The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans

In metazoans, most pre-messenger RNAs contain introns that are removed by splicing. The spliced mRNAs are then exported to the cytoplasm. Recent studies showed that splicing promotes efficient mRNA export, but the mechanism for coupling these two processes is not known. Here we show that Aly, the metazoan homologue of the yeast mRNA export factor Yralp (ref. 2), is recruited to messenger ribonucleoprotein (mRNP) complexes generated by splicing. In contrast, Aly does not associate with mRNPs assembled on identical mRNAs that already have no introns or with heterogenous nuclear RNP (hnRNP) complexes. Aly is recruited during spliceosome assembly, and then becomes tightly associated with the spliced mRNP. Aly shuttles between the nucleus and cytoplasm, and excess recombinant Aly increases both the rate and efficiency of mRNA export in vivo. Consistent with its splicing-dependent recruitment, Aly co-localizes with splicing factors in the nucleus. We conclude that splicing is required for efficient mRNA export as a result of coupling between the splicing and the mRNA export machineries.     

An early evolutionary origin for the minor spliceosome

The minor spliceosome is a ribonucleoprotein complex that catalyses the removal of an atypical class of spliceosomal introns (U12-type) from eukaryotic messenger RNAs. It was first identified and characterized in animals, where it was found to contain several unique RNA constituents that share structural similarity with and seem to be functionally analogous to the small nuclear RNAs (snRNAs) contained in the major spliceosome. Subsequently, minor spliceosomal components and U12-type introns have been found in plants but not in fungi. Unlike that of the major spliceosome, which arose early in the eukaryotic lineage, the evolutionary history of the minor spliceosome is unclear because there is evidence of it in so few organisms. Here we report the identification of homologues of minor-spliceosome-specific proteins and snRNAs, and U12-type introns, in distantly related eukaryotic microbes (protists) and in a fungus (Rhizopus oryzae). Cumulatively, our results indicate that the minor spliceosome had an early origin: several of its characteristic constituents are present in representative organisms from all eukaryotic supergroups for which there is any substantial genome sequence information. In addition, our results reveal marked evolutionary conservation of functionally important sequence elements contained within U12-type introns and snRNAs.     

An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins

Plasma membrane receptors for hormones, drugs, neurotransmitters and sensory stimuli are coupled to guanine nucleotide regulatory proteins. Recent cloning of the genes and/or cDNAs for several of these receptors including the visual pigment rhodopsin, the adenylate-cyclase stimulatory beta-adrenergic receptor and two subtypes of muscarinic cholinergic receptors has suggested that these are homologous proteins with several conserved structural and functional features. Whereas the rhodopsin gene consists of five exons interrupted by four introns, surprisingly the human and hamster beta-adrenergic receptor genes contain no introns in either their coding or untranslated sequences. We have cloned and sequenced a DNA fragment in the human genome which cross-hybridizes with a full-length beta 2-adrenergic receptor probe at reduced stringency. Like the beta 2-adrenergic receptor this gene appears to be intronless, containing an uninterrupted long open reading frame which encodes a putative protein with all the expected structural features of a G-protein-coupled receptor.     

The gain of two chloroplast tRNA introns marks the green algal ancestors of land plants

The relationship of green algae to land plants has greatly interested botanists for more than a century. In recent years, several characters, particularly ultrastructural ones, have been used to define a green algal group (Charophyceae) from which land plants are thought to have arisen. Here we provide the first molecular genetic evidence in support of the charophycean origin of land plants. Group II introns have previously been found in both the tRNAAla and tRNAIle genes of all land plant chloroplast DNAs examined, whereas all algae and eubacteria examined have uninterrupted genes. The distribution of these introns in Coleochaete, Nitella and Spirogyra, members of the Charophyceae, confirms that these taxa are part of the lineage that gave rise to land plants. Furthermore, the intron data place Coleochaete and Nitella closer to land plants than Spirogyra. These introns were most probably acquired by the chloroplast genome more than 400-500 million years ago, the time of land plant origin.     

An intron with a constitutive transport element is retained in a Tap messenger RNA

Alternative splicing is a key factor contributing to genetic diversity and evolution. Intron retention, one form of alternative splicing, is common in plants but rare in higher eukaryotes, because messenger RNAs with retained introns are subject to cellular restriction at the level of cytoplasmic export and expression. Often, retention of internal introns restricts the export of these mRNAs and makes them the targets for degradation by the cellular nonsense-mediated decay machinery if they contain premature stop codons. In fact, many of the database entries for complementary DNAs with retained introns represent them as artefacts that would not affect the proteome. Retroviruses are important model systems in studies of regulation of RNAs with retained introns, because their genomic and mRNAs contain one or more unspliced introns. For example, Mason-Pfizer monkey virus overcomes cellular restrictions by using a cis-acting RNA element known as the constitutive transport element (CTE). The CTE interacts directly with the Tap protein (also known as nuclear RNA export factor 1, encoded by NXF1), which is thought to be a principal export receptor for cellular mRNA, leading to the hypothesis that cellular mRNAs with retained introns use cellular CTE equivalents to overcome restrictions to their expression. Here we show that the Tap gene contains a functional CTE in its alternatively spliced intron 10. Tap mRNA containing this intron is exported to the cytoplasm and is present in polyribosomes. A small Tap protein is encoded by this mRNA and can be detected in human and monkey cells. Our results indicate that Tap regulates expression of its own intron-containing RNA through a CTE-mediated mechanism. Thus, CTEs are likely to be important elements that facilitate efficient expression of mammalian mRNAs with retained introns.     

Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium

Genetic transformation of plant cells by Agrobacterium represents a unique case of trans-kingdom DNA transfer. During this process, Agrobacterium exports its transferred (T) DNA and several virulence (Vir) proteins into the host cell, within which T-DNA nuclear import is mediated by VirD2 (ref. 3) and VirE2 (ref. 4) and their host cell interactors AtKAP-alpha and VIP1 (ref. 6), whereas its integration is mediated mainly by host cell proteins. The factors involved in the uncoating of T-DNA from its cognate proteins, which occurs before integration into the host genome, are still unknown. Here, we report that VirF-one of the few known exported Vir proteins whose function in the host cell remains unknown-is involved in targeted proteolysis of VIP1 and VirE2. We show that VirF localizes to the plant cell nucleus and interacts with VIP1, a nuclear protein. VirF, which contains an F-box motif, significantly destabilizes both VIP1 and VirE2 in yeast cells. Destabilization of VIP1 in the presence of VirF was then confirmed in planta. These results suggest that VIP1 and its cognate VirE2 are specifically targeted by the VirF-containing Skp1-Cdc53-cullin-F-box complex for proteolysis. The critical role of proteasomal degradation in Agrobacterium-mediated genetic transformation was also evident from inhibition of T-DNA expression by a proteasomal inhibitor.     

果蝇胚胎不同表达水平基因内含子序列特征的比较分析

对果蝇胚胎低表达和高表达水平基因内含子的序列结构进行分析,发现2种表达水平的基因内含子序列特征有明显差异.高表达基因的内含子一般比低表达基因的长,其中高表达基因第1内含子的平均长度是低表达基因的2.62倍,第2内含子的平均长度是低表达基因的1.79倍.两类基因第1内含子中的CpG岛含量最高,并且高表达基因内含子中CpG岛含量要高于低表达基因.此外,与低表达基因相比,TATA box、CAAT box和GC box在高表达基因内含子中出现的频数明显要高些,尤其是在第1内含子中.作者还提取出果蝇胚胎2种表达水平基因第1内含子中高频出现的6-mer简单重复序列,发现一些重复序列与实验得到的转录因子结合位点相符合.这些结果提示内含子特别是第1内含子有可能调控果蝇胚胎基因的转录从而影响基因的表达水平.     

Self-splicing introns in tRNA genes of widely divergent bacteria.

The organization of eukaryotic genes into exons separated by introns has been considered as a primordial arrangement but because it does not exist in eubacterial genomes it may be that introns are relatively recent acquisitions. A self-splicing group I intron has been found in cyanobacteria at the same position of the same gene (that encoding leucyl transfer RNA, UAA anticodon) as a similar group I intron of chloroplasts, which indicates that this intron predates the invasion of eukaryotic cells by cyanobacterial endosymbionts. But it is not clear from this isolated example whether introns are more generally present in different genes or in more diverse branches of the eubacteria. Many mitochondria have intron-rich genomes and were probably derived from the alpha subgroup of the purple bacteria (or Proteobacteria), so ancient introns might also have been retained in these bacteria. We describe here the discovery of two small (237 and 205 nucleotides) self-splicing group I introns in members of two proteobacterial subgroups, Agrobacterium tumefaciens (alpha) and Azoarcus sp. (beta). The introns are inserted in genes for tRNA(Arg) and tRNA(Ile), respectively, after the third anticodon nucleotide. Their occurrence in different genes of phylogenetically diverse bacteria indicates that group I introns have a widespread distribution among eubacteria.     

Unusual sequences in the murine immunoglobulin mu-delta heavy-chain region

The delta heavy (H) chain of mouse immunoglobulin D (IgD) is unusual both in its structure and in its differential expression relative to immunoglobulin M (IgM; reviewed in ref. 1). The region of DNA between IgM and IgD H-chain constant-region genes is probably implicated in this control. So far only fragments of the area have been sequenced. Now, however, we present the complete sequence as well as the sequence of the introns of the C delta gene. We have found several interesting features (Fig. 1), including an open reading frame (ORF) between Cmu and C delta which encodes 146 amino acids that might represent a previously unsuspected domain-like protein; three blocks of simple repetitive sequences; a 162-base pair (bp) unique-sequence inverted repeat; and a domain-like pseudogene in the large intron of C delta. We have not found, however, any sequence 5' of C delta resembling the switch (S) recombination sequences associated with class switching in other heavy chains. Moreover, we have determined the 3' deletion end point of an IgD-producing myeloma and find no sequences reminiscent of switch sites nearby.     

Spontaneous shuffling of domains between introns of phage T4

The three self-splicing introns in phage T4 (in the td, sunY and nrdB genes) (Fig. 1a) each have the conserved group I catalytic RNA core structure (Fig. 1b), out of which is looped an open reading frame. Although the core sequences are very similar (approximately 60% identity), the open reading frames seem to be unrelated. Single crossover recombination events between homologous core sequences in the closely linked td and nrdB introns have led to 'exon shuffling. Here we describe spontaneous double crossovers between the unlinked td and sun Y introns that result in shuffling of an intron structure element, P7.1 (refs 3 and 4). The intron domain-switch variants were isolated as genetic suppressors of a splicing-defective P7.1 deletion in the td intron. This unprecedented example of suppression through inter-intron sequence substitution indicates that the introns are in a state of genetic flux and implies the functional interchangeability of the two analogous but nonidentical P7.1 elements. The implications of such recombination events are discussed in the light of the evolution of the introns themselves as well as that of their host genomes.     

Reverse self-splicing of group II intron RNAs in vitro.

Group II introns, which are classed together on the basis of a conserved secondary structure, are found in organellar genes of lower eukaryotes and plants. Like introns in nuclear pre-messenger RNA, they are excised by a two-step splicing reaction to generate branched circular RNAs, the so-called lariats. A remarkable feature of group II introns is their self-splicing activity in vitro. In the absence of a nucleotide cofactor, the intron RNAs catalyse two successive transesterification reactions which lead to autocatalytic excision of the lariat IVS from pre-mRNA and concomitantly to exon ligation. By virtue of its ability to specifically bind the 5' exon, the intron can also catalyse such reactions on exogenous RNA substrates. This sequence-specific attachment could enable group II introns to integrate into unrelated RNAs by reverse splicing, in a process similar to that described for the self-splicing Tetrahymena group I intron. Here we report that group II lariat IVS can indeed reintegrate itself into an RNA composed of the ligated exons in vitro. This occurs by a process of self-splicing that completely reverses both transesterification steps of the forward reaction: it involves a transition of the 2'-5' phosphodiester bond of the lariat RNA into the 3'-5' bond of the reconstituted 5' splice junction.     

Translational control of intron splicing in eukaryotes

Most eukaryotic genes are interrupted by non-coding introns that must be accurately removed from pre-messenger RNAs to produce translatable mRNAs. Splicing is guided locally by short conserved sequences, but genes typically contain many potential splice sites, and the mechanisms specifying the correct sites remain poorly understood. In most organisms, short introns recognized by the intron definition mechanism cannot be efficiently predicted solely on the basis of sequence motifs. In multicellular eukaryotes, long introns are recognized through exon definition and most genes produce multiple mRNA variants through alternative splicing. The nonsense-mediated mRNA decay (NMD) pathway may further shape the observed sets of variants by selectively degrading those containing premature termination codons, which are frequently produced in mammals. Here we show that the tiny introns of the ciliate Paramecium tetraurelia are under strong selective pressure to cause premature termination of mRNA translation in the event of intron retention, and that the same bias is observed among the short introns of plants, fungi and animals. By knocking down the two P. tetraurelia genes encoding UPF1, a protein that is crucial in NMD, we show that the intrinsic efficiency of splicing varies widely among introns and that NMD activity can significantly reduce the fraction of unspliced mRNAs. The results suggest that, independently of alternative splicing, species with large intron numbers universally rely on NMD to compensate for suboptimal splicing efficiency and accuracy.     

Style self-incompatibility gene products of Nicotiana alata are ribonucleases

Self-incompatibility in flowering plants is often controlled by a single nuclear gene (the S-gene) having several alleles. This gene prevents fertilization by self-pollen or by pollen bearing either of the two S-alleles expressed in the style. Sequence analysis shows that three alleles of the S gene of Nicotiana alata encode style glycoproteins with regions of defined homology. Two of the homologous regions also show precise homology with ribonucleases T2 (ref. 4) and Rh (ref. 5). We report here that glycoproteins corresponding to the S1, S2, S3, S6 and S7 alleles isolated from style extracts of N. alata are ribonucleases. These style S-gene-encoded glycoproteins account for most of the ribonuclease activity recovered from style extracts. The ribonuclease specific activity of style extracts of the self-incompatible species N. alata is 100-1,000-fold higher than that of the related self-compatible species N. tabacum. These observations implicate ribonuclease activity in the mechanism of gametophytic self-incompatibility.     

Mouse Cmu heavy chain immunoglobulin gene segment contains three intervening sequences separating domains

The IgM molecule is composed of subunits made up of two light chain and two heavy chain (mu) polypeptides. The mu chain is encoded by several gene segments--variable (V), joining (J) and constant (Cmu). The Cmu gene segment is of particular interest for several reasons. First, the mu chain must exist in two very different environments--as an integral membrane protein in receptor IgM molecules (micrometer) and as soluble serum protein in IgM molecules into the blood (mus). Second, the Cmu region in mus is composed of four homology units or domains (Cmu1, Cmu2, Cmu3 and Cmu4) of approximately 110 amino acid residues plus a C-terminal tail of 19 residues. We asked two questions concerning the organisation of the Cmu gene segment. (1) Are the homology units separated by intervening DNA sequences as has been reported for alpha (ref. 5), gamma 1 (ref. 6) and gamma 2b (ref. 7) heavy chain genes? (2) Is the C-terminal tail separated from the Cmu4 domain by an intervening DNA sequence? If so, DNA rearrangements or RNA splicing could generate hydrophilic and hydrophobic C-terminal tails for the mus and micrometer polypeptides, respectively. We demonstrate here that intervening DNA sequences separate each of the four coding regions for Cmu domains, and that the coding regions for the Cmu4 domains and the C-terminal tail are directly contiguous.     

Sequence organization and genomic complexity of primate theta 1 globin gene, a novel alpha-globin-like gene

The alpha-like and beta-like globin genes have provided a paradigm for the study of molecular evolution and regulation of multigene families in eukaryotes. The human alpha-globin gene cluster, which is on chromosome 16 (ref. 1), consists of six genes arranged in the order 5'-zeta(embryonic)-psi zeta-psi alpha 2-psi alpha 1-alpha 2(adult)-alpha 1(adult)-3'. DNA sequencing data have demonstrated that zeta (ref. 6) and alpha 2 (or alpha 1, refs 7-9) are the embryonic and adult genes, respectively, while psi zeta (ref. 6), psi alpha 2 (ref. 5) psi alpha 1 (ref. 10) are all inactive pseudogenes. Restriction mapping analysis has shown that the structure of this locus in several anthropoid primates is nearly identical to that of the human. Recently, we have isolated the adult alpha-globin gene region from orang-utan, olive baboon and rhesus macaque by molecular cloning. We report here the complete nucleotide sequence of a gene located immediately downstream from the adult alpha 1-globin gene of the orang-utan, along with its flanking DNA. We designate this gene as theta 1, and show that it contains the essential sequence elements required for an expressive gene. The putative polypeptide is 141 amino acids long, identical to that of the alpha- or zeta-globin, but its predicted amino-acid sequence is nearly as different from the orang-utan alpha-globin (55 differences) as the human zeta-globin is from the human alpha-globin (59 differences), suggesting an ancient history for the theta 1-globin gene. Results of blot hybridization experiments using the cloned orang-utan theta 1 gene sequence as probe demonstrate a similar alpha 2-alpha 1-theta 1 linkage map existing in the human genome. Furthermore, multiple copies of sequences homologous to the theta 1 gene are detected in both human and orang-utan. These results cast a new light on the primate alpha-globin gene family, and have intriguing implications for the existence of previously unreported, functional globin-like gene(s) in the primate genomes.     

Correlations between recombination rate and intron distributions along chromosomes of C. elegans

Generally speaking, the intron size positively correlates with recombination rate in Caenorhabditis elegans genome. Here, we analyze the correlations between recombination rate and some measures of different intron lengths so as to know whether the recombination influences the introns of different lengths in the same way. Results show that the correlation between the recombination rate and the percentage of short introns (<100 bp) is negative, but the correlation between the recombination rate and the percentage of introns that are larger than 500 bp is positive. Average intron length correlates positively with the recombination rate for introns whose length is in the range of 100–1000 bp. We speculate that the recombination mainly exerts impact on introns whose length ranges from 100–1000 bp. We also show that the average intron number per gene correlates negatively with the recombination rate.     

A chloride channel widely expressed in epithelial and non-epithelial cells.

Chloride channels have several functions, including the regulation of cell volume, stabilizing membrane potential, signal transduction and transepithelial transport. The plasma membrane Cl- channels already cloned belong to different structural classes: ligand-gated channels, voltage-gated channels, and possibly transporters of the ATP-binding-cassette type (if the cystic fibrosis transmembrane regulator is a Cl- channel). The importance of chloride channels is illustrated by the phenotypes that can result from their malfunction: cystic fibrosis, in which transepithelial transport is impaired, and myotonia, in which ClC-1, the principal skeletal muscle Cl- channel, is defective. Here we report the properties of ClC-2, a new member of the voltage-gated Cl- channel family. Its sequence is approximately 50% identical to either the Torpedo electroplax Cl- channel, ClC-0 (ref. 8), or the rat muscle Cl- channel, ClC-1 (ref. 9). Isolated initially from rat heart and brain, it is also expressed in pancreas, lung and liver, for example, and in pure cell lines of fibroblastic, neuronal, and epithelial origin, including tissues and cells affected by cystic fibrosis. Expression in Xenopus oocytes induces Cl- currents that activate slowly upon hyperpolarization and display a linear instantaneous current-voltage relationship. The conductivity sequence is Cl- greater than or equal to Br- greater than I-. The presence of ClC-2 in such different cell types contrasts with the highly specialized expression of ClC-1 (ref. 9) and also with the cloned cation channels, and suggests that its function is important for most cells.     

Intron-dependent evolution of chicken glyceraldehyde phosphate dehydrogenase gene

The function of introns in the evolution of genes can be explained in at least two ways: either introns appeared late in evolution and therefore could not have participated in the construction of primordial genes, or RNA splicing and introns existed in the earliest organisms but were lost during the evolution of the modern prokaryotes. The latter alternative allows the possibility of intron participation in the formation of primordial genes before the divergence of modern prokaryotes and eukaryotes. Blake suggested that evidence for intron-facilitated evolution of a gene might be found by comparing the borders of functional protein domains with the placement of introns. We therefore examined glyceraldehyde phosphate dehydrogenase (GAPDH), a glycolytic enzyme, because it is the first protein for which the following data are available: X-ray crystallographic studies demonstrating structurally independent protein 'domains' which were highly conserved during the divergence of prokaryotes and eukaryotes; and a study of genomic organization which mapped introns in the gene. Sequencing of the chicken GAPDH gene revealed 11 introns. We report here that sites of three of the introns (IV, VI and XI) correspond closely with the borders of the NAD-binding, catalytic and helical tail domains of the enzyme, supporting the hypothesis that introns did have a role in the evolution of primitive genes. In addition, other biochemical and structural data were used to construct a model of the intron-mediated assembly of the GAPDH gene that explains the existence of 10 introns.     

南方红豆杉紫杉烷7β-羟基化酶基因全长序列的克隆和进化分析

 从南方红豆杉Taxus wallichiana var.mairei的新鲜嫩叶中提取基因组DNA作为模板,利用三组特异引物进行PCR扩增,然后克隆测序得到紫杉烷7β-羟基化酶的基因全长。该基因编码区起始密码子为ATG,终止密码子为TGA,全长1 692 bp;碱基组成为490 A (29.0%),351 C (20.7%),362 G (21.4%)和489 T (28.9%)。将紫杉烷7β-羟基化酶基因全长序列与细胞色素P450基因家族的其它三个成员进行比对,发现它与紫杉烷2α-羟基化酶基因、紫杉烷10β-羟基化酶基因及紫杉烷13α-羟基化酶基因的一致性分别为74%、68%及76%。它们的外显子和内含子的连接区均具保守的GT-AG结构,内含子区的变异性明显高于外显子区。进一步以红豆杉属的13个紫杉烷羟基化酶基因家族成员为对象,利用位点间可变ω(非同义替换率dN和同义替换率dS的比值) 模型对该基因家族的适应性进化进行分析。分支模型、位点模型以及分支－位点模型的分析表明：紫杉烷羟基化酶基因家族的少数分支处于正选择压力下(ω>1),但未检测到正选择位点;而绝大部分位点受强烈的负选择作用(ω<1)。