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Query: UNIPROT:P06889 (Mol)
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Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of U1 small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of U1 snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slow-migrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact U1 and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. U1 snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by U1-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H cleavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions.
Mol Cell Biol 1988 Feb
PMID:Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles. 283 38

We have cloned the gene encoding a novel small cytoplasmic RNA from the fission yeast Schizosaccharomyces pombe. Four lines of evidence support the idea that this RNA is a homolog of the 7SL RNA component of mammalian signal recognition particle (SRP), which targets presecretory proteins to the endoplasmic reticulum membrane. First, it shares limited but significant primary sequence homology with previously identified 7SL RNAs and can be folded into a similar secondary structure. Second, it possesses the 5' triphosphate characteristic of unprocessed RNA polymerase III transcripts, and moreover, it is the only fission yeast RNA in this size range with such a terminus. Third, its behavior in cell fractionation experiments suggests that it is part of a small ribonucleoprotein which forms salt-labile contacts with larger structures. Fourth, the particle containing S. pombe 7SL RNA resembles mammalian SRP in both size (11S) and affinity for DEAE-Sepharose. Disruption of the single-copy gene, designated slr1+, reveals that the RNA is indispensable for growth in fission yeast. This result is not surprising, since secretion is an essential cellular process.
Mol Cell Biol 1988 Apr
PMID:Identification of an essential Schizosaccharomyces pombe RNA homologous to the 7SL component of signal recognition particle. 283 48

We have cloned and sequenced a protamine gene from the chum salmon (Oncorhynchus keta). This gene sequence is highly homologous to one found in the rainbow trout (Salmo gairdneri), including the conservation of two structurally different repetitive elements. One of these repeats resembles a nonviral retroposon and the second is similar to a retroviral-like transposable element. The degree of sequence divergence between the O. keta and S. gairdneri genes is much less within the transcription unit than in the repetitive elements or the remainder of the flanking DNA, suggesting that since the coding and the untranslated regions are highly conserved, both contribute significantly to the structure and stability of protamine mRNA (or its cognate messenger ribonucleoprotein) and this may be important for the translational control of protamine synthesis.
J Mol Evol 1988
PMID:Characterization of a protamine gene from the chum salmon (Oncorhynchus keta). 283 40

We have studied the ultrastructural organization of nucleoli in benign naevi and malignant melanomas. In benign naevus cells the nucleoli displayed a compact ribonucleoprotein distribution, with one or two large fibrillar centres. In malignant melanoma cells the nucleoli were large with an irregular, nucleolonema-like ribonucleoprotein distribution and they exhibited numerous, small fibrillar centres. Statistical evaluation of the size of fibrillar centres indicated a mean value of 0.482 micron 2 +/- 0.136 SD for naevi and 0.221 micron 2 +/- 0.128 SD for malignant melanomas. These features, together with the more dispersed chromatin pattern of malignant melanoma nuclei compared with those of benign naevus cells, are proposed as diagnostic parameters which differentiate benign naevi from malignant melanomas at the ultrastructural level.
Virchows Arch B Cell Pathol Incl Mol Pathol 1986
PMID:Ultrastructural organization of nucleoli in benign naevi and malignant melanomas. 288 58

The small nuclear RNAs U4 and U6 display extensive sequence complementarity and co-exist in a single ribonucleoprotein particle. We have investigated intermolecular base-pairing between both RNAs by psoralen cross-linking, with emphasis on the native U4/U6 ribonucleoprotein complex. A mixture of small nuclear ribonucleoproteins U1 to U6 from HeLa cells, purified under non-denaturing conditions by immune affinity chromatography with antibodies specific for the trimethylguanosine cap of the small nuclear RNAs was treated with aminomethyltrioxsalen. A psoralen cross-linked U4/U6 RNA complex could be detected in denaturing polyacrylamide gels. Following digestion of the cross-linked U4/U6 RNA complex with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate cross-linked fragments. These fragments were analysed by chemical sequencing methods and their positions identified within RNAs U4 and U6. Two overlapping fragments of U4 RNA, spanning positions 52 to 65, were cross-linked to one fragment of U6 RNA (positions 51 to 59). These fragments show complementarity over a contiguous stretch of eight nucleotides. From these results, we conclude that in the native U4/U6 ribonucleoprotein particle, both RNAs are base-paired via these complementary regions. The small nuclear RNAs U4 and U6 became cross-linked in the deproteinized U4/U6 RNA complex also, provided that small nuclear ribonucleoproteins were phenolized at 0 degree C. When the phenolization was performed at 65 degrees C, no cross-linking could be detected upon reincubation of the dissociated RNAs at lower temperature. These results indicate that proteins are not required to stabilize the mutual interactions between both RNAs, once they exist. They further suggest, however, that proteins may well be needed for exposing the complementary RNA regions for proper intermolecular base-pairing in the course of the assembly of the U4/U6 RNP complex from isolated RNAs. Our results are discussed also in terms of the different secondary structures that the small nuclear RNAs U4 and U6 may adopt in the U4/U6 ribonucleoprotein particle as opposed to the isolated RNAs.
J Mol Biol 1985 Oct 20
PMID:Localization of a base-paired interaction between small nuclear RNAs U4 and U6 in intact U4/U6 ribonucleoprotein particles by psoralen cross-linking. 293 55

Polyclonal antibodies have been produced which react with a nuclear protein having a molecular weight of 107kD and a pI of 8.7-8.8 (designated p107). This protein is shown to be a component of the residual ribonucleoprotein (RNP) network of the nuclear matrix. P107 localized exclusively to the nuclear interior but not within nucleolar or chromatin domains. We have taken advantage of this unique probe to examine whether the RNP network of the isolated nuclear matrix has a physical counterpart in situ. We show that RNA, p107, divalent cations and the 28 kD Sm antigen of U-snRNPs are components of in situ macromolecular assemblies. While the morphology and intranuclear distribution of these assemblies are insensitive to the removal of chromatin, they are markedly altered by degradation of RNA. Digestion in situ of RNA in the presence of EDTA followed by extraction with high ionic strength buffers solubilized the components of these assemblies. Electron microscopic and immunobiochemical data are presented which support the concept that the residual RNP network of the nuclear matrix is an isolate of a pre-existing structure, and that perturbations in this internal network can be created by RNA degradation, depletion of essential metal ions and proteolysis.
Mol Cell Biochem 1986 May
PMID:Macromolecular domains containing nuclear protein p107 and U-snRNP protein p28: further evidence for an in situ nuclear matrix. 294 79

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.
Mol Cell Biol 1985 Sep
PMID:Transcription boundaries of U1 small nuclear RNA. 294 63

Small nuclear ribonucleoproteins (snRNPs) containing U1 and U5 snRNAs from HeLa cells have been fractionated using a combination of isopycnic centrifugation in cesium chloride and ion-exchange chromatography on DEAE-Sepharose. The procedure is based on the extreme stability conferred upon snRNPs by Mg2+ enabling them to withstand the very high ionic strength that prevails in cesium chloride. U1 snRNP prepared by this method contains all nine major proteins (68K, A, B, B', C, D, E, F, G) corresponding to those previously identified by immunoprecipitation and is therefore precipitable by anti-RNP and anti-Sm antibodies. U5 snRNP purified in this way contains the common D to G proteins and is also enriched in a 25 X 10(3) Mr protein that may be U5 snRNP-specific. The core-resistant U5 snRNA sequence (nucleotide 84 to 3' OH) covered by D to G proteins is extended by only six nucleotides. A similar situation is seen in U4-U6 snRNP, which we have obtained in a sufficiently pure form to examine protected sequences. However, the core-resistant sequence of U4 (nucleotide 116 to 3' OH) in U4-U6 snRNP is extended by 37 nucleotides, suggesting that the protein composition of this particle could be more complex than that of U5 snRNP. The ribonucleoprotein organization of snRNPs is summarized and discussed in view of our current knowledge on snRNA sequences protected by proteins.
J Mol Biol 1986 Jun 05
PMID:RNA-protein organization of U1, U5 and U4-U6 small nuclear ribonucleoproteins in HeLa cells. 294 70

The ribonucleoprotein (RNP) structures of the pre-mRNA and RNA processing products generated during in vitro splicing of an SP6/beta-globin pre-mRNA were characterized by sucrose gradient sedimentation analysis. Early, during the initial lag phase of the splicing reaction, the pre-mRNA sedimented heterogeneously but was detected in both 40S and 60S RNP complexes. An RNA substrate lacking a 3' splice site consensus sequence was not assembled into the 60S RNP complex. The two splicing intermediates, the first exon RNA species and an RNA species containing the intron and the second exon in a lariat configuration (IVS1-exon 2 RNA species), were found exclusively in a 60S RNP complex. These two splicing intermediates cosedimented under a variety of conditions, indicating that they are contained in the same RNP complex. The products of the splicing reaction, accurately spliced RNA and the excised IVS1 lariat RNA species, are released from the 60S RNP complex and detected in smaller RNP complexes. Sequence-specific RNA-factor interactions within these RNP complexes were evidenced by the preferential protection of the pre-mRNA branch point from RNase A digestion and protection of the 2'-5' phosphodiester bond of the lariat RNA species from enzymatic debranching. The various RNP complexes were further characterized and could be distinguished by immunoprecipitation with anti-Sm and anti-(U1)RNP antibodies.
Mol Cell Biol 1986 Jul
PMID:Ribonucleoprotein complex formation during pre-mRNA splicing in vitro. 294 39

The human U3 ribonucleoprotein (RNP) has been analyzed to determine its protein constituents, sites of protein-RNA interaction, and RNA secondary structure. By using anti-U3 RNP antibodies and extracts prepared from HeLa cells labeled in vivo, the RNP was found to contain four nonphosphorylated proteins of 36, 30, 13, and 12.5 kilodaltons and two phosphorylated proteins of 74 and 59 kilodaltons. U3 nucleotides 72-90, 106-121, 154-166, and 190-217 must contain sites that interact with proteins since these regions are immunoprecipitated after treatment of the RNP with RNase A or T1. The secondary structure was probed with specific nucleases and by chemical modification with single-strand-specific reagents that block subsequent reverse transcription. Regions that are single stranded (and therefore potentially able to interact with a substrate RNA) include an evolutionarily conserved sequence at nucleotides 104-112 and nonconserved sequences at nucleotides 65-74, 80-84, and 88-93. Nucleotides 159-168 do not appear to be highly accessible, thus making it unlikely that this U3 sequence base pairs with sequences near the 5.8S rRNA-internal transcribed spacer II junction, as previously proposed. Alternative functions of the U3 RNP are discussed, including the possibility that U3 may participate in a processing event near the 3' end of 28S rRNA.
Mol Cell Biol 1987 Aug
PMID:Structural analysis of the human U3 ribonucleoprotein particle reveal a conserved sequence available for base pairing with pre-rRNA. 295 55


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