Rat homolog of PinX1 is a nucleolar protein involved in the regulation of telomere length

Human PinX1 involves in regulation of telomere length. Here, we describe the function of a rat homolog of PinX1. Rat PinX1 (rPinX1) was cloned from WB-F344, a rat hepatic stem-like epithelial cell. It encodes a protein of 331 amino acids with 70% homology to human PinX1 and 91% homology to mouse. Northern analysis revealed that rPinX1 is expressed in both somatic and germ tissues, most abundantly in heart, liver and testis. Co-localization with a nucleolar protein, fibrillarin, showed that rPinX1 resides in the nucleolus. Analysis of truncated mutants revealed that an internal K,E/D region seems to be important for nucleolar localization. A stable cell line expressing rPinX1 was established in NIH3T3, a mouse-transformed embryonic fibroblast cell line, and stable cells were subcultured for more than 150 population doublings. The growth of stable rPinX1 cells slowed down at late passages, and a fraction of these cells exhibited increased size and stained positively for senescence-associated β-galactosidase. Overexpression of rPinX1 in NIH3T3 cells resulted in gradual telomere shortening over successive passages. However, the telomeric 3′ overhang was not altered by PinX1 expression. This study demonstrates that a rat homolog of human PinX1 is a nucleolar protein, and that overexpression of rPinX1 induces cellular senescence and telomere shortening, but has no effect on 3′ overhang length. The function of PinX1 in regulating telomere length is conserved in rodents, and this study may provide insight into the mechanism by which a nucleolar protein can regulate telomere length.     

RNA B is the major nucleolar trimethylguanosine-capped small nuclear RNA associated with fibrillarin and pre-rRNAs in Trypanosoma brucei.

RNA B is one of three abundant trimethylguanosine-capped U small nuclear RNAs (snRNAs) of Trypanosoma brucei which is not strongly identified with other U snRNAs by sequence homology. We show here that RNA B is a highly diverged U3 snRNA homolog likely involved in pre-rRNA processing. Sequence identity between RNA B and U3 snRNAs is limited; only two of four boxes of homology conserved between U3 snRNAs are obvious in RNA B. These are the box A homology, specific for U3 snRNAs, and the box C homology, common to nucleolar snRNAs and required for association with the nucleolar protein, fibrillarin. A 35-kDa T. brucei fibrillarin homolog was identified by using an anti-Physarum fibrillarin monoclonal antibody. RNA B and fibrillarin were localized in nucleolar fractions of the nucleus which contained pre-rRNAs and did not contain nucleoplasmic snRNAs. Fibrillarin and RNA B were precipitated by scleroderma patient serum S4, which reacts with fibrillarins from diverse organisms; RNA B was the only trimethylguanosine-capped RNA precipitated. Furthermore, RNA B sedimented with pre-rRNAs in nondenaturing sucrose gradients, similarly to U3 and other nucleolar snRNAs, suggesting that RNA B is hydrogen bonded to rRNA intermediates and might be involved in their processing.     

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

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.     

Human RBM28 protein is a specific nucleolar component of the spliceosomal snRNPs

The biogenesis of spliceosomal small nuclear RNAs (snRNAs) involves organized translocations between the cytoplasm and certain nuclear domains, such as Cajal bodies and nucleoli. Here we identify human RBM28 protein as a novel snRNP component, based on affinity selection of U6 small nuclear ribonucleoprotein (snRNP). As shown by immunofluorescence, RBM28 is a nucleolar protein. Anti-RBM28 immunoprecipitation from HeLa cell lysates revealed that this protein specifically associates with U1, U2, U4, U5, and U6 snRNAs. Our data provide the first evidence that RBM28 is a common nucleolar component of the spliceosomal ribonucleoprotein complexes, possibly coordinating their transition through the nucleolus.     

Mpp10p, a U3 small nucleolar ribonucleoprotein component required for pre-18S rRNA processing in yeast.

We have isolated and characterized Mpp10p, a novel protein component of the U3 small nucleolar ribonucleoprotein (snoRNP) from the yeast Saccharomyces cerevisiae. The MPP10 protein was first identified in human cells by its reactivity with an antibody that recognizes specific sites of mitotic phosphorylation. To study the functional role of MPP10 in pre-rRNA processing, we identified the yeast protein by performing a GenBank search. The yeast Mpp10p homolog is 30% identical to the human protein over its length. Antibodies to the purified yeast protein recognize a 110-kDa polypeptide in yeast extracts and immunoprecipitate the U3 snoRNA, indicating that Mpp10p is a specific protein component of the U3 snoRNP in yeast. As a first step in the genetic analysis of Mpp10p function, diploid S. cerevisiae cells were transformed with a null allele. Sporulation and tetrad analysis indicate that MPP10 is an essential gene. A strain was constructed where Mpp10p is expressed from a galactose-inducible, glucose- repressible promoter. After depletion of Mpp10p by growth in glucose, cell growth is arrested and levels of 18S and its 20S precursor are reduced or absent while the 23S and 35S precursors accumulate. This pattern of accumulation of rRNA precursors suggests that Mpp10p is required for cleavage at sites A0, A1, and A2. Pulse-chase analysis of newly synthesized pre-rRNAs in Mpp10p-depleted yeast confirms that little mature 18S rRNA formed. These results reveal a novel protein essential for ribosome biogenesis and further elucidate the composition of the U3 snoRNP.     

U3, U8 and U13 comprise a new class of mammalian snRNPs localized in the cell nucleolus.

Using anti-(U3)RNP autoantibodies, we have isolated and characterized two additional small nucleolar RNAs from HeLa cells, which are less abundant than U3 RNA. Both RNAs possess a trimethylguanosine cap as judged by precipitation with anti-TMG antibody, but are not precipitated by either anti-Sm or anti-La antibodies. In addition, both RNAs are not precipitable by anti-Th serum, which recognizes another nucleolar RNP autoantigen. Sequence analysis revealed that one of these RNAs, 136 nucleotides long, is the human U8 homolog; while the other, 105 nucleotides long, represents a novel species which we designate U13. Both RNAs share with U3 two conserved sequences (boxes C and D). The role of one or both of these boxes in binding the common 34 kd antigenic protein, otherwise known as fibrillarin, is discussed. Fractionation of HeLa cells revealed that U8 and U13, like U3, reside in the nucleolus. In glycerol gradients both RNAs cosediment with larger structures possibly representing ribosomal precursors. We propose that U3, U8 and U13 comprise a new subset of mammalian snRNPs whose roles in ribosome biogenesis are discussed.     

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.     

Isolation and characterization of a human U3 small nucleolar RNA gene

U3 RNA is an abundant, capped, small nucleolar RNA, implicated in the processing of preribosomal RNA. In this study, a DNA clone coding for U3 RNA (clone U3-1) was isolated from a human genomic library and characterized. The DNA sequence was identical to that of human U3 RNA isolated from HeLa cells. The flanking regions showed homology to the enhancer, promoter, and 3'-processing signal found in U1 and U2 snRNA genes. Further, the recently identified "U3 box" (GATTGGCTGCN10TATGTTAATTATGG) of rat U3 genes (Stroke and Weiner, (1985) J. Mol. Biol. 184, 183-193), was also found in the human U3 gene. This gene was transcribed in Xenopus oocytes; it is the first cloned true human U3 gene.     

cDNA Cloning and Characterization of the Human U3 Small Nucleolar Ribonucleoprotein Complex-Associated 55-Kilodalton Protein

The eukaryotic nucleolus contains a large number of small RNA molecules (snoRNAs) which, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. The most abundant and one of the best-conserved snoRNAs is the U3 RNA. So far, only one human U3 snoRNA-associated protein, fibrillarin, has been characterized. Previously, the U3 snoRNPwas purified from CHO cells, and three proteins of 15, 50, and 55 kDa were found to copurify with the U3 snoRNA (B. Lübben, C. Marshallsay, N. Rottmann, and R. Lührmann, Nucleic Acids Res. 21:5377–5385, 1993). Here we report the cDNA cloning and characterization of the human U3 snoRNP-associated 55-kDa protein. The isolated cDNA codes for a novel nucleolar protein which is specifically associated with the U3 snoRNA. This protein, referred to as hU3-55k, is the first characterized U3 snoRNP-specific protein from humans. hU3-55k is a new member of the family of WD-40 repeat proteins and is conserved throughout evolution. It appears that the C-terminal end of hU3-55k is required for nucleolar localization and U3 snoRNA binding.     

The intron-containing L3 ribosomal protein gene (RPL3): sequence analysis and identification of U43 and of two novel intronic small nucleolar RNAs

Isolation and sequencing of bovine and human intron-containing L3 ribosomal protein genes are here reported. They exhibit very similar organisation, both comprising 10 exons and nine introns. A polymorphic locus, involving a 19-bp deletion, was found in intron 6 of the human gene. The frequency of the two alleles has been estimated in 200 haploid genomes. In bovine and human genes intron sequences are rather different, except for limited regions, located in corresponding positions, which show a surprisingly high degree of identity. All these regions contain conserved features defining the box C/D class of small nucleolar RNAs. Demonstration is given that U43 small nucleolar RNA is encoded within the first intron of both bovine and human genes. Single nucleotide sequences, encoding two novel species of small nucleolar RNAs (U82, U83a and U83b), are located in introns 3, 5 and 7. Their expression has been investigated and a possible role of these molecules in 2'-O-ribose methylation of rRNAs is discussed.     

The yeast homolog of human PinX1 is involved in rRNA and small nucleolar RNA maturation,not in telomere elongation inhibition

In human cells, PinX1 protein has recently been shown to regulate telomere length by repressing the telomerase. In this work, we show that the putative yeast homolog of PinX1, encoded by the YGR280c open reading frame (ORF), is a new component of the ribosomal RNA processing machinery. The protein has a KK(E/D) C-terminal domain typical of nucleolar proteins and bears a putative RNA interacting domain widespread in eukaryotes called the G-patch. The protein was hence renamed Gno1p (G-patch nucleolar protein). GNO1 deletion results in a large growth defect due to the inhibition of the pre-ribosomal RNA processing first cleavage steps at sites A(0), A(1), and A(2). Furthermore, Gno1p is involved in the final 3'-end trimming of U18 and U24 small nucleolar RNAs. A mutational analysis showed that the G-patch of Gno1p is essential for both functions, whereas the KK(E/D) repeats are only required for U18 small nucleolar RNA maturation. We found that PinX1 complemented the gno1-Delta mutation, suggesting that it has a dual function in telomere length regulation and ribosomal RNA maturation in agreement with its telomeric and nucleolar localization in human cells. Conversely, we found that Gno1p does not exhibit the in vivo telomerase inhibitor activity of PinX1.     

The yeast homologue of U3 snRNA.

snR17, one of the most abundant capped small nuclear RNAs of Saccharomyces cerevisiae, is equivalent to U3 snRNA of other eukaryotes. It is 328 nucleotides in length, 1.5 times as long as other U3 RNAs, but shares significant homology both in nucleotide sequence and in predicted secondary structure. Human scleroderma antiserum specific to nucleolar U3 RNP can enrich snR17 from sonicated yeast nuclear extracts. Unlike other yeast snRNAs which are encoded by single copy genes, snR17 is encoded by two genetically unlinked genes: SNR17A and SNR17B. The RNA snR17A is more abundant than snR17B. Deleting one or other of the genes has no obvious phenotypic effect, except that the steady-state level of snR17B is increased in snr17a- strains. Haploid strains with both genes deleted are inviable, therefore yeast U3 is essential.     

A weak C' box renders U3 snoRNA levels dependent on hU3-55K binding

The rate of ribosome biogenesis, which is downregulated in terminally differentiated cells and upregulated in most cancers, regulates the growth rate and is linked to the cell's proliferative potential. The U3 box C/D small nucleolar RNP (snoRNP) is an integral component of the small subunit (SSU) processome and is essential for 18S rRNA processing. We show that U3 snoRNP assembly, and therefore U3 snoRNA accumulation, is regulated through the U3-specific protein hU3-55K. Furthermore, we report that the levels of several SSU processome components, including the U3 snoRNA but not other box C/D snoRNAs, are specifically downregulated during human lung (CaCo-2) and colon (CaLu-3) epithelial cell differentiation. c-Myc is reported to play an integral role in regulating ribosome production by controlling the expression of many ribosome biogenesis factors. Our data, however, indicate that this regulation is not dependent on c-Myc since the level of this protein does not change during epithelial cell differentiation. In addition, depletion of c-Myc had only a mild affect on the levels of SSU processome proteins. CaCo-2 cells are colon adenocarcinoma epithelial cells that are believed to revert to their precancerous state during differentiation. This suggests a significant increase in the levels of specific SSU processome components during tumorogenesis.     

Nucleolar proteins identified in human cells as antigens by sera from dogs with autoimmune disorders

In the course of a systematic screening of sera from dogs suffering from autoimmune disorders, three sera were shown by indirect immunofluorescence to characteristically label the nucleoli and nucleoplasm of human cell lines (Hep-2 and HeLa). This pattern of staining persisted throughout the cell cycle, except for mitosis when the fluorescence was localized in extrachromosomal areas. By immunoblotting nuclear and subnuclear fractions, three polypeptides of 110,000, 95,000, and 45,000 Da apparent molecular weight were identified, which reacted with all three sera. By means of affinity purification, it was shown that an antibody specific for any one of the three proteins also reacts with the two others. This antigenic cross-reactivity suggested regions of structural homology shared by the three proteins. Indeed, treatment of nucleoli with high concentrations of DNase I containing residual proteolytic activity resulted in the disappearance of the 110- and 95-kDa proteins and the concomitant appearance of a doublet of 45-kDa proteins. Subnuclear localization studies indicated that all three polypeptides were located in both nucleoli and nucleoplasm. Significantly, the 110-kDa protein differs from the major nucleolar protein, nucleolin, by its electrophoretic mobility in two-dimensional gels, its location in nucleoli and in nucleoplasm, its absence in nucleolar organizer regions of chromosomes, and its differential solubility of DNase I. Therefore, the three antigenically related species reported in this study constitute a new class of nucleolar proteins.     

Human 1A6/DRIM, the homolog of yeast Utp20, functions in the 18S rRNA processing

1A6/DRIM is a nucleolar protein with a nucleolar targeting sequence in its 3'-terminus. Bioinformatic analysis indicated that human 1A6/DRIM shares 23% identity and 43% similarity with yeast Utp20, which has been reported as a component of U3 snoRNA protein complex and has been implicated in 18S rRNA processing. In the present study, we found, by utilizing RT-PCR with RNA extracted from anti-1A6/DRIM immunoprecipitates and Northern blotting, that 1A6/DRIM is associated with U3 snoRNA. Pulse-chase labeling assays showed that silencing of 1A6/DRIM expression in HeLa cells resulted in a delayed 18S rRNA processing. Furthermore, immunoprecipitations revealed that 1A6/DRIM was also associated with fibrillarin, another U3 RNP component in HeLa cells. These results indicate that 1A6/DRIM is involved in 18S rRNA processing and is the bona fide mammalian Utp20.     

Different forms of U15 snoRNA are encoded in the introns of the ribosomal protein S1 gene of Xenopus laevis.

Recent cloning and sequencing of one of the two Xenopus gene copies (S1b) coding for the ribosomal protein S1 has revealed that its introns III, V and VI carry a region of about 150 nt that shares an identity of 60%. We show here the presence in Xenopus oocytes and cultured cells of a 143-147 nt long RNA species encoded by these three repeated sequences on the same strand as the S1 mRNA and by at least one repeat present in the S1 a copy of the r-protein gene. We identify these RNAs as forms of the small nucleolar RNA U15 (U15 snoRNA) because of their sequence homology with an already described human U15 RNA encoded in the first intron of the human r-protein S3 gene, which is homologous to Xenopus S1. Comparison of the various Xenopus and human U15 RNA forms shows a very high conservation in some regions, but considerable divergence in others. In particular the most conserved sequences include two box C and two box D motifs, typical of most snoRNAs interacting with the nucleolar protein fibrillarin. Adjacent to the two D boxes there are two sequences, 9 and 10 nt in length, which are perfectly complementary to an evolutionary conserved sequence of the 28S rRNA. Modeling the possible secondary structure of Xenopus and human U15 RNAs reveals that, in spite of the noticeable sequence diversity, a high structural conservation in some cases may be maintained by compensatory mutations. We show also that the different Xenopus U15 RNA forms are expressed at comparable levels, localized in the nucleoli and produced by processing of the intronic sequences, as recently described for other snoRNAs.     

The 65 and 110 kDa SR-related proteins of the U4/U6.U5 tri-snRNP are essential for the assembly of mature spliceosomes

The association of the U4/U6.U5 tri-snRNP with pre-spliceosomes is a poorly understood step in the spliceosome assembly pathway. We have identified two human tri-snRNP proteins (of 65 and 110 kDa) that play an essential role in this process. Characterization by cDNA cloning of the 65 and 110 kDa proteins revealed that they are likely orthologues of the yeast spliceosomal proteins Sad1p and Snu66p, respectively. Immunodepletion of either protein from the HeLa cell nuclear extracts inhibited pre-mRNA splicing due to a block in the formation of mature spliceosomes, but had no effect on the integrity of the U4/U6.U5 tri-snRNP. Spliceosome assembly and splicing catalysis could be restored to the respective depleted extract by the addition of recombinant 65 or 110 kDa protein. Our data demonstrate that both proteins are essential for the recruitment of the tri-snRNP to the pre-spliceosome but not for the maintenance of the tri-snRNP stability. Moreover, since both proteins contain an N-terminal RS domain, they could mediate the association of the tri-snRNP with pre-spliceosomes by interaction with members of the SR protein family.