply reflect its nucleolar localization. A fragment of U3 containing the box B/C motif is not localized to nucleoli but retained in coiled bodies. Thus, nuclear retention and nucleolar localization are distinct processes with differing sequence requirements.

scholarly journals Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy

2018 ◽  
Vol 115 (17) ◽  
pp. E3969-E3977 ◽  
Author(s):  
Sasikumar Rajoo ◽  
Pascal Vallotton ◽  
Evgeny Onischenko ◽  
Karsten Weis
Keyword(s):  
Nuclear Pore Complex ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Altered Expression ◽  
Copy Numbers ◽  
Quantitative Image ◽  
Multiple Copies ◽  
Almost All ◽  
High Degree

The nuclear pore complex (NPC) is an eightfold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ∼30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure. However, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed nuclear rim intensity measurement (NuRIM), to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC, suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM, we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188, that their altered expression leads to significant changes in the NPC stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution.

scholarly journals The G-Protein α-Subunit GasC Plays a Major Role in Germination in the Dimorphic FungusPenicillium marneffei

Genetics ◽  
2003 ◽  
Vol 164 (2) ◽  
pp. 487-499 ◽  
Author(s):  
Sophie Zuber ◽  
Michael J Hynes ◽  
Alex Andrianopoulos
Keyword(s):  
G Protein ◽  
Germination Rate ◽  
Yeast Cells ◽  
Class Iii ◽  
Temperature Dependent ◽  
Gene Encoding ◽  
Α Subunit ◽  
G Protein Α Subunit ◽  
Opportunistic Human Pathogen

AbstractThe opportunistic human pathogen Penicillium marneffei exhibits a temperature-dependent dimorphic switch. At 25°, multinucleate, septate hyphae that can undergo differentiation to produce asexual spores (conidia) are produced. At 37° hyphae undergo arthroconidiation to produce uninucleate yeast cells that divide by fission. This work describes the cloning of the P. marneffei gasC gene encoding a G-protein α-subunit that shows high homology to members of the class III fungal Gα-subunits. Characterization of a ΔgasC mutant and strains carrying a dominant-activating gasCG45R or a dominant-interfering gasCG207R allele show that GasC is a crucial regulator of germination. A ΔgasC mutant is severely delayed in germination, whereas strains carrying a dominant-activating gasCG45R allele show a significantly accelerated germination rate. Additionally, GasC signaling positively affects the production of the red pigment by P. marneffei at 25° and negatively affects the onset of conidiation and the conidial yield, showing that GasC function overlaps with functions of the previously described Gα-subunit GasA. In contrast to the S. cerevisiae ortholog Gpa2, our data indicate that GasC is not involved in carbon or nitrogen source sensing and plays no major role in either hyphal or yeast growth or in the switch between these two forms.

scholarly journals The Small Subunit Processome Is Required for Cell Cycle Progression at G1

2004 ◽  
Vol 15 (11) ◽  
pp. 5038-5046 ◽  
Author(s):  
Kara A. Bernstein ◽  
Susan J. Baserga
Keyword(s):  
Cell Cycle ◽  
Ribosome Biogenesis ◽  
Cell Cycle Progression ◽  
Small Subunit ◽  
Cellular Growth ◽  
Yeast Cells ◽  
G1 Phase ◽  
Relay Cell ◽  
Ssu Processome ◽  
Nucleolar Rna

Without ribosome biogenesis, translation of mRNA into protein ceases and cellular growth stops. We asked whether ribosome biogenesis is cell cycle regulated in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and we determined that it is not regulated in the same manner as in metazoan cells. We therefore turned our attention to cellular sensors that relay cell size information via ribosome biogenesis. Our results indicate that the small subunit (SSU) processome, a complex consisting of 40 proteins and the U3 small nucleolar RNA necessary for ribosome biogenesis, is not mitotically regulated. Furthermore, Nan1/Utp17, an SSU processome protein, does not provide a link between ribosome biogenesis and cell growth. However, when individual SSU processome proteins are depleted, cells arrest in the G1 phase of the cell cycle. This arrest was further supported by the lack of staining for proteins expressed in post-G1. Similarly, synchronized cells depleted of SSU processome proteins did not enter G2. This suggests that when ribosomes are no longer made, the cells stall in the G1. Therefore, yeast cells must grow to a critical size, which is dependent upon having a sufficient number of ribosomes during the G1 phase of the cell cycle, before cell division can occur.

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