Evolutionary conservation of a 2-kb intronic sequence flanking a tissue-specific alternative exon in the PTBP2 gene

Rahman, Lambratu; Bliskovski, Valery; Kaye, Frederic J.; Zajac-Kaye, Maria

doi:10.1016/s0888-7543(03)00207-6

Cited by 24 publications

(24 citation statements)

References 40 publications

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“…Space has not allowed for a detailed discussion of another phenomenon known to be associated with unusual levels of cross-species conservation; that of alternative splicing (e.g., Sorek and Ast 2003;Bejerano et al 2004b;Rahman et al 2004;Sugnet et al 2004). Alternative splicing might (as noted by a reviewer of this work) provide some useful clues about how unusual noncoding conservation arises.…”

Section: Discussionmentioning

confidence: 99%

Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes

Siepel¹,

Bejerano²,

Pedersen³

et al. 2005

Genome Res.

3,624

3,757

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We have conducted a comprehensive search for conserved elements in vertebrate genomes, using genome-wide multiple alignments of five vertebrate species (human, mouse, rat, chicken, and Fugu rubripes). Parallel searches have been performed with multiple alignments of four insect species (three species of Drosophila and Anopheles gambiae), two species of Caenorhabditis, and seven species of Saccharomyces. Conserved elements were identified with a computer program called phastCons, which is based on a two-state phylogenetic hidden Markov model (phylo-HMM). PhastCons works by fitting a phylo-HMM to the data by maximum likelihood, subject to constraints designed to calibrate the model across species groups, and then predicting conserved elements based on this model. The predicted elements cover roughly 3%-8% of the human genome (depending on the details of the calibration procedure) and substantially higher fractions of the more compact Drosophila melanogaster (37%-53%), Caenorhabditis elegans (18%-37%), and Saccharaomyces cerevisiae (47%-68%) genomes. From yeasts to vertebrates, in order of increasing genome size and general biological complexity, increasing fractions of conserved bases are found to lie outside of the exons of known protein-coding genes. In all groups, the most highly conserved elements (HCEs), by log-odds score, are hundreds or thousands of bases long. These elements share certain properties with ultraconserved elements, but they tend to be longer and less perfectly conserved, and they overlap genes of somewhat different functional categories. In vertebrates, HCEs are associated with the 3Ј UTRs of regulatory genes, stable gene deserts, and megabase-sized regions rich in moderately conserved noncoding sequences. Noncoding HCEs also show strong statistical evidence of an enrichment for RNA secondary structure.

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Section: Discussionmentioning

confidence: 99%

Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes

Siepel¹,

Bejerano²,

Pedersen³

et al. 2005

Genome Res.

3,624

3,757

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“…To isolate genomic DNA from cells, the QIAamp DNA blood kit (Qiagen) was used according to the manufacturer's instructions. Vector copy numbers of transduced cells were determined by quantitative PCR on an Applied Biosystems (Darmstadt, Germany) Step One Plus real-time PCR system using the QuantiTect SYBR green kit (Qiagen) with a reference sample known to contain 9 integrations (clone B, internal standard; kindly provided by U. Modlich, Hannover, Germany) and primers for the vector-specific PRE and the PTBP2 intron (evolutionarily conserved PTBP2 intronic sequence [28]) (19). From PCR efficiencies and by cross-point deviations from the reference sample, the vector copy numbers of unknown samples were determined according to the work of Pfaffl (27).…”

Section: Vol 84 2010 Sin Alpharetroviral Vectors 6627mentioning

confidence: 99%

Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design

Suerth

Maetzig

Galla

et al. 2010

J Virol

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Accidental insertional activation of proto-oncogenes and potential vector mobilization pose serious challenges for human gene therapy using retroviral vectors. Comparative analyses of integration sites of different retroviral vectors have elucidated distinct target site preferences, highlighting vectors based on the alpharetrovirus Rous sarcoma virus (RSV) as those with the most neutral integration spectrum. To date, alpharetroviral vector systems are based mainly on single constructs containing viral coding sequences and intact long terminal repeats (LTR). Even though they are considered to be replication incompetent in mammalian cells, the transfer of intact viral genomes is unacceptable for clinical applications, due to the risk of vector mobilization and the potentially immunogenic expression of viral proteins, which we minimized by setting up a split-packaging system expressing the necessary viral proteins in trans. Moreover, intact LTRs containing transcriptional elements are capable of activating cellular genes. By removing most of these transcriptional elements, we were able to generate a self-inactivating (SIN) alpharetroviral vector, whose LTR transcriptional activity is strongly reduced and whose transgene expression can be driven by an internal promoter of choice. Codon optimization of the alpharetroviral Gag/Pol expression construct and further optimization steps allowed the production of high-titer self-inactivating vector particles in human cells. We demonstrate proof of principle for the versatility of alpharetroviral SIN vectors for the genetic modification of murine and human hematopoietic cells at a low multiplicity of infection.

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“…The ultraconserved elements associated with alternative splicing events often contain small coding exons that are skipped in the mRNA in some tissues, but the elements extend well into the flanking intronic regions on one or both sides of the exon. Such is the case for one explicitly studied ultraconserved element (uc.33) in PTBP2, a polypyrimidine tract binding protein (23). PTBP2 contains a 312bp ultraconserved segment that is mostly intronic, but includes a small (34bp) exon that is included in the mRNA only in brain tissue.…”

mentioning

confidence: 99%

“…The PTBP2 element may form an RNA structure in the pre-mRNA that participates in the regulation of splicing through interactions with the spliceosome (23). We used RNAfold (24) to further assess the potential of this and other ultraconserved elements to form an RNA secondary structure, comparing the energy of the best folded structure for both the positive and negative strand element to that of 10,000 random permutations of the same sequence (table S4).…”

mentioning

confidence: 99%

“…in regions of coding exons that also bind splicing factors, or in regions of ribosomal RNA that must form RNA structures as well as bind proteins. If the exonic ultraconserved elements form pre-mRNA structures that are under selection to preserve interaction with the spliceosome or editing machinery (23,27), then these interactions must be extremely constraining over hundreds of bases of DNA, much like those of the anciently derived ribosomal RNAs, making them potentially quite novel objects for molecular study. The same holds true if the conservation in the non-exonic elements is associated with selection for molecular interactions involved in the regulation of transcription, which could be in cis over long genomic distances, or in trans, perhaps also involving RNA (29,32,33).…”

mentioning

confidence: 99%

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Ultraconserved Elements in the Human Genome

Bejerano

Pheasant

Makunin

et al. 2004

Science

1,561

1,607

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There are 481 segments longer than 200 bp that are absolutely conserved (100% identity with no insertions or deletions) between orthologous regions of the human, rat and mouse genomes. Nearly all of these segments are also conserved in the chicken and dog genomes, with an average of 95% and 99% identity, respectively. Many are also significantly conserved in fish. These ultraconserved elements of the human genome are most often located either overlapping exons in genes involved in RNA processing or in introns or nearby genes involved in regulation of transcription and development. Along with more than 5,000 sequences of over 100bp that are absolutely conserved among the three sequenced mammals, these represent a class of genetic elements whose functions and evolutionary origins are yet to be determined, but which are more highly conserved between these species than proteins, and appear to be essential for the ontogeny of mammals and other vertebrates.Although only about 1.2% of the human genome appears to code for protein (1-3), it has been estimated that as much as 5% is more conserved than expected from neutral evolution since the split with rodents, and hence may be under negative or "purifying" selection (4-6). Several studies have found specific non-coding segments in the human genome that appear to be under selection, using a threshold for conservation of 70% or 80% identity with mouse over more than 100bp (7-13). A study of these elements on human chromosome 21 found that those that were very highly conserved in multiple species contained significant numbers of non-coding elements (13). Similar results were found comparing the human, mouse and rat (14, 15) in a study of the 1.8 Mb CFTR region (16,17), and in a functional study of the SIM2 locus in a number of mammalian species (18). We determined the longest segments of the human genome that are maximally conserved with orthologous segments in rodents: those showing 100% identity and with no insertions or deletions in their alignment with mouse and rat. Exclusive of ribosomal RNA regions, there are 481 such segments longer than 200bp that we call ultraconserved elements (table S1). They are widely distributed in the genome (on all chromosomes except chromosomes 21 and Y), and are often found in clusters (Fig. 1). The probability is less than 10 -22 of finding even one such element in 2.9 billion bases under a simple model of neutral evolution with independent substitutions at each site, using the slowest neutral substitution rate that is observed for any 1 Mb region of the genome (supporting text, section S1). Nearly all of these elements also exhibited extremely high levels of conservation with orthologous regions in the chicken genome (467/481 = 97% of the elements aligning at an average of 95.7% identity, 29 at 100% identity), and about two-thirds of them with the fugu genome as well (324/481 = 67.3% of the elements aligning at an average of 76.8% identity), despite the fact that only about 4% of the human genome can be reliably aligned to the chicken ...

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Evolutionary conservation of a 2-kb intronic sequence flanking a tissue-specific alternative exon in the PTBP2 gene

Cited by 24 publications

References 40 publications

Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes

Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes

Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design

Ultraconserved Elements in the Human Genome

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