Showing papers on "Intron published in 2003"

Mechanisms of Alternative Pre-Messenger RNA Splicing

Abstract: Alternative pre-mRNA splicing is a central mode of genetic regulation in higher eukaryotes. Variability in splicing patterns is a major source of protein diversity from the genome. In this review, I describe what is currently known of the molecular mechanisms that control changes in splice site choice. I start with the best-characterized systems from the Drosophila sex determination pathway, and then describe the regulators of other systems about whose mechanisms there is some data. How these regulators are combined into complex systems of tissue-specific splicing is discussed. In conclusion, very recent studies are presented that point to new directions for understanding alternative splicing and its mechanisms.

Genome-Wide Analysis of NBS-LRR–Encoding Genes in Arabidopsis

Abstract: The Arabidopsis genome contains ∼200 genes that encode proteins with similarity to the nucleotide binding site and other domains characteristic of plant resistance proteins. Through a reiterative process of sequence analysis and reannotation, we identified 149 NBS-LRR–encoding genes in the Arabidopsis (ecotype Columbia) genomic sequence. Fifty-six of these genes were corrected from earlier annotations. At least 12 are predicted to be pseudogenes. As described previously, two distinct groups of sequences were identified: those that encoded an N-terminal domain with Toll/Interleukin-1 Receptor homology (TIR-NBS-LRR, or TNL), and those that encoded an N-terminal coiled-coil motif (CC-NBS-LRR, or CNL). The encoded proteins are distinct from the 58 predicted adapter proteins in the previously described TIR-X, TIR-NBS, and CC-NBS groups. Classification based on protein domains, intron positions, sequence conservation, and genome distribution defined four subgroups of CNL proteins, eight subgroups of TNL proteins, and a pair of divergent NL proteins that lack a defined N-terminal motif. CNL proteins generally were encoded in single exons, although two subclasses were identified that contained introns in unique positions. TNL proteins were encoded in modular exons, with conserved intron positions separating distinct protein domains. Conserved motifs were identified in the LRRs of both CNL and TNL proteins. In contrast to CNL proteins, TNL proteins contained large and variable C-terminal domains. The extant distribution and diversity of the NBS-LRR sequences has been generated by extensive duplication and ectopic rearrangements that involved segmental duplications as well as microscale events. The observed diversity of these NBS-LRR proteins indicates the variety of recognition molecules available in an individual genotype to detect diverse biotic challenges.

Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies

Abstract: The spliced alignment of expressed sequence data to genomic sequence has proven a key tool in the comprehensive annotation of genes in eukaryotic genomes. A novel algorithm was developed to assemble clusters of overlapping transcript alignments (ESTs and full-length cDNAs) into maximal alignment assemblies, thereby comprehensively incorporating all available transcript data and capturing subtle splicing variations. Complete and partial gene structures identified by this method were used to improve The Institute for Genomic Research Arabidopsis genome annotation (TIGR release v.4.0). The alignment assemblies permitted the automated modeling of several novel genes and >1000 alternative splicing variations as well as updates (including UTR annotations) to nearly half of the ~27 000 annotated protein coding genes. The algorithm of the Program to Assemble Spliced Alignments (PASA) tool is described, as well as the results of automated updates to Arabidopsis gene annotations.

Pre-mRNA splicing and human disease

Abstract: The precision and complexity of intron removal during pre-mRNA splicing still amazes even 26 years after the discovery that the coding information of metazoan genes is interrupted by introns (Berget et al. 1977; Chow et al. 1977). Adding to this amazement is the recent realization that most human genes express more than one mRNA by alternative splicing, a process by which functionally diverse protein isoforms can be expressed according to different regulatory programs. Given that the vast majority of human genes contain introns and that most pre-mRNAs undergo alternative splicing, it is not surprising that disruption of normal splicing patterns can cause or modify human disease. The purpose of this review is to highlight the different mechanisms by which disruption of pre-mRNA splicing play a role in human disease. Several excellent reviews provide detailed information on splicing and the regulation of splicing (Burge et al. 1999; Hastings and Krainer 2001; Black 2003). The potential role of splicing as a modifier of human disease has also recently been reviewed (NissimRafinia and Kerem 2002).

CLIP identifies Nova-regulated RNA networks in the brain.

Abstract: Nova proteins are neuron-specific antigens targeted in paraneoplastic opsoclonus myoclonus ataxia (POMA), an autoimmune neurologic disease characterized by abnormal motor inhibition Nova proteins regulate neuronal pre-messenger RNA splicing by directly binding to RNA To identify Nova RNA targets, we developed a method to purify protein-RNA complexes from mouse brain with the use of ultraviolet cross-linking and immunoprecipitation (CLIP)Thirty-four transcripts were identified multiple times by Nova CLIPThree-quarters of these encode proteins that function at the neuronal synapse, and one-third are involved in neuronal inhibitionSplicing targets confirmed in Nova-/- mice include c-Jun N-terminal kinase 2, neogenin, and gephyrin; the latter encodes a protein that clusters inhibitory gamma-aminobutyric acid and glycine receptors, two previously identified Nova splicing targetsThus, CLIP reveals that Nova coordinately regulates a biologically coherent set of RNAs encoding multiple components of the inhibitory synapse, an observation that may relate to the cause of abnormal motor inhibition in POMA

LINE-mediated retrotransposition of marked Alu sequences

Abstract: Alu elements are the most successful transposons in humans. They are 300-bp non-coding sequences transcribed by RNA polymerase III (Pol III) and are expected to retrotranspose with the aid of reverse transcriptases of cellular origin. We previously showed that human LINEs can generate cDNA copies of any mRNA transcript by means of a retroposition process involving reverse transcription and integration by the LINE-encoded endonuclease and reverse transcriptase. Here we show mobility of marked Alu sequences in human HeLa cells with the canonical features of a retrotransposition process, including splicing out of an autocatalytic intron introduced into the marked sequence, target site duplications of varying lengths and integrations into consensus A-rich sequences. We further show that the poly-A stretch at the Alu 3' end is essential for mobility, that LINEs are required for transposition and that the rate of retroposition is 100-1,000 times higher for Alu transcripts than for control mRNAs, thus accounting for the high mutational activity of these elements observed in humans.

Molecular evolution of the insect chemoreceptor gene superfamily in Drosophila melanogaster.

Abstract: The insect chemoreceptor superfamily in Drosophila melanogaster is predicted to consist of 62 odorant receptor (Or) and 68 gustatory receptor (Gr) proteins, encoded by families of 60 Or and 60 Gr genes through alternative splicing. We include two previously undescribed Or genes and two previously undescribed Gr genes; two previously predicted Or genes are shown to be alternative splice forms. Three polymorphic pseudogenes and one highly defective pseudogene are recognized. Phylogenetic analysis reveals deep branches connecting multiple highly divergent clades within the Gr family, and the Or family appears to be a single highly expanded lineage within the superfamily. The genes are spread throughout the Drosophila genome, with some relatively recently diverged genes still clustered in the genome. The Gr5a gene on the X chromosome, which encodes a receptor for the sugar trehalose, has transposed from one such tandem cluster of six genes at cytological location 64, as has Gr61a, and all eight of these receptors might bind sugars. Analysis of intron evolution suggests that the common ancestor consisted of a long N-terminal exon encoding transmembrane domains 1-5 followed by three exons encoding transmembrane domains 6-7. As many as 57 additional introns have been acquired idiosyncratically during the evolution of the superfamily, whereas the ancestral introns and some of the older idiosyncratic introns have been lost at least 48 times independently. Altogether, these patterns of molecular evolution suggest that this is an ancient superfamily of chemoreceptors, probably dating back at least to the origin of the arthropods.

Challenging the dogma: the hidden layer of non-protein-coding RNAs in complex organisms

Abstract: The central dogma of biology holds that genetic information normally flows from DNA to RNA to protein. As a consequence it has been generally assumed that genes generally code for proteins, and that proteins fulfil not only most structural and catalytic but also most regulatory functions, in all cells, from microbes to mammals. However, the latter may not be the case in complex organisms. A number of startling observations about the extent of non-protein-coding RNA (ncRNA) transcription in the higher eukaryotes and the range of genetic and epigenetic phenomena that are RNA-directed suggests that the traditional view of the structure of genetic regulatory systems in animals and plants may be incorrect. ncRNA dominates the genomic output of the higher organisms and has been shown to control chromosome architecture, mRNA turnover and the developmental timing of protein expression, and may also regulate transcription and alternative splicing. This paper re-examines the available evidence and suggests a new framework for considering and understanding the genomic programming of biological complexity, autopoletic development and phenotypic variation. BioEssays 25:930-939,2003. (C) 2003 Wiley Periodicals, Inc.

Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss

Abstract: One of the most interesting opportunities in comparative genomics is to compare not only genome sequences but additional phenomena, such as alternative splicing, using orthologous genes in different genomes to find similarities and differences between organisms. Recently, genomics studies have suggested that 40-60% of human genes are alternatively spliced and have catalogued up to 30,000 alternative splice relationships in human genes. Here we report an analysis of 9,434 orthologous genes in human and mouse, which indicates that alternative splicing is associated with a large increase in frequency of recent exon creation and/or loss. Whereas most exons in the mouse and human genomes are strongly conserved in both genomes, exons that are only included in alternative splice forms (as opposed to the constitutive or major transcript form) are mostly not conserved and thus are the product of recent exon creation or loss events. A similar comparison of orthologous exons in rat and human validates this pattern. Although this says nothing about the complex question of adaptive benefit, it does indicate that alternative splicing in these genomes has been associated with increased evolutionary change.

MicroRNAs: at the root of plant development?

Abstract: Although most genes use RNA in the form of mRNA as a coding intermediate for protein production, there are many genes whose final products are RNA. These noncoding RNAs range from the familiar transfer and ribosomal RNAs to the more recently discovered regulatory RNAs. One type of regulatory RNA was

Splicing double: insights from the second spliceosome

Abstract: Almost 20 years after the discovery of introns and RNA splicing, a second spliceosome was uncovered. Although this new spliceosome is structurally and functionally analogous to the well-characterized major-class splicing apparatus, it mediates the excision of a minor class of evolutionarily conserved introns that have non-canonical consensus sequences. This unanticipated diversity in the splicing machinery is refining both the mechanistic understanding and evolutionary models of RNA splicing.

A quantitative analysis of intron effects on mammalian gene expression

Abstract: In higher eukaryotes, intron-containing and intronless versions of otherwise identical genes can exhibit dramatically different expression profiles. Introns and the act of their removal by the spliceosome can affect gene expression at many different levels, including transcription, polyadenylation, mRNA export, translational efficiency, and the rate of mRNA decay. However, the extent to which each of these steps contributes to the overall effect of any one intron on gene expression has not been rigorously tested. Here we report construction and initial characterization of a luciferase-based reporter system for monitoring the effects of individual introns and their position within the gene on protein expression in mammalian cells. Quantitative analysis of constructs containing human TPI intron 6 at two different positions within the Renilla luciferase open reading frame revealed that this intron acts primarily to enhance mRNA accumulation. Spliced mRNAs also exhibited higher translational yields than did intronless transcripts. However, nucleocytoplasmic mRNA distribution and mRNA stability were largely unaffected. These findings were extended to two other introns in a TCR-beta minigene.

The human genome encodes 10 α-crystallin-related small heat shock proteins: HspB1-10

Abstract: To obtain an inventory of all human genes that code for alpha-crystallin-related small heat shock proteins (sHsps), the databases available from the public International Human Genome Sequencing Consortium (IHGSC) and the private Celera human genome project were exhaustively searched. Using the human Hsp27 protein sequence as a query in the protein databases, which are derived from the predicted genes in the human genome, 10 sHsp-like proteins were retrieved, including Hsp27 itself. Repeating the search procedure with all 10 proteins and with a variety of more distantly related animal sHsps, no further human sHsps were detected, as was the case when searches were performed at deoxyribonucleic acid level. The 10 retrieved proteins comprised the 9 earlier recognized human sHsps (Hsp27/HspB1, HspB2, HspB3, alphaA-crystallin/HspB4, alphaB-crystallin/HspB5, Hsp20/HspB6, cvHsp/HspB7, H11/HspB8, and HspB9) and a sperm tail protein known since 1993 as outer dense fiber protein 1 (ODF1). Although this latter protein probably serves a structural role and has a high cysteine content (14%), it clearly contains an alpha-crystallin domain that is characteristic for sHsps. ODF1 can as such be designated as HspB10. The expression of all 10 human sHsp genes was confirmed by expressed sequence tag (EST) searches. For Hsp27/HspB1, 2 retropseudogenes were detected. The HspB1-10 genes are dispersed over 9 chromosomes, reflecting their ancient origin. Two of the genes (HspB3 and HspB9) are intronless, and the others have 1 or 2 introns at various positions. The transcripts of several sHsp genes, notably HspB7, display low levels of alternative splicing, as supported by EST evidence, which may result in minor amounts of isoforms at the protein level.

The spliceosome: the most complex macromolecular machine in the cell?

Abstract: The primary transcripts, pre-mRNAs, of almost all protein-coding genes in higher eukaryotes contain multiple non-coding intervening sequences, introns, which must be precisely removed to yield translatable mRNAs. The process of intron excision, splicing, takes place in a massive ribonucleoprotein complex known as the spliceosome. Extensive studies, both genetic and biochemical, in a variety of systems have revealed that essential components of the spliceosome include five small RNAs–U1, U2, U4, U5 and U6, each of which functions as a RNA, protein complex called an snRNP (small nuclear ribonucleoprotein). In addition to snRNPs, splicing requires many non-snRNP protein factors, the exact nature and number of which has been unclear. Technical advances, including new affinity purification methods and improved mass spectrometry techniques, coupled with the completion of many genome sequences, have now permitted a number of proteomic analyses of purified spliceosomes. These studies, recently reviewed by Jurica and Moore,1 reveal that the spliceosome is composed of as many as 300 distinct proteins and five RNAs, making it among the most complex macromolecular machines known. BioEssays 25:1147–1149, 2003. © 2003 Wiley Periodicals, Inc.

The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana

Abstract: The entire mitochondrial genome of rapeseed (Brassica napus L.) was sequenced and compared with that of Arabidopsis thaliana. The 221 853 bp genome contains 34 protein-coding genes, three rRNA genes and 17 tRNA genes. This gene content is almost identical to that of Arabidopsis. However the rps14 gene, which is a pseudo-gene in Arabidopsis, is intact in rapeseed. On the other hand, five tRNA genes are missing in rapeseed compared to Arabidopsis, although the set of mitochondrially encoded tRNA species is identical in the two Cruciferae. RNA editing events were systematically investigated on the basis of the sequence of the rapeseed mitochondrial genome. A total of 427 C to U conversions were identified in ORFs, which is nearly identical to the number in Arabidopsis (441 sites). The gene sequences and intron structures are mostly conserved (more than 99% similarity for protein-coding regions); however, only 358 editing sites (83% of total editings) are shared by rapeseed and Arabidopsis. Non-coding regions are mostly divergent between the two plants. One-third (about 78.7 kb) and two-thirds (about 223.8 kb) of the rapeseed and Arabidopsis mitochondrial genomes, respectively, cannot be aligned with each other and most of these regions do not show any homology to sequences registered in the DNA databases. The results of the comparative analysis between the rapeseed and Arabidopsis mitochondrial genomes suggest that higher plant mitochondria are extremely conservative with respect to coding sequences and somewhat conservative with respect to RNA editing, but that non-coding parts of plant mitochondrial DNA are extraordinarily dynamic with respect to structural changes, sequence acquisition and/or sequence loss.

trans and cis Splicing in Trypanosomatids: Mechanism, Factors, and Regulation

Abstract: mRNA maturation in trypanosomes differs from the process in most eukaryotes mainly because protein-coding genes are transcribed into polycistronic RNAs in this organism ([78][1]). Studies from the ongoing genome project suggest that the entire chromosome may be transcribed as large transcripts, but

RSEARCH: finding homologs of single structured RNA sequences.

Abstract: For many RNA molecules, secondary structure rather than primary sequence is the evolutionarily conserved feature. No programs have yet been published that allow searching a sequence database for homologs of a single RNA molecule on the basis of secondary structure.

The transcriptional activity of human Chromosome 22

Abstract: A DNA microarray representing nearly all of the unique sequences of human Chromosome 22 was constructed and used to measure global-transcriptional activity in placental poly(A) + RNA. We found that many of the known, related and predicted genes are expressed. More importantly, our study reveals twice as many transcribed bases as have been reported previously. Many of the newly discovered expressed fragments were verified by RNA blot analysis and a novel technique called differential hybridization mapping (DHM). Interestingly, a significant fraction of these novel fragments are expressed antisense to previously annotated introns. The coding potential of these novel expressed regions is supported by their sequence conservation in the mouse genome. This study has greatly increased our understanding of the biological information encoded on a human chromosome. To facilitate the dissemination of these results to the scientific community, we have developed a comprehensive Web resource to present the findings of this study and other features of human Chromosome 22 at http://array.mbb.yale.edu/chr22.

Intronic Sequences Flanking Alternatively Spliced Exons Are Conserved Between Human and Mouse

Abstract: Comparison of the sequences of mouse and human genomes revealed a surprising number of nonexonic, nonexpressed conserved sequences, for which no function could be assigned. To study the possible correlation between these conserved intronic sequences and alternative splicing regulation, we developed a method to identify exons that are alternatively spliced in both human and mouse. We compiled two exon sets: one of alternatively spliced conserved exons and another of constitutively spliced conserved exons. We found that 77% of the conserved alternatively spliced exons were flanked on both sides by long conserved intronic sequences. In comparison, only 17% of the conserved constitutively spliced exons were flanked by such conserved intronic sequences. The average length of the conserved intronic sequences was 103 bases in the upstream intron and 94 bases in the downstream intron. The average identity levels in the immediately flanking intronic sequences were 88% and 80% for the upstream and downstream introns, respectively, higher than the conservation levels of 77% that were measured in promoter regions. Our results suggest that the function of many of the intronic sequence blocks that are conserved between human and mouse is the regulation of alternative splicing.

Genomic Organization and Expression Analysis of B7-H4, an Immune Inhibitory Molecule of the B7 Family

Abstract: B7-H4 is a recently identified B7 family member that negatively regulates T cell immunity by the inhibition of T cell proliferation, cytokine production, and cell cycle progression. In this study, we report that the genomic DNA of human B7-H4 is mapped on chromosome 1 comprised of six exons and five introns spanning 66 kb, of which exon 6 is used for alternative splicing to generate two different transcripts. Similar B7-H4 structure is also found in mouse genomic DNA in chromosome 3. A human B7-H4 pseudogene is identified in chromosome 20p11.1 with a single exon and two stop codons in the coding region. Immunohistochemistry analysis using B7-H4-specific mAb demonstrates that B7-H4 is not expressed on the majority of normal human tissues. In contrast, up to 85% (22 of 26) of ovarian cancer and 31% (5 of 16) of lung cancer tissues constitutively express B7-H4. Our results indicate a tight regulation of B7-H4 expression in the translational level in normal peripheral tissues and a potential role of B7-H4 in the evasion of tumor immunity.

Molecular Architecture of the Multiprotein Splicing Factor SF3b

Abstract: The splicing factor SF3b is a multiprotein complex essential for the accurate excision of introns from pre-messenger RNA. As an integral component of the U2 small nuclear ribonucleoprotein (snRNP) and the U11/U12 di-snRNP, SF3b is involved in the recognition of the pre-messenger RNA's branch site within the major and minor spliceosomes. We have determined the three-dimensional structure of the human SF3b complex by single-particle electron cryomicroscopy at a resolution of less than 10 angstroms, allowing identification of protein domains with known structural folds. The best fit of a modeled RNA-recognition motif indicates that the protein p14 is located in the central cavity of the complex. The 22 tandem helical repeats of the protein SF3b155 are located in the outer shell of the complex enclosing p14.

Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing

Abstract: In Arabidopsis thaliana, cis-regulatory sequences of the floral homeotic gene AGAMOUS (AG) are located in the second intron. This 3-kb intron contains binding sites for two direct activators of AG, LEAFY (LFY) and WUSCHEL (WUS), along with other putative regulatory elements. We have used phylogenetic footprinting and the related technique of phylogenetic shadowing to identify putative cis-regulatory elements in this intron. Among 29 Brassicaceae species, several other motifs, but not the LFY and WUS binding sites identified previously, are largely invariant. Using reporter gene analyses, we tested six of these motifs and found that they are all functionally important for the activity of AG regulatory sequences in A. thaliana. Although there is little obvious sequence similarity outside the Brassicaceae, the intron from cucumber AG has at least partial activity in A. thaliana. Our studies underscore the value of the comparative approach as a tool that complements gene-by-gene promoter dissection but also demonstrate that sequence-based studies alone are insufficient for a complete identification of cis-regulatory sites.

Transcriptome and genome conservation of alternative splicing events in humans and mice.

Abstract: Combining mRNA and EST data in splicing graphs with whole genome alignments, we discover alternative splicing events that are conserved in both human and mouse transcriptomes. 1,964 of 19,156 (10%) loci examined contain one or more such alternative splicing events, with 2,698 total events. These events represent a lower bound on the amount of alternative splicing in the human genome. Also, as these alternative splicing events are conserved between the human and mouse transcriptomes they should be enriched for functionally significant alternative splicing events, free from much of the noise found in the EST libraries. Further classification of these alternative splicing events reveals that 1,037 (38.4%) are due to exon skipping, 497 (18.4%) are due to alternative 3' splice sites, 214 (7.9%) are due to alternative 5' splice sites, 75 (2.8%) are due to intron retention and the other 875 (32.4%) are due to other, more complicated, alternative splicing events. In addition, genomic sequences nearby these alternative splicing events display increased sequence conservation. Both the alternatively spliced exons and the proximal intron show increased levels of genomic conservation relative to constitutively spliced exons. For exon skipping events both intron regions flanking the exon are conserved while for alternative 5' and 3' splicing events the conservation is greater near the alternative splice site.