Mutations in the U5 snRNA result in altered splicing of subsets of pre-mRNAs and reduced stability of Prp8

The U5 snRNA loop 1 aligns the 5′ and 3′ exons for ligation during the second step of pre-mRNA splicing. U5 is intimately associated with Prp8, which mediates pre-mRNA repositioning within the catalytic core of the spliceosome and interacts directly with U5 loop 1. The genome-wide effect of three U5 loop 1 mutants has been assessed by microarray analysis. These mutants exhibited impaired and improved splicing of subsets of pre-mRNAs compared to wild-type U5. Analysis of pre-mRNAs that accumulate revealed a change in base prevalence at specific positions near the splice sites. Analysis of processed pre-mRNAs exhibiting mRNA accumulation revealed a bias in base prevalence at one position within the 5′ exon. While U5 loop 1 can interact with some of these positions the base bias is not directly related to sequence changes in loop 1. All positions that display a bias in base prevalence are at or next to positions known to interact with Prp8. Analysis of Prp8 in the presence of the three U5 loop 1 mutants revealed that the most severe mutant displayed reduced Prp8 stability. Depletion of U5 snRNA in vivo also resulted in reduced Prp8 stability. Our data suggest that certain mutations in U5 loop 1 perturb the stability of Prp8 and may affect interactions of Prp8 with a subset of pre-mRNAs influencing their splicing. Therefore, the integrity of U5 is important for the stability of Prp8 during splicing and provides one possible explanation for why U5 loop 1 and Prp8 are so highly conserved.     

粗糙脉孢菌snRNA基因的克隆及其表达调控研究

Pre‐m RNA(precursor m RNA)的剪接是真核基因表达中的重要一环,由剪接体复合物(spliceosome)催化完成。小核RNA(small nuclear RNAs,sn RNAs)是剪接体的重要结构和功能组分。本工作首次鉴定了粗糙脉孢菌的U1、U2、U4、U5和U6等sn RNA基因,这些基因除U5为单一拷贝外,其余为多拷贝基因且表达量存在差异。对各基因的近端序列元件(proximal sequence elements,PSEs)的分析显示在大部分基因都存在一段回文的保守序列GTGCAC,荧光素酶报告基因实验证实该序列具有调控部分sn RNA基因转录的功能。我们还通过温度梯度实验检测了stk‐16第三内含子的剪接情况变化,结果提示可变剪接对调节生物可能对不同温度环境的适应具有重要作用。     

Structural basis for the bifunctionality of the U5 snRNP 52K protein (CD2BP2)

The bifunctional protein U5-52K is associated with the spliceosomal 20 S U5 snRNP, and it also plays a role in immune response as CD2 receptor binding protein 2 (CD2BP2). U5-52K binds to the CD2 receptor via its GYF-domain specifically recognizing a proline-rich motif on the cytoplasmic surface of the receptor. The GYF-domain is also mediating the interaction of the proteins U5-52K and U5-15K within the spliceosomal U5 snRNP. Here we report the crystal structure of the complex of GYF-domain and U5-15K protein revealing the structural basis for the bifunctionality of the U5-52K protein. The complex structure unveils novel interaction sites on both proteins, as neither the polyproline-binding site of the GYF-domain nor the common ligand-binding cleft of thioredoxin-like proteins, to which U5-15K belongs, are involved in the interaction of U5-15K and U5-52K.     

Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly

Pseudouridine (Ψ) is the most abundant internal modification identified in RNA, and yet little is understood of its effects on downstream reactions. Yeast U2 snRNA contains three conserved Ψs (Ψ35, Ψ42, and Ψ44) in the branch site recognition region (BSRR), which base pairs with the pre‐mRNA branch site during splicing. Here, we show that blocks to pseudouridylation at these positions reduce the efficiency of pre‐mRNA splicing, leading to growth‐deficient phenotypes. Restoration of pseudouridylation at these positions using designer snoRNAs results in near complete rescue of splicing and cell growth. These Ψs interact genetically with Prp5, an RNA‐dependent ATPase involved in monitoring the U2 BSRR‐branch site base‐pairing interaction. Biochemical analysis indicates that Prp5 has reduced affinity for U2 snRNA that lacks Ψ42 and Ψ44 and that Prp5 ATPase activity is reduced when stimulated by U2 lacking Ψ42 or Ψ44 relative to wild type, resulting in inefficient spliceosome assembly. Furthermore, in vivo DMS probing analysis reveals that pseudouridylated U2, compared to U2 lacking Ψ42 and Ψ44, adopts a slightly different structure in the branch site recognition region. Taken together, our results indicate that the Ψs in U2 snRNA contribute to pre‐mRNA splicing by directly altering the binding/ATPase activity of Prp5.     

玉米雌穗发育期基因差异表达与杂种优势的研究

杂种优势在提高粮食作物特别是玉米的产量方面具有重要的作用。然而,杂种优势的原理却仍然是一个世界性的难题。用12个玉米自交系及其按不完全双列杂交组配的33个杂交种为材料,分4个不同发育时期取杂交种及其亲本的雌穗组织,利用差异显示技术,分析杂种与亲本的基因差异表达类型及其与7个主要农艺性状的杂种表现和杂种优势的相关关系。发现1):在5种表达类型中单态表达(基因在杂交种和双亲中同时表达的类型)的数量最大,这说明杂种优势的形成不仅与基因的表达与否相关,还与大量基因的上调或下调表达相关;2):在玉米雌幼穗的发育初期杂交种与双亲的基因表达差异最大,这可能与雌穗发育初期器官的形成和发育相关,因此这一时期差异表达(在质的方面)的基因对产量性状和杂种优势的形成具有密切关系;3):综合各种基因表达类型与产量性状和杂种优势的关系,发现某些基因在杂种中的沉默表达可以促进籽粒的发育和抑制幼穗中小花的发育。     

DExD/H-box Prp5 protein is in the spliceosome during most of the splicing cycle

The DExD/H-box Prp5 protein (Prp5p) is an essential, RNA-dependent ATPase required for pre-spliceosome formation during nuclear pre-mRNA splicing. In order to understand how this protein functions, we used in vitro, biochemical assays to examine its association with the spliceosome from Saccharomyces cerevisiae. GST-Prp5p in splicing assays pulls down radiolabeled pre-mRNA as well as splicing intermediates and lariat product, but reduced amounts of spliced mRNA. It cosediments with active spliceosomes isolated by glycerol gradient centrifugation. In ATP-depleted extracts, GST-Prp5p associates with pre-mRNA even in the absence of spliceosomal snRNAs. Maximal selection in either the presence or absence of ATP requires a pre-mRNA with a functional intron. Prp5p is present in the commitment complex and functions in subsequent pre-spliceosome formation. Reduced Prp5p levels decrease levels of commitment, pre-spliceosomal and spliceosomal complexes. Thus Prp5p is most likely an integral component of the spliceosome, being among the first splicing factors associating with pre-mRNA and remaining until spliceosome disassembly. The results suggest a model in which Prp5p recruits the U2 snRNP to pre-mRNA in the commitment complex and then hydrolyzes ATP to promote stable association of U2 in the pre-spliceosome. They also suggest that Prp5p could have multiple ATP-independent and ATP-dependent functions at several stages of the splicing cycle.     

Structure, organization and expression of the eukaryotic translation initiation factor 5, eIF-5, gene in Zea mays

The maize genomic DNA sequence encoding the eukaryotic translation initiation factor 5 (eIF-5) has been isolated from genomic library of maize seedlings and the exon–intron structure determined (accession number AJ132240). The length of genomic DNA sequenced was about 7 kb and contained two exons with the translation start site in exon 2. The only intron is located in the non-coding 5′ region and it is 1298 bp long with the splice acceptor and donor sites conforming to the AG/GT rules. Repetitive sequence fragments are located in the 5′ and 3′ intergenic region. The accumulation of eIF-5 mRNA was studied by RNA blot and in situ hybridization. The observed distribution of mRNA may correlate with the function of the protein, as it appears to be highly abundant in tissues where the proportion of cells actively dividing is very high, such as meristematic regions.     

Direct probing of RNA structure and RNA-protein interactions in purified HeLa cell's and yeast spliceosomal U4/U6.U5 tri-snRNP particles

The U4/U6.U5 tri-snRNP is a key component of spliceosomes. By using chemical reagents and RNases, we performed the first extensive experimental analysis of the structure and accessibility of U4 and U6 snRNAs in tri-snRNPs. These were purified from HeLa cell nuclear extract and Saccharomyces cerevisiae cellular extract. U5 accessibility was also investigated. For both species, data demonstrate the formation of the U4/U6 Y-shaped structure. In the human tri-snRNP and U4/U6 snRNP, U6 forms the long range interaction, that was previously proposed to be responsible for dissociation of the deproteinized U4/U6 duplex. In both yeast and human tri-snRNPs, U5 is more protected than U4 and U6, suggesting that the U5 snRNP-specific protein complex and other components of the tri-snRNP wrapped the 5' stem-loop of U5. Loop I of U5 is partially accessible, and chemical modifications of loop I were identical in yeast and human tri-snRNPs. This reflects a strong conservation of the interactions of proteins with the functional loop I. Only some parts of the U4/U6 Y-shaped motif (the 5' stem-loop of U4 and helix II) are protected. Due to difference of protein composition of yeast and human tri-snRNP, the U6 segment linking the 5' stem-loop to the Y-shaped structure and the U4 central single-stranded segment are more accessible in the yeast than in the human tri-snRNP, especially, the phylogenetically conserved ACAGAG sequence of U6. Data are discussed taking into account knowledge on RNA and protein components of yeast and human snRNPs and their involvement in splicesome assembly.     

Regulation of the maize ubiquitin (Ubi-1) promoter in developing maize (Zea mays L.) seeds examined using transient gene expression in kernels grown in vitro

Kernel culture was assessed for evaluating novel gene expression in developing maize (Zea mays L.) seeds by comparing the transient expression of maize ubiquitin (Ubi-1) promoter-driven β-glucuronidase (GUS) delivered by particle bombardment in kernels grown in culture with those grown in planta. With kernels from either source, GUS expression, as determined by histochemical staining, was widespread in young, actively growing kernels, but it diminished with kernel age and by 25 days after pollination was found only in the embryo. Transient expression of Ubi-1 in kernels grown in vitro was not affected by wounding, ethylene treatment, pathogen invasion, or heat shock. In contrast, the plant hormones indole-3-acetic acid and kinetin both stimulated transient Ubi-1 expression in the endosperm, particularly at the periphery. Transient gene expression in developing maize seeds grown in vitro should allow for facile and rapid evaluation of the tissue-specificity and environmental responses of novel gene constructs in developing maize seeds. Received: 31 July 1997 / Revision received: 13 October 1997 / Accepted: 31 October 1997