M Phase Phosphoprotein 10 Is a Human U3 Small Nucleolar Ribonucleoprotein Component

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.

Download Full-text

Nuclear coiled bodies and the nucleolus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100167834 ◽

1994 ◽

Vol 52 ◽

pp. 20-21

Author(s):

K. Brasch ◽

J. Williams ◽

D. Gallo ◽

T. Lee ◽

R. L. Ochs

Keyword(s):

Mouse Liver ◽

Nuclear Body ◽

Nuclear Bodies ◽

Model Systems ◽

Coiled Body ◽

Rrna Processing ◽

Malignant Cells ◽

Sm Proteins ◽

Coiled Bodies ◽

Unique Protein

Though first described in 1903 by Ramon-y-Cajal as silver-staining “accessory bodies” to nucleoli, nuclear bodies were subsequently rediscovered by electron microscopy about 30 years ago. Nuclear bodies are ubiquitous, but seem most abundant in hyperactive and malignant cells. The best studied type of nuclear body is the coiled body (CB), so termed due to characteristic morphology and content of a unique protein, p80-coilin (Fig.1). While no specific functions have as yet been assigned to CBs, they contain spliceosome snRNAs and proteins, and also the nucleolar protein fibrillarin. In addition, there is mounting evidence that CBs arise from or are generated near the nucleolus and then migrate into the nucleoplasm. This suggests that as yet undefined links may exist, between nucleolar pre-rRNA processing events and the spliceosome-associated Sm proteins in CBs.We are examining CB and nucleolar changes in three diverse model systems: (1) estrogen stimulated chick liver, (2) normal and neoplastic cells, and (3) polyploid mouse liver.

Download Full-text

Association of Nonribosomal Nucleolar Proteins in Ribonucleoprotein Complexes during Interphase and Mitosis

Molecular Biology of the Cell ◽

10.1091/mbc.10.1.77 ◽

1999 ◽

Vol 10 (1) ◽

pp. 77-90 ◽

Cited By ~ 66

Author(s):

Serafı́n Piñol-Roma

Keyword(s):

Ribosomal Proteins ◽

Rna Binding ◽

Rrna Synthesis ◽

28S Rrna ◽

Ribosomal Subunits ◽

Interphase Cell ◽

Ribonucleoprotein Complexes ◽

Nucleolar Proteins ◽

M Phase ◽

Nucleolar Rna

rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin—a major nucleolar RNA-binding protein—contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits.

Download Full-text

Interactions of U2 Gene Loci and Their Nuclear Transcripts with Cajal (Coiled) Bodies: Evidence for PreU2 within Cajal Bodies

Molecular Biology of the Cell ◽

10.1091/mbc.11.9.2987 ◽

2000 ◽

Vol 11 (9) ◽

pp. 2987-2998 ◽

Cited By ~ 40

Author(s):

Kelly P. Smith ◽

Jeanne Bentley Lawrence

Keyword(s):

Potential Role ◽

Coiled Body ◽

Oligonucleotide Probes ◽

Primary Transcript ◽

U2 Snrna ◽

Rna Foci ◽

Coiled Bodies ◽

Snrna Gene

The Cajal (coiled) body (CB) is a structure enriched in proteins involved in mRNA, rRNA, and snRNA metabolism. CBs have been shown to interact with specific histone and snRNA gene loci. To examine the potential role of CBs in U2 snRNA metabolism, we used a variety of genomic and oligonucleotide probes to visualize in situ newly synthesized U2 snRNA relative to U2 loci and CBs. Results demonstrate that long spacer sequences between U2 coding repeats are transcribed, supporting other recent evidence that U2 transcription proceeds past the 3′ box. The presence of bright foci of this U2 locus RNA differed between alleles within the same nucleus; however, this did not correlate with the loci's association with a CB. Experiments with specific oligonucleotide probes revealed signal for preU2 RNA within CBs. PreU2 was also detected in the locus-associated RNA foci, whereas sequences 3′ of preU2 were found only in these foci, not in CBs. This suggests that a longer primary transcript is processed before entry into CBs. Although this work shows that direct contact of a U2 locus with a CB is not simply correlated with RNA at that locus, it provides the first evidence of new preU2 transcripts within CBs. We also show that, in contrast to CBs, SMN gems do not associate with U2 gene loci and do not contain preU2. Because other evidence indicates that preU2 is processed in the cytoplasm before assembly into snRNPs, results point to an involvement of CBs in modification or transport of preU2 RNA.

Download Full-text

Arabidopsis small nucleolar RNA monitors the efficient pre-rRNA processing during ribosome biogenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614852113 ◽

2016 ◽

Vol 113 (42) ◽

pp. 11967-11972 ◽

Cited By ~ 7

Author(s):

Pan Zhu ◽

Yuqiu Wang ◽

Nanxun Qin ◽

Feng Wang ◽

Jia Wang ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Higher Plants ◽

Ribosomal Subunit ◽

Rrna Processing ◽

Developmental Defects ◽

Protein Mutants ◽

5.8S Rrna ◽

Important Regulator ◽

Nucleolar Rna

Ribosome production in eukaryotes requires the complex and precise coordination of several hundred assembly factors, including many small nucleolar RNAs (snoRNAs). However, at present, the distinct role of key snoRNAs in ribosome biogenesis remains poorly understood in higher plants. Here we report that a previously uncharacterized C (RUGAUGA)/D (CUGA) type snoRNA, HIDDEN TREASURE 2 (HID2), acts as an important regulator of ribosome biogenesis through a snoRNA–rRNA interaction. Nucleolus-localized HID2 is actively expressed in Arabidopsis proliferative tissues, whereas defects in HID2 cause a series of developmental defects reminiscent of ribosomal protein mutants. HID2 associates with the precursor 45S rRNA and promotes the efficiency and accuracy of pre-rRNA processing. Intriguingly, disrupting HID2 in Arabidopsis appears to impair the integrity of 27SB, a key pre-rRNA intermediate that generates 25S and 5.8S rRNA and is known to be vital for the synthesis of the 60S large ribosomal subunit and also produces an imbalanced ribosome profile. Finally, we demonstrate that the antisense-box of HID2 is both functionally essential and highly conserved in eukaryotes. Overall, our study reveals the vital and possibly conserved role of a snoRNA in monitoring the efficiency of pre-rRNA processing during ribosome biogenesis.

Download Full-text

The dynamics of coiled bodies in the nucleus of adenovirus-infected cells.

Molecular Biology of the Cell ◽

10.1091/mbc.7.7.1137 ◽

1996 ◽

Vol 7 (7) ◽

pp. 1137-1151 ◽

Cited By ~ 34

Author(s):

L Rebelo ◽

F Almeida ◽

C Ramos ◽

K Bohmann ◽

A I Lamond ◽

...

Keyword(s):

Protein Synthesis ◽

Hela Cells ◽

Host Protein ◽

Coiled Body ◽

Adenovirus Infection ◽

Protein Synthesis Inhibitors ◽

Coiled Bodies ◽

Infected Cells ◽

Host Protein Synthesis ◽

Small Nuclear Ribonucleoproteins

The coiled body is a specific intranuclear structure of unknown function that is enriched in splicing small nuclear ribonucleoproteins (snRNPs). Because adenoviruses make use of the host cell-splicing machinery and subvert the normal subnuclear organization, we initially decided to investigate the effect of adenovirus infection on the coiled body. The results indicate that adenovirus infection induces the disassembly of coiled bodies and that this effect is probably secondary to the block of host protein synthesis induced by the virus. Furthermore, coiled bodies are shown to be very labile structures, with a half-life of approximately 2 h after treatment of HeLa cells with protein synthesis inhibitors. After blocking of protein synthesis, p80 coilin was detected in numerous microfoci that do not concentrate snRNP. These structures may represent precursor forms of the coiled body, which goes through a rapid cycle of assembly/disassembly in the nucleus and requires ongoing protein synthesis to reassemble.

Download Full-text

Rrp47p Is an Exosome-Associated Protein Required for the 3′ Processing of Stable RNAs

Molecular and Cellular Biology ◽

10.1128/mcb.23.19.6982-6992.2003 ◽

2003 ◽

Vol 23 (19) ◽

pp. 6982-6992 ◽

Cited By ~ 116

Author(s):

Philip Mitchell ◽

Elisabeth Petfalski ◽

Rym Houalla ◽

Alexandre Podtelejnikov ◽

Matthias Mann ◽

...

Keyword(s):

Mrna Decay ◽

Nuclear Protein ◽

Rrna Processing ◽

Small Nucleolar Rna ◽

Whole Cell ◽

Cell Lysates ◽

Small Nuclear Rnas ◽

Nuclear Exosome ◽

Nucleolar Rna

ABSTRACT Related exosome complexes of 3′→5′ exonucleases are present in the nucleus and the cytoplasm. Purification of exosome complexes from whole-cell lysates identified a Mg2+-labile factor present in substoichiometric amounts. This protein was identified as the nuclear protein Yhr081p, the homologue of human C1D, which we have designated Rrp47p (for rRNA processing). Immunoprecipitation of epitope-tagged Rrp47p confirmed its interaction with the exosome and revealed its association with Rrp6p, a 3′→5′ exonuclease specific to the nuclear exosome fraction. Northern analyses demonstrated that Rrp47p is required for the exosome-dependent processing of rRNA and small nucleolar RNA (snoRNA) precursors. Rrp47p also participates in the 3′ processing of U4 and U5 small nuclear RNAs (snRNAs). The defects in the processing of stable RNAs seen in rrp47-Δ strains closely resemble those of strains lacking Rrp6p. In contrast, Rrp47p is not required for the Rrp6p-dependent degradation of 3′-extended nuclear pre-mRNAs or the cytoplasmic 3′→5′ mRNA decay pathway. We propose that Rrp47p functions as a substrate-specific nuclear cofactor for exosome activity in the processing of stable RNAs.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.18.3.1181 ◽

1998 ◽

Vol 18 (3) ◽

pp. 1181-1189 ◽

Cited By ~ 124

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.

Download Full-text

Small nucleolar RNA

Biochemistry and Cell Biology ◽

10.1139/o95-092 ◽

1995 ◽

Vol 73 (11-12) ◽

pp. 845-858 ◽

Cited By ~ 27

Author(s):

Susan A. Gerbi

Keyword(s):

Rna Polymerase Ii ◽

Binding Sites ◽

Ribosome Biogenesis ◽

Internal Transcribed Spacers ◽

28S Rrna ◽

Small Nucleolar Rnas ◽

Rrna Processing ◽

Conserved Sequence ◽

Nucleolar Rna ◽

Nucleolar Rnas

A growing list of small nucleolar RNAs (snoRNAs) has been characterized in eukaryotes. They are transcribed by RNA polymerase II or III; some snoRNAs are encoded in the introns of other genes. The nonintronic polymerase II transcribed snoRNAs receive a trimethylguanosine cap, probably in the nucleus, and move to the nucleolus. snoRNAs are complexed with proteins, sometimes including fibrillarin. Localization and maintenance in the nucleolus of some snoRNAs requires the presence of initial precursor rRNA (pre-rRNA). Many snoRNAs have conserved sequence boxes C and D and a 3′ terminal stem; the roles of these features are discussed. Functional assays done for a few snoRNAs indicate their roles in rRNA processing for cleavage of the external and internal transcribed spacers (ETS and ITS). U3 is the most abundant snoRNA and is needed for cleavage of ETS1 and ITS1; experimental results on U3 binding sites in pre-rRNA are reviewed. 18S rRNA production also needs U14, U22, and snR30 snoRNAs, whereas U8 snoRNA is needed for 5.8S and 28S rRNA production. Other snoRNAs that are complementary to 18S or 28S rRNA might act as chaperones to mediate RNA folding. Whether snoRNAs join together in a large rRNA processing complex (the "processome") is not yet clear. It has been hypothesized that such complexes could anchor the ends of loops in pre-rRNA containing 18S or 28S rRNA, thereby replacing base-paired stems found in pre-rRNA of prokaryotes.Key words: RNA processing, small nucleolar RNAs, nucleolus, ribosome biogenesis, rRNA processing complex.

Download Full-text

The rRNA-processing function of the yeast U14 small nucleolar RNA can be rescued by a conserved RNA helicase-like protein.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.4124 ◽

1997 ◽

Vol 17 (7) ◽

pp. 4124-4132 ◽

Cited By ~ 44

Author(s):

W Q Liang ◽

J A Clark ◽

M J Fournier

Keyword(s):

Mutational Analysis ◽

Rna Helicase ◽

Suppressor Gene ◽

Rrna Processing ◽

Base Pairing ◽

Small Nucleolar Rna ◽

Stem Loop ◽

Dead Box ◽

Extragenic Suppressor ◽

Nucleolar Rna

The phylogenetically conserved U14 small nucleolar RNA is required for processing of rRNA, and this function involves base pairing with conserved complementary sequences in 18S RNA. With a view to identifying other important U14 interactions, a stem-loop domain required for activity of Saccharomyces cerevisiae U14 RNAs (the Y domain) was first subjected to detailed mutational analysis. The mapping results showed that most nucleotides of the Y domain can be replaced without affecting function, except for loop nucleotides conserved among five different yeast species. Defective variants were then used to identify both intragenic and extragenic suppressor mutations. All of the intragenic mutations mapped within six nucleotides of the primary mutation, suggesting that suppression involves a change in conformation and that the loop element is involved in an essential intermolecular interaction rather than intramolecular base pairing. A high-copy extragenic suppressor gene, designated DBP4 (DEAD box protein 4), encodes an essential, putative RNA helicase of the DEAD-DEXH box family. Suppression by DBP4 (initially CA4 [T.-H. Chang, J. Arenas, and J. Abelson, Proc. Natl. Acad. Sci. USA 87:1571-1575, 1990]) restores the level of 18S rRNA and is specific for the Y domain but is not allele specific. DBP4 is predicted to function either in assembly of the U14 small nucleolar RNP or, more likely, in its interaction with other components of the rRNA processing apparatus. Mediating the interaction of U14 with precursor 18S RNA is an especially attractive possibility.

Download Full-text

Design, Synthesis and Biological Evaluation of Morpholinated Isatin-Quinoline Hybrids as Potential Anti-Breast Cancer Agents.

10.21203/rs.3.rs-595432/v1 ◽

2021 ◽

Author(s):

Atamjit Singh ◽

Komalpreet Kaur ◽

Jaspreet Kaur ◽

Puja Gulati ◽

Amit Duggal ◽

...

Keyword(s):

Breast Cancer ◽

Estrogen Receptor Alpha ◽

Fibroblast Cell ◽

Biological Evaluation ◽

Breast Cancer Cell Lines ◽

Design Synthesis ◽

M Phase ◽

Almost All ◽

Morpholine Derivatives ◽

Mcf 7

Abstract Keeping in view the emerging need of potent and safer anti-breast cancer agents as well as pharmacological attributes of isatin, quinoline and morpholine derivatives, novel hydrazone linked morpholinated isatin-quinoline hybrids has been designed, synthesized and evaluated as anti-breast cancer agents. Synthesized hybrid compounds were preliminary screened against two breast cancer cell lines (MCF-7 and MDA-MB-231). Almost all synthetics showed potent inhibitory potential against hormone positive MCF-7 cells while inactive against hormone negative MDA-MB-231 cells. Potent compounds were further evaluated against L929 (noncancerous skin fibroblast) cell line and found highly selective for MCF-7 cells over L929 cells. Cell cycle analysis confirmed that most potent compound AS-4 (MCF-7: GI50 = 4.36 µM) cause mitotic arrest at G2/M-phase. Due to higher selectivity toward estrogen receptor alpha (ERα) dependent MCF-7 cells various binding interactions of AS-4 with ERα are also streamlined, suggesting the capability of AS-4 in completely blocking ERα. Overall study suggest that, AS-4 can act as a potential lead for further development of potent and safer anti-breast cancer agents.

Download Full-text