Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome

The splicing process, which removes intervening sequences from messenger RNA (mRNA) precursors is essential to gene expression in eukaryotic cells. This site-specific process requires precise sequence recognition at the boundaries of an intervening sequence, but the mechanism of this recognition is not understood. The splicing of mRNA precursors occurs in a multicomponent complex termed the spliceosome. Such an assembly of components is likely to play a key role in specifying those sequences to be spliced. In order to analyze spliceosome structure, a stringent approach was developed to obtain splicing complexes free of cellular contaminants. This approach is a form of affinity chromatography based on the high specificity of the biotin-streptavidin interaction. A minimum of three subunits: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles were identified in the 35S spliceosome structure, which also contains the bipartite RNA intermediate of splicing. A 25S presplicing complex contained only the U2 particle. The multiple subunit structure of the spliceosome has implications for the regulation of a splicing event and for its possible catalysis by ribozyme or ribozymes.     

Both catalytic steps of nuclear pre-mRNA splicing are reversible

Nuclear pre-messenger RNA (pre-mRNA) splicing is an essential processing step for the production of mature mRNAs from most eukaryotic genes. Splicing is catalyzed by a large ribonucleoprotein complex, the spliceosome, which is composed of five small nuclear RNAs and more than 100 protein factors. Despite the complexity of the spliceosome, the chemistry of the splicing reaction is simple, consisting of two consecutive transesterification reactions. The presence of introns in spliceosomal RNAs of certain fungi has suggested that splicing may be reversible; however, this has never been demonstrated experimentally. By using affinity-purified spliceosomes, we have shown that both catalytic steps of splicing can be efficiently reversed under appropriate conditions. These results provide considerable insight into the catalytic flexibility of the spliceosome.     

RNA required for import of precursor proteins into mitochondria

A cytoplasmic RNA moiety is necessary for posttranslational uptake of nuclear-encoded mammalian proteins destined for the mitochondrial matrix. Post-translational addition of ribonuclease to a reticulocyte lysate-programmed cell-free translation mixture inhibited subsequent import of six different mitochondrial matrix enzyme precursors into rat liver mitochondria. The required RNA is highly protected, as indicated by the high concentrations of ribonuclease necessary to produce this inhibition. The dependence of the inhibitory effect on temperature, duration of exposure to ribonuclease, and availability of divalent cations is characteristic of the nuclease susceptibility of ribonucleoproteins. The ribonuclease-sensitive component was found in a 400-kilodalton fraction which contains the mitochondrial protein precursors.     

Differential control of U1 small nuclear RNA expression during mouse development

During normal mouse development the relative amounts of two types of U1 small nuclear RNA's (U1 RNA) change significantly. Fetal tissues have comparable levels of the two major types of mouse U1 RNA's, mU1a and mU1b, whereas most differentiated adult tissues contain only mU1a RNA's. Those adult tissues that also accumulate detectable amounts of embryonic (mU1b) RNA's (for example, testis, spleen, and thymus) contain a significant proportion of stem cells capable of further differentiation. Several strains of mice express minor sequence variants of U1 RNA's that are subject to the same developmental controls as the major types of adult and embryonic U1 RNA. The differential accumulation of embryonic U1 RNA's may influence the pattern of gene expression during early development and differentiation.     

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.     

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).     

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.     

A small viral RNA is required for in vitro packaging of bacteriophage phi 29 DNA

A small RNA of Bacillus subtilis bacteriophage phi 29 is shown to have a novel and essential role in viral DNA packaging in vitro. This requirement for RNA in the encapsidation of viral DNA provides a new dimension of complexity to the attendant protein-DNA interactions. The RNA is a constituent of the viral precursor shell of the DNA-packaging machine but is not a component of the mature virion. Studies of the sequential interactions involving this RNA molecule are likely to provide new insight into the structural and possible catalytic roles of small RNA molecules. The phi 29 assembly in extracts and phi 29 DNA packaging in the defined in vitro system were strongly inhibited by treatment with the ribonucleases A or T1. However, phage assembly occurred normally in the presence of ribonuclease A that had been treated with a ribonuclease inhibitor. An RNA of approximately 120 nucleotides co-purified with the phi 29 precursor protein shell (prohead), and this particle was the target of ribonuclease action. Removal of RNA from the prohead by ribonuclease rendered it inactive for DNA packaging. By RNA-DNA hybridization analysis, the RNA was shown to originate from a viral DNA segment very near the left end of the genome, the end packaged first during in vitro assembly.     

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.     

2种荔枝RNA提取方法的比较

比较利用TRIZOL法和改进SDS法2种方法提取荔枝叶片总RNA的可行性。通过琼脂糖凝胶电泳及紫外分光光度计检测,对提取所得RNA的完整性、纯度及浓度进行分析。试验结果表明,改进SDS法适合荔枝叶片总RNA的提取,能有效获得纯度高、完整性好的RNA样品。     

Structure of PTB bound to RNA: specific binding and implications for splicing regulation

The polypyrimidine tract binding protein (PTB) is a 58-kilodalton RNA binding protein involved in multiple aspects of messenger RNA metabolism, including the repression of alternative exons. We have determined the solution structures of the four RNA binding domains (RBDs) of PTB, each bound to a CUCUCU oligonucleotide. Each RBD binds RNA with a different binding specificity. RBD3 and RBD4 interact, resulting in an antiparallel orientation of their bound RNAs. Thus, PTB will induce RNA looping when bound to two separated pyrimidine tracts within the same RNA. This leads to structural models for how PTB functions as an alternative-splicing repressor.     

2种方法从大鼠晶状体中提取总RNA及评测

[目的]测评2种方法从大鼠晶体中提取总RNA的效果[方法]采用2种方法从大鼠晶状体中提取总RNA,通过测定总RNA的浓度以及A260/A280、A260/A230、琼脂糖凝胶电泳、RT-PCR方法对其进行评价。[结果]使用柱式法从大鼠晶状体中提取总RNA,其28S、18S亚基清晰可见,Nano Drop2000测定显示A260/A280、A260/A230均在1.8~2.0,可用于反转录聚合酶链反应等下游试验。Trizol法提取结果显示总RNA降解较多,不适合进行下游试验。[结论]柱式法操作简便,全程可在常温下进行,总RNA质量高,适宜于实验教学及科研。Trizol法提取总RNA操作要求相对较高,且易受到苯酚污染及乙醇残留,但适合抽提大量样本的总RNA,对于实验教学有一定难度。