RESUMO
Three different base paired stems form between U2 and U6 snRNA over the course of the mRNA splicing reaction (helices I, II and III). One possible function of U2/U6 helix II is to facilitate subsequent U2/U6 helix I and III interactions, which participate directly in catalysis. Using an in vitro trans-splicing assay, we investigated the function of sequences located just upstream from the branch site (BS). We find that these upstream sequences are essential for stable binding of U2 to the branch region, and for U2/U6 helix II formation, but not for initial U2/BS pairing. We also show that non-functional upstream sequences cause U2 snRNA stem-loop IIa to be exposed to dimethylsulfate modification, perhaps reflecting a U2 snRNA conformational change and/or loss of SF3b proteins. Our data suggest that initial binding of U2 snRNP to the BS region must be stabilized by an interaction with upstream sequences before U2/U6 helix II can form or U2 stem-loop IIa can participate in spliceosome assembly.
Assuntos
Pareamento de Bases/genética , RNA Nuclear Pequeno/genética , Sequências Reguladoras de Ácido Nucleico/genética , Trans-Splicing/genética , Sequência de Bases , Extratos Celulares , Éxons/genética , Células HeLa , Humanos , Íntrons/genética , Dados de Sequência Molecular , Mutação/genética , Estabilidade de RNA/genética , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/metabolismo , Ribonuclease H/metabolismo , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/genética , Ésteres do Ácido Sulfúrico/metabolismoRESUMO
The genes encoding primate U2 snRNA are organized as a nearly perfect tandem array (the RNU2 locus) that has been evolving concertedly for >35 Myr since the divergence of baboons and humans. Thus the repeat units of the tandem array are essentially identical within each species, but differ between species. Homogeneity is maintained because any change in one repeat unit is purged from the array or fixed in all other repeats. Intriguingly, the cytological location of RNU2 has remained unchanged despite concerted evolution of the tandem array. We had found previously that junction sequences between the U2 tandem array and flanking DNA were subject to remodeling over a region of 200-300 bp during the past 5 Myr in the hominid lineage. Here we show that the junctions between the U2 tandem array and flanking DNA have undergone dramatic rearrangements over a region of 1 to >10 kbp in the 35 Myr since divergence of the Old World Monkey and hominid lineages. We argue that these rearrangements reflect the high level of genetic activity required to sustain concerted evolution, and propose a model to explain why maintenance of homogeneity within a tandemly repeated multigene family would lead to junctional diversity.
Assuntos
Cromossomos/genética , Evolução Molecular , Papio/genética , RNA Nuclear Pequeno/genética , Recombinação Genética/genética , Sequências de Repetição em Tandem/genética , Elementos Alu/genética , Animais , Clonagem Molecular , Sequência Conservada/genética , Retrovirus Endógenos/genética , Variação Genética/genética , Gorilla gorilla/genética , Humanos , Modelos Genéticos , Família Multigênica/genética , Pan troglodytes/genética , Provírus/genética , Especificidade da Espécie , Sequências Repetidas Terminais/genéticaRESUMO
Retrovirus-like sequences and their solitary (solo) long terminal repeats (LTRs) are common repetitive elements in eukaryotic genomes. We reported previously that the tandemly arrayed genes encoding U2 snRNA (the RNU2 locus) in humans and apes contain a solo LTR (U2-LTR) which was presumably generated by homologous recombination between the two LTRs of an ancestral provirus that is retained in the orthologous baboon RNU2 locus. We have now sequenced the orthologous U2-LTRs in human, chimpanzee, gorilla, orangutan, and baboon and examined numerous homologs of the U2-LTR that are dispersed throughout the human genome. Although these U2-LTR homologs have been collectively referred to as LTR13 in the literature, they do not display sequence similarity to any known retroviral LTRs; however, the structure of LTR13 closely resembles that of other retroviral LTRs with a putative promoter, polyadenylation signal, and a tandemly repeated 53-bp enhancer-like element. Genomic blotting indicates that LTR13 is primate-specific; based on sequence analysis, we estimate there are about 2,500 LTR13 elements in the human genome. Comparison of the primate U2-LTR sequences suggests that the homologous recombination event that gave rise to the solo U2-LTR occurred soon after insertion of the ancestral provirus into the ancestral U2 tandem array. Phylogenetic analysis of the LTR13 family confirms that it is diverse, but the orthologous U2-LTRs form a coherent group in which chimpanzee is closest to the humans; orangutan is a clear outgroup of human, chimpanzee, and gorilla; and baboon is a distant relative of human, chimpanzee, gorilla, and orangutan. We compare the LTR13 family with other known LTRs and consider whether these LTRs might play a role in concerted evolution of the primate RNU2 locus.
Assuntos
Evolução Molecular , Genoma Humano , Sequências Repetitivas de Ácido Nucleico , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/genética , Animais , Sequência de Bases , Elementos de DNA Transponíveis , Elementos Facilitadores Genéticos , Gorilla gorilla , Humanos , Repetições de Microssatélites , Dados de Sequência Molecular , Pan troglodytes , Papio , Filogenia , Pongo pygmaeus , Recombinação Genética , Retroviridae/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
The multigene family encoding human U2 small nuclear RNA (snRNA) is organized as a single large tandem array containing 5 to 25 copies of a 6.1-kb repeat unit (the RNU2 locus). Remarkably, each of the repeat units within an individual U2 tandem array appears to be identical except for an irregular dinucleotide tract, known as the CT microsatellite, which exhibits minor length and sequence polymorphism. Using a somatic cell genetic assay, we previously noticed that the CT microsatellite appeared to stabilize artificial tandem arrays of U2 snRNA genes. We now demonstrate that the CT microsatellite is required to establish large tandem arrays of transcriptionally active U2 genes, increasing both the average and maximum size of the resulting arrays. In contrast, the CT microsatellite has no effect on the average or maximal size of artificial arrays containing transcriptionally inactive U2 genes that lack key promoter elements. Our data reinforce the connection between recombination and transcription. Active U2 transcription interferes with establishment or maintenance of the U2 tandem array, and the CT microsatellite opposes these effects, perhaps by binding GAGA or GAGA-related factors which alter local chromatin structure. We speculate that the mechanisms responsible for maintenance of tandem arrays containing active promoters may differ from those that maintain tandem arrays of transcriptionally inactive sequences.
Assuntos
Repetições de Microssatélites , RNA Nuclear Pequeno/genética , Recombinação Genética , Adenina , Citosina , Repetições de Dinucleotídeos , Guanina , Humanos , Timidina , Transcrição Gênica , Células Tumorais CultivadasRESUMO
We have surveyed the tandemly repeated genes encoding U2 snRNA in a diverse panel of humans. We found only two polymorphisms within the U2 repeat unit: a SacI polymorphism (alleles SacI+ or SacI-) and a CT microsatellite polymorphism (alleles CT+ or CT-). Surprisingly, individual U2 tandem arrays are entirely SacI+ or SacI-, and entirely CT+ or CT-, although the SacI and CT alleles can occur in any combination. We also found that polymorphisms in the left and right junction regions flanking the tandem array fall into only two haplotypes (JL+ and JL-, JR+ and JR-). Most surprisingly, JL+ is always associated with JR+, and JL- with JR-. Thus individual U2 arrays do not exchange flanking markers, despite independent assortment and subsequent homogenization of the SacI and CT alleles within the U2 repeat units. We propose that the primary driving force for concerted evolution of the tandem U2 genes is intrachromosomal homogenization; interchromosomal genetic exchanges are much rarer, and reciprocal nonsister chromatid exchange apparently does not occur. Thus concerted evolution of the U2 tandem array occurs in situ along a chromosome lineage, and linkage disequilibrium between sequences flanking the U2 array may persist for long periods of time.
Assuntos
Cromossomos/metabolismo , RNA Nuclear Pequeno/genética , Sequências Repetitivas de Ácido Nucleico/genética , Animais , Sequência de Bases , Cromossomos/genética , Clonagem Molecular , DNA Satélite/genética , Eletroforese em Gel de Ágar , Evolução Molecular , Marcadores Genéticos/genética , Haplótipos/genética , Humanos , Desequilíbrio de Ligação/genética , Dados de Sequência Molecular , Linhagem , Polimorfismo Genético/genética , Polimorfismo de Fragmento de Restrição , Primatas/genética , Recombinação Genética/genética , Análise de SequênciaRESUMO
Infection of human cells with oncogenic adenovirus type 12 (Ad12) induces four specific chromosome fragile sites. Remarkably, three of these sites appear to colocalize with tandem arrays of genes encoding small, abundant, ubiquitously expressed structural RNAs--the RNU1 locus encoding U1 small nuclear RNA (snRNA), the RNU2 locus encoding U2 snRNA, and the RN5S locus encoding 5S rRNA. Recently, an artificial tandem array of the natural 5.8-kb U2 repeat unit has been shown to generate a new Ad12-inducible fragile site (Y.-P. Li, R. Tomanin, J. R. Smiley, and S. Bacchetti, Mol. Cell. Biol. 13:6064-6070, 1993), demonstrating that the U2 repeat unit alone is sufficient for virally induced fragility. To identify elements within the U2 repeat unit that are required for virally induced fragility, we generated cell lines containing artificial tandem arrays of the entire 5.8-kb repeat unit, an 834-bp fragment spanning the U2 gene alone, or the same 834-bp fragment from which key U2 transcriptional regulatory elements had been deleted. The U2 snRNA coding regions within each artificial array were marked by an innocuous single base change (U to C at position 87) so that the relative expression of supernumerary and endogenous U2 genes could be monitored by a primer extension assay. We find that artificial arrays of both the 5.8- and the 0.8-kb U2 repeat units are fragile but that arrays lacking either the distal sequence element or both the distal and the proximal sequence elements of the promoter are not. Surprisingly, variations in repeat copy number and/or transcriptional activity of the artificial arrays do not appear to correlate with the degree of Ad12-inducible fragility. We conclude that U2 transcriptional regulatory elements are required for virally induced fragility but not necessarily U2 snRNA transcription per se.
Assuntos
Infecções por Adenovirus Humanos/genética , Adenovírus Humanos/genética , Fragilidade Cromossômica , Dano ao DNA , Regiões Promotoras Genéticas , RNA Nuclear Pequeno/genética , Sequência de Aminoácidos , Linhagem Celular , Sítios Frágeis do Cromossomo , Genes , Ligação de Hidrogênio , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Sequências Repetitivas de Ácido NucleicoRESUMO
In primates, the tandemly repeated genes encoding U2 small nuclear RNA evolve concertedly, i.e. the sequence of the U2 repeat unit is essentially homogeneous within each species but differs somewhat between species. Using chromosome painting and the NGFR gene as an outside marker, we show that the U2 tandem array (RNU2) has remained at the same chromosomal locus (equivalent to human 17q21) through multiple speciation events over > 35 million years leading to the Old World monkey and hominoid lineages. The data suggest that the U2 tandem repeat, once established in the primate lineage, contained sequence elements favoring perpetuation and concerted evolution of the array in situ, despite a pericentric inversion in chimpanzee, a reciprocal translocation in gorilla and a paracentric inversion in orang utan. Comparison of the 11 kb U2 repeat unit found in baboon and other Old World monkeys with the 6 kb U2 repeat unit in humans and other hominids revealed that an ancestral U2 repeat unit was expanded by insertion of a 5 kb retrovirus bearing 1 kb long terminal repeats (LTRs). Subsequent excision of the provirus by homologous recombination between the LTRs generated a 6 kb U2 repeat unit containing a solo LTR. Remarkably, both junctions between the human U2 tandem array and flanking chromosomal DNA at 17q21 fall within the solo LTR sequence, suggesting a role for the LTR in the origin or maintenance of the primate U2 array.
