Saccharomyces cerevisiae has a U1-like small nuclear RNA with unexpected properties

Previous experiments indicated that only a small subset of the approximately equal to 24 small nuclear RNAs (snRNAs) in Saccharomyces cerevisiae have binding sites for the Sm antigen, a hallmark of metazoan small nuclear ribonucleoproteins (snRNPs) involved in pre-messenger RNA splicing. Antibodies from human serum to Sm proteins were used to show that four snRNAs (snR7, snR14, snR19, and snR20) can be immunoprecipitated from yeast extracts. Three of these four, snR7, snR14, and snR20, have been shown to be analogs of mammalian U5, U4, and U2, respectively. Several regions of significant homology to U1 (164 nucleotides) have now been found in cloned and sequenced snR19 (568 nucleotides). These include ten out of ten matches to the 5' end of U1, the site known to interact with the 5' splice site of mammalian introns. Surprisingly, the precise conservation of this sequence precludes perfect complementarity between snR19 and the invariant yeast 5' junction (GTATGT), which differs from the mammalian consensus at the fourth position (GTPuAGT).     

An essential signaling role for the m3G cap in the transport of U1 snRNP to the nucleus

The major small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4 + U6, and U5 have to be transported from the cytoplasm, where they are synthesized, to the nucleus, where they splice pre-messenger RNAs. Since the free core snRNP proteins in the cytoplasm do not enter the nucleus on their own, the nuclear location signal must either reside on the snRNA or be created as a result of snRNA-protein interaction. Here the involvement by the 5'-terminal cap of snRNA molecules in the nucleo-cytoplasmic transport of UsnRNPs has been studied by microinjection of synthetic U1 RNA molecules into frog oocytes; the U1 RNA bore either the normal cap (m3G) or a chemical derivative. Antibodies in the cytoplasm against the m3G cap inhibited the nuclear uptake of U1 snRNP. U1 RNA that was uncapped or contained an unnatural ApppG cap did not enter the nucleus, even though it carried a normal complement of protein molecules. When the ribose ring of the m3G cap was oxidized with periodate, nuclear transport of U1 snRNPs was severely inhibited. Finally, microinjection of m3G cap alone (but not m7G cap) into oocytes severely inhibited the transport of U1 snRNPs to the nucleus. These data suggest that one step in the nuclear uptake of U1 snRNPs involves the m3G cap structure.     

U1 small nuclear RNA genes are subject to dosage compensation in mouse cells

Multiple copies of a gene that encodes human U1 small nuclear RNA were introduced into mouse C127 cells with bovine papilloma virus as the vector. For some recombinant constructions, the human U1 gene copies were maintained extrachromosomally on the viral episome in an unrearranged fashion. The relative abundance of human and mouse U1 small nuclear RNA varied from one cell line to another, but in some lines human U1 RNA accounted for as much as one-third of the total U1. Regardless of the level of human U1 expression, the total amount of U1 RNA (both mouse and human) in each cell line was nearly the same relative to endogenous mouse 5S or U2 RNA. This result was obtained whether measurements were made of total cellular U1 or of only the U1 in small nuclear ribonucleoprotein particles that could be precipitated with antibody directed against the Sm antigen. The data suggest that the multigene families encoding mammalian U1 RNA are subject to some form of dosage compensation.     

Association of TALS developmental disorder with defect in minor splicing component U4atac snRNA

The spliceosome, a ribonucleoprotein complex that includes proteins and small nuclear RNAs (snRNAs), catalyzes RNA splicing through intron excision and exon ligation to produce mature messenger RNAs, which, in turn serve as templates for protein translation. We identified four point mutations in the U4atac snRNA component of the minor spliceosome in patients with brain and bone malformations and unexplained postnatal death [microcephalic osteodysplastic primordial dwarfism type 1 (MOPD 1) or Taybi-Linder syndrome (TALS); Mendelian Inheritance in Man ID no. 210710]. Expression of a subgroup of genes, possibly linked to the disease phenotype, and minor intron splicing were affected in cell lines derived from TALS patients. Our findings demonstrate a crucial role of the minor spliceosome component U4atac snRNA in early human development and postnatal survival.     

Aggregation of lysine-containing zeins into protein bodies in Xenopus oocytes

Zeins, the storage proteins of maize, are totally lacking in the essential amino acids lysine and tryptophan. Lysine codons and lysine- and tryptophan-encoding oligonucleotides were introduced at several positions into a 19-kilodalton zein complementary DNA by oligonucleotide-mediated mutagenesis. A 450-base pair open reading frame from a simian virus 40 (SV40) coat protein was also engineered into the zein coding region. Messenger RNAs for the modified zeins were synthesized in vitro with an SP6 RNA polymerase system and injected into Xenopus laevis oocytes. The modifications did not affect the translation, signal peptide cleavage, or stability of the zeins. The ability of the modified zeins to assemble into structures similar to maize protein bodies was assayed by two criteria: assembly into membrane-bound vesicles resistant to exogenously added protease, and ability to self-aggregate into dense structures. All of the modified zeins were membrane-bound; only the one containing a 17-kilodalton SV40 protein fragment was unable to aggregate. These findings suggest that it may be possible to create high-lysine corn by genetic engineering.     

Essential role for the SMN complex in the specificity of snRNP assembly

The Survival of Motor Neurons (SMN) protein, the product of the spinal muscular atrophy-determining gene, is part of a large macromolecular complex (SMN complex) that functions in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Using cell extracts and purified components, we demonstrated that the SMN complex is necessary and sufficient to mediate the ATP-dependent assembly of the core of seven Sm proteins on uridine-rich, small nuclear ribonucleic acids (U snRNAs). In vitro experiments revealed strict requirements for ordered binding of the Sm proteins and the U snRNAs to the SMN complex. Importantly, the SMN complex is necessary to ensure that Sm cores assemble only on correct RNA targets and prevent their otherwise promiscuous association with other RNAs. Thus, the SMN complex functions as a specificity factor essential for the efficient assembly of Sm proteins on U snRNAs and likely protects cells from illicit, and potentially deleterious, nonspecific binding of Sm proteins to RNAs.     

Mutations in U4atac snRNA, a component of the minor spliceosome, in the developmental disorder MOPD I

Small nuclear RNAs (snRNAs) are essential factors in messenger RNA splicing. By means of homozygosity mapping and deep sequencing, we show that a gene encoding U4atac snRNA, a component of the minor U12-dependent spliceosome, is mutated in individuals with microcephalic osteodysplastic primordial dwarfism type I (MOPD I), a severe developmental disorder characterized by extreme intrauterine growth retardation and multiple organ abnormalities. Functional assays showed that mutations (30G>A, 51G>A, 55G>A, and 111G>A) associated with MOPD I cause defective U12-dependent splicing. Endogenous U12-dependent but not U2-dependent introns were found to be poorly spliced in MOPD I patient fibroblast cells. The introduction of wild-type U4atac snRNA into MOPD I cells enhanced U12-dependent splicing. These results illustrate the critical role of minor intron splicing in human development.     

Isolation of mutants of an animal virus in bacteria

Mutants of animal viruses can be isolated in bacteria by recombinant DNA methods. Since no viral functions are required for propagation of recombinants in bacteria, viral mutants with lethal changes in cis- or trans-acting elements can be isolated, as well as partially or conditionally defective mutants. In the cases of viruses with small DNA genomes, such as the tumorigenic simian virus 40 (SV40), the entire viral DNA can be inserted into the bacterial plasmid pBR322 and cloned in Escherichia coli. Recombinant plasmids with a single copy of SV40 DNA cause morphological transformation of mouse cells in culture with the same efficiency as SV40 DNA isolated from virus-infected monkey cells, but the recombinant DNA is noninfectious and replicates poorly in permissive cells. However, SV40 DNA excised from the plasmid replicates as well as authentic viral DNA and is fully infectious. SV40 mutants with small deletions or base substitutions have been isolated by in vitro site-specific or random local mutagenesis of recombinant DNA followed by cloning in E. coli. Many of the mutants thus isolated are defective in specific viral functions.     

An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly

Splicing of nuclear precursor messenger RNA (pre-mRNA) occurs on a large ribonucleoprotein complex, the spliceosome. Several small nuclear ribonucleoproteins (snRNP's) are subunits of this complex that assembles on the pre-mRNA. Although the U1 snRNP is known to recognize the 5' splice site, its roles in spliceosome formation and splice site alignment have been unclear. A new affinity purification method for the spliceosome is described which has provided insight into the very early stages of spliceosome formation in a yeast in vitro splicing system. Surprisingly, the U1 snRNP initially recognizes sequences at or near both splice junctions in the intron. This interaction must occur before the other snRNP's (U2, U4, U5, and U6) can join the complex. The results suggest that interaction of the two splice site regions occurs at an early stage of spliceosome formation and is probably mediated by U1 snRNP and perhaps other factors.     

Small nuclear RNA U2 is base-paired to heterogeneous nuclear RNA

Eukaryotic cells contain a set of low molecular weight nuclear RNA's. One of the more abundant of these is termed U2 RNA. The possibility that U2 RNA is hydrogen-bonded to complementary sequences in other nuclear RNA's was investigated. Cultured human (HeLa) cells were treated with a psoralen derivative that cross-links RNA chains that are base-paired with one another. High molecular weight heterogeneous nuclear RNA was isolated under denaturing conditions, and the psoralen cross-links were reversed. Electrophoresis of the released RNA and hybridization with a human cloned U2 DNA probe revealed that U2 is hydrogen-bonded to complementary sequences in heterogeneous nuclear RNA in vivo. In contrast, U2 RNA is not base-paired with nucleolar RNA, which contains the precursors of ribosomal RNA. The results suggest that U2 RNA participates in messenger RNA processing in the nucleus.     

Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein

The removal of introns from eukaryotic messenger RNA precursors shares mechanistic characteristics with the self-splicing of certain introns, prompting speculation that the catalytic reactions of nuclear pre-messenger RNA splicing are fundamentally RNA-based. The participation of five small nuclear RNAs (snRNAs) in splicing is now well documented. Genetic analysis in yeast has revealed the requirement, in addition, for several dozen proteins. Some of these are tightly bound to snRNAs to form small nuclear ribonucleoproteins (snRNPs); such proteins may promote interactions between snRNAs or between an snRNA and the intron. Other, non-snRNP proteins appear to associate transiently with the spliceosome. Some of these factors, which include RNA-dependent adenosine triphosphatases, may promote the accurate recognition of introns.     

猪卵泡液和胎牛血清对猪小腔卵泡卵母细胞体外成熟的影响

本研究探讨了猪卵泡液(pFF)和胎牛血清(FCS)对猪小腔卵泡卵母细胞体外成熟的影响。将猪小腔卵泡(直径<2mm)卵母细胞-卵丘复合体(COCs)以15～35枚为一组随机置于添加不同浓度(0、5%、10%、20%)pFF(试验一)或不同浓度(0、5%、10%、20%)FCS(试验二)的改良TCM-199成熟液中培养44～48h,观察卵母细胞的核成熟率和卵丘扩散情况。试验一结果显示,成熟液中添加10%pFF与对照组和添加5%、20%pFF组相比,显著提高猪小腔卵泡卵母细胞的核成熟率(28 2%比20 5%、20 9%、22 3%;p<0 05);添加5%和20%pFF组与对照组相比,它们的小腔卵泡卵母细胞核成熟率之间差异不显著(p>0 05);添加5%pFF组的COCs卵丘细胞呈现第3类扩散,添加10%和20%pFF组的COCs卵丘细胞呈现第4类扩散,而不添加pFF的对照组的COCs卵丘细胞呈现第2类扩散。试验二结果显示,成熟液中添加10%FCS与对照组和添加5%、20%FCS组相比,明显提高猪小腔卵泡卵细胞的核成熟率(60 0%比37 5%、40 2%、43 9%;p<0 05);添加5%和20%FCS组与对照组相比,它们的小腔卵泡卵细胞核成熟率之间差异不显著(p<0 05);FCS在COCs体外成熟培养的前22～24h促进了卵丘扩散(3个处理组的COCs卵丘细胞呈现第3类扩散,而对照组COCs的卵丘细胞呈现第2类扩散)。研究结果表明:(1)成熟液中添加pFF可促使猪小     

Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing

U6 is one of the five small nuclear RNA's (snRNA's) that are required for splicing of nuclear precursor messenger RNA (pre-mRNA). The size and sequence of U6 RNA are conserved among organisms as diverse as yeast and man, and so it has been proposed that U6 RNA functions as a catalytic element in splicing. A procedure for in vitro reconstitution of functional yeast U6 small nuclear ribonucleoproteins (snRNP's) with synthetic U6 RNA was applied in an attempt to elucidate the function of yeast U6 RNA. Two domains in U6 RNA were identified, each of which is required for in vitro splicing. Single nucleotide substitutions in these two domains block splicing either at the first or the second step. Invariably, U6 RNA mutants that block the first step of splicing do not enter the spliceosome. On the other hand, those that block the second step of splicing form a spliceosome but block cleavage at the 3' splice site of the intron. In both domains, the positions of base changes that block the second step of splicing correspond exactly to the site of insertion of pre-mRNA-type introns into the U6 gene of two yeast species, providing a possible explanation for the mechanism of how these introns originated and adding further evidence for the proposed catalytic role of U6 RNA.     

U1-specific protein C needed for efficient complex formation of U1 snRNP with a 5' splice site

One of the functions of U1 small nuclear ribonucleoprotein (snRNP) in the splicing reaction of pre-mRNA molecules is the recognition of the 5' splice site. U1 snRNP proteins as well as base-pair interactions between U1 snRNA and the 5' splice site are important for the formation of the snRNP-pre-mRNA complex. To determine which proteins are needed for complex formation, the ability of U1 snRNPs gradually depleted of the U1-specific proteins C, A, and 70k to bind to an RNA molecule containing a 5' splice site sequence was studied in a nitrocellulose filter binding assay. The most significant effect was always observed when protein C was removed, either alone or together with other U1-specific proteins; the binding was reduced by 50 to 60%. Complementation of protein C-deficient U1 snRNPs with purified C protein restored their 5' splice site binding activity. These data suggest that protein C may potentiate the base-pair interaction between U1 RNA and the 5' splice site.     

An origin unwinding activity regulates initiation of DNA replication during mammalian cell cycle

An in vitro assay was developed to study the positive factors that regulate the onset of DNA replication during the mammalian cell cycle. Extracts prepared from cells at defined positions in the cell cycle were used to examine the replication of SV40 DNA in a cell free system. Extracts prepared from S phase cells were ten times more efficient at initiating replication at the SV40 origin than were extracts from G1 cells, whereas elongation rates were similar in G1 and S reactions. At a discrete point in the cell cycle, just before the cell's entry into S, an activity appeared that was required, in conjunction with SV40 T antigen, for site specific initiation at the SV40 origin. This factor had a role in unwinding DNA at the replication origin.     

Transmission of integrated sea urchin histone genes by nuclear transplantation in Xenopus laevis

Sea urchin histone genes contained in a recombinant plasmid pSp102 were microinjected into the cytoplasm of fertilized eggs of Xenopus laevis. By the late blastula stage, plasmid DNA sequences were detected comigrating with the high molecular weight cellular DNA (greater than 48 kilobases). Analysis of the DNA from injected embryos digested with various restriction endonuclease demonstrated that the injected DNA was integrated into the frog genome. Clones of embryos containing the pSp102 DNA sequences were produced by means of nuclear transplantation. Individuals of the same clone contain the pSp102 sequences integrated into similar chromosomal locations. These sites vary between different clones.     

Nuclear factors in B lymphoma enhance splicing of mouse membrane-bound mu mRNA in Xenopus oocytes

Regulation of the synthesis of membrane-bound and secreted immunoglobulin mu heavy chains at the level of RNA processing is an important element for B cell development. The precursor mu RNA is either polyadenylated at the upstream poly(A) site (for the secreted form) or spliced (for the membrane-bound form) in a mutually exclusive manner. When the mouse mu gene linked to the SV40/HSV-TK hybrid promoter was microinjected into Xenopus oocytes, the mu messenger RNA (mRNA) was altered by coinjection of nuclei of mouse surface IgM-bearing B-lymphoma cells to include the synthesis of the membrane-bound form. An increase in the membrane-bound form was not observed when nuclei of IgM-secreting hybridoma cells or fibroblast cells were coinjected. Deletion of the upstream poly(A) site did not eliminate the effect of B-lymphoma nuclei suggesting that membrane-specific splicing is stimulated. Further, splicing of other mu gene introns was not affected by coinjection of B-lymphoma nuclei. These results suggest that mature B cells contain one or more transacting nuclear factors that stimulate splicing specific for membrane-bound mu mRNA.     

绵羊胚胎细胞核移植的研究

采用国产药品和试剂对绵羊进行超数排卵和胚胎细胞核移植。以 McGrath Solter 和Willadsen 两种去(注)核方法,将8-细胞胚胎细胞核经电融合植入去核成熟卵母细胞内,并经琼脂包埋和中间受体培养,使移核胚胎发育。实验结果表明:(1)用国产激素和化学药品进行绵羊胚胎细胞核移植可使移核胚胎在中间受体内发育为囊胚或桑椹胚;(2)去核时卵母细胞质剩得过少可导致移核胚过早(6-细胞期)发生致密化或形成内细胞团细胞数目较少的小囊胚;(3)Willadsen 去(注)核方法的去核和注核操作成功率明显优于 McGrath-Solter 方法;应用McGrath Solter 法的去核率仅为60%;(4)强度为0.63KV/cm,持续160微秒(μs)的一次电脉冲获得的胚胎融合率最高,达71.2%;以同样电场条件刺激未去核卵母细胞的孤雌活化率在71.4%。     

Antibodies to Asp-Asp-Glu-Asp can inhibit transport of nuclear proteins into the nucleus

The signal sequence of simian virus 40 (SV40) large T-antigen for translocation into the nucleus is composed of positively charged amino acids Lys-Lys-Lys-Arg-Lys. Rabbit antibodies to a synthetic peptide containing the negatively charged amino acid sequence Asp-Asp-Asp-Glu-Asp were obtained. Indirect immunofluorescence of the antigens recognized by the antibody was punctate at the nuclear rim or the nuclear surface, depending on the plane of focus. The antibody blocked transport of nuclear proteins into the nucleus. The antigens recognized by the antibody were predominantly localized to the nuclear pores.     

Control of alternative splicing by the differential binding of U1 small nuclear ribonucleoprotein particle.

Cellular factors controlling alternative splicing of precursor messenger RNA are largely unknown, even though this process plays a central role in specifying the diversity of proteins in the eukaryotic cell. For the identification of such factors, a segment of the rat preprotachykinin gene was used in which differential expression of neuropeptides gamma and K is dependent on alternative splicing of the fourth exon (E4). Sequence variants of the three-exon segment, (E3-E4-E5) were created, resulting in a sensitive assay for factors mediating the splicing switch between E4-skipping and E4-inclusion. A dinucleotide mutation in the 5' splice site of E4 that increase base-pairing of this site to U1 small nuclear RNA resulted in uniform selection of E4, whereas a control mutation that destroyed base-pairing resulted in uniform E4-skipping. Affinity selection of spliceosomes formed on these functionally distinct substrates revealed that the extreme difference in splicing was mediated by differential binding of the U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site of E4. These data show that, apart from its established role in selecting 5' splice sites, U1 snRNP plays a fundamental role in 3' exon selection and provides insight into possible mechanisms of alternative splicing.