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1.
Science ; 271(5256): 1716-8, 1996 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-8596930

RESUMEN

A conserved sequence element in a minor class of eukaryotic pre-messenger RNA (pre-mRNA) introns was previously proposed to base pair with a complementary sequence in the U12 small nuclear RNA (snRNA) in a manner analogous to the pairing of US snRNA with the branch site sequence of the major class of introns. Here, mutations generated in this conserved sequence element block the splicing of a member of this minor intron class in vivo. The block was relieved by coexpression of a U12 snRNA containing compensatory mutations that restore the proposed base pairing interaction. These results show that this minor class of pre-mRNA introns is a distinct class existing alongside the major class of introns in animal genomes, and these results also establish an in vivo function for U12 snRNA.


Asunto(s)
Intrones , Precursores del ARN/genética , Empalme del ARN , ARN Nuclear Pequeño/genética , Animales , Composición de Base , Secuencia de Bases , Células CHO , Secuencia Conservada , Cricetinae , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Precursores del ARN/metabolismo , ARN Nuclear Pequeño/metabolismo
2.
Science ; 266(5191): 1685-8, 1994 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-7527587

RESUMEN

The stereochemical specificities and reaction courses for both self-splicing steps of a group II intron have been determined by phosphorothioate substitution at the 5' and 3' splice site phosphodiester bonds. Both steps of the splicing reaction proceeded with a phosphorothioate in the Sp configuration but were blocked by the Rp diastereomer. Both steps also proceeded with inversion of stereochemical configuration around phosphorus, consistent with a concerted transesterification reaction. These results are identical to those found for nuclear precursor mRNA (pre-mRNA) splicing and provide support for the hypothesis that group II introns and nuclear pre-mRNA introns share a common evolutionary history.


Asunto(s)
Intrones , Conformación de Ácido Nucleico , Empalme del ARN , ARN/química , Secuencia de Bases , Exones , Datos de Secuencia Molecular , Oligorribonucleótidos/química , Oxígeno/química , Fósforo/química , ARN/genética , Azufre/química , Tionucleótidos/química
3.
Science ; 225(4665): 898-903, 1984 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-6206566

RESUMEN

The splicing of messenger RNA precursors in vitro proceeds through an intermediate that has the 5' end of the intervening sequence joined to a site near the 3' splice site. This lariat structure, which has been characterized for an adenovirus 2 major late transcript, has a branch point, with 2'-5' and 3'-5' phosphodiester bonds emanating from a single adenosine residue. The excised intervening sequence retains the branch site and terminates in a guanosine residue with a 3' hydroxyl group. The phosphate group at the splice junction between the two exons originates from the 3' splice site at the precursor.


Asunto(s)
Precursores de Ácido Nucleico/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , ARN Viral/metabolismo , ARN/metabolismo , Adenovirus Humanos/metabolismo , Secuencia de Bases , Fenómenos Químicos , Química , Conformación de Ácido Nucleico , Precursores de Ácido Nucleico/análisis , Oligorribonucleótidos/metabolismo , Fosfatos/metabolismo , ARN/análisis , Precursores del ARN , ARN Mensajero/análisis , ARN Viral/análisis
4.
Mol Cell Biol ; 15(8): 4466-78, 1995 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-7542746

RESUMEN

We have previously shown, using phosphorothioate substitutions at splice site, that both transesterification steps of group II intron self-splicing proceed, by stereochemical inversion, with an Sp but not an Rp phosphorothioate. Under alternative reaction conditions or with various intron fragments, group II introns can splice following hydrolysis at the 5' splice site and can also hydrolyze the bond between spliced exons (the spliced-exon reopening reaction). In this study, we have determined the stereochemical specificities of all of the major model hydrolytic reactions carried out by the aI5 gamma intron from Saccharomyces cerevisiae mitochondria. For all substrates containing exon 1 and most of the intron, the stereospecificity of hydrolysis is the same as for the step 1 transesterification reaction. In contrast, the spliced-exon reopening reaction proceeds with an Rp but not an Sp phosphorothioate at the scissile bond, as does true reverse splicing. Thus, by stereochemistry, this reaction appears to be related to the reverse of step 2 of self-splicing. Finally, a substrate RNA that contains the first exon and nine nucleotides of the intron, when reacted with the intron ribozyme, releases the first exon regardless of the configuration of the phosphorothioate at the 5' splice site, suggesting that this substrate can be cleaved by either the step 1 or the step 2 reaction site. Our findings clarify the relationships of these model reactions to the transesterification reactions of the intact self-splicing system and permit new studies to be interpreted more rigorously.


Asunto(s)
Intrones/genética , Mitocondrias/genética , Empalme del ARN , Saccharomyces cerevisiae/genética , Exones/genética , Hidrólisis , Conformación Molecular , ARN/metabolismo , Recombinación Genética , Estereoisomerismo , Especificidad por Sustrato , Tionucleótidos/metabolismo
5.
Mol Cell Biol ; 2(3): 302-7, 1982 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-6180303

RESUMEN

Dispersed, highly repeated DNA sequences were found within and near the Syrian hamster gene coding for the multifunctional protein CAD. Most of the repeated sequences were homologous to each other and had similar properties. They hybridized to many cytoplasmic polyadenylated RNAs and to 7S and 4.5S cytoplasmic non-polyadenylated RNAs. Cloned DNA fragments containing repeated sequences were transcribed in vitro by RNA polymerase III. The repeated sequences from Syrian hamsters share many properties with the Alu family of repetitive DNA from humans. The hamster sequences were homologous to total repetitive human DNA but only very weakly homologous to two cloned members of the human Alu family.


Asunto(s)
Complejos Multienzimáticos/genética , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Aspartato Carbamoiltransferasa/genética , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Células Cultivadas , Cricetinae , ADN Recombinante , Dihidroorotasa/genética , Humanos , Riñón , Mesocricetus , Hibridación de Ácido Nucleico , ARN/análisis , ARN Polimerasa III/metabolismo , Transcripción Genética
6.
Mol Cell Biol ; 2(3): 293-301, 1982 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-6125880

RESUMEN

Two adjacent fragments of genomic DNA spanning the gene for CAD, which encodes the first three enzymes of UMP biosynthesis, were cloned from a mutant Syrian hamster cell line containing multiple copies of this gene. The mutant was selected for resistance to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, the second enzyme in the pathway. The sizes and positions of about 37 intervening sequences within the 25-kilobase CAD gene were mapped by electron microscopy, and the locations of the 5' and 3' ends of the 7.9-kilobase CAD mRNA were established by electron microscopy and by other hybridization methods. The coding sequences are small (100 to 400 bases), as are most of the intervening sequences (50 to 300 bases). However, there are also several large intervening sequences of up to 5,000 bases each. Two small cytoplasmic polyadenylated RNAs are transcribed from a region just beyond the 5' end of the CAD gene, and their abundance reflects the degree of gene amplification.


Asunto(s)
Amidohidrolasas/genética , Aspartato Carbamoiltransferasa/genética , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Dihidroorotasa/genética , Ligasas/genética , Complejos Multienzimáticos/genética , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Cricetinae , Elementos Transponibles de ADN , Riñón , Mesocricetus , Hibridación de Ácido Nucleico , Secuencias Repetitivas de Ácidos Nucleicos , Uridina Monofosfato/biosíntesis
7.
Mol Cell Biol ; 21(6): 1942-52, 2001 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11238930

RESUMEN

U12-dependent introns containing alterations of the 3' splice site AC dinucleotide or alterations in the spacing between the branch site and the 3' splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5' splice site AU dinucleotide, any nucleotide could serve as the 3'-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3' splice site function. A branch site-to-3' splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3' splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3' splice site but that formation of the prespliceosomal complex A requires an active 3' splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3' splice site.


Asunto(s)
Intrones , Empalme del ARN , Nucleótidos de Adenina , Animales , Secuencia de Bases , Células CHO , Cricetinae , Datos de Secuencia Molecular , Mutación , Sitios de Empalme de ARN
8.
Mol Cell Biol ; 2(3): 308-19, 1982 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-6180304

RESUMEN

Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional protein CAD, overproduce this protein as a result of amplification of the CAD gene. We have used a sensitive in situ hybridization technique to localize CAD genomes in spreads of metaphase chromosomes from several independent PALA-resistant lines and from wild-type PALA-sensitive cells. The amplified genes were always found within chromosomes, usually in an expanded region of the short arm of chromosome B9. In wild-type cells, the CAD gene was also on the short arm of chromosome B9. In one mutant line, 90 to 100 CAD genes were found within an expanded B9 chromosome and 10 to 15 more were near the distal end of one arm of several different chromosomes. Another line contained most the genes in a telomeric chromosome or large chromosome fragment. The amplified genes were in chromosomal regions that were stained in a banded pattern by trypsin-Giemsa. A few double minute chromosomes were observed in a very small fraction of the total spreads examined. The it situ hybridizations were performed in the presence of 10% dextral sulfate 500, which increases the signal by as much as 100-fold. Using recombinant DNA plasmids nick-translated with [125I]dCTP to high specific radioactivity, 10 CAD genes in a single chromosomal region were revealed after 1 week of autoradiographic exposure, and the position of the unique gene could be seen after 1 month.


Asunto(s)
Mapeo Cromosómico , Complejos Multienzimáticos/genética , Hibridación de Ácido Nucleico , Animales , Ácido Aspártico/análogos & derivados , Ácido Aspártico/farmacología , Células Cultivadas , Aberraciones Cromosómicas , Cricetinae , ADN/análisis , ADN Recombinante , Sulfato de Dextran , Dextranos/farmacología , Resistencia a Medicamentos , Amplificación de Genes , Cariotipificación , Riñón , Mesocricetus , Ácido Fosfonoacético/análogos & derivados , Ácido Fosfonoacético/farmacología
9.
J Mol Biol ; 239(3): 357-65, 1994 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-8201617

RESUMEN

Eukaryotic nuclear genomes contain a rare class of pre-mRNA introns with consensus sequence features that differ markedly from most pre-mRNA introns. Four genes have so far been identified that contain one copy each of this rare intron class in addition to several standard introns. These introns and homologous introns from several species were compared to identify conserved sequence elements and to establish consensus sequences for these elements. The only well-conserved elements are found at the 5' and 3' ends of the introns. The 5' splice site sequence is ATATCCTT beginning with the first nucleotide of the intron and is invariant in the introns examined to date. The 3' splice site consensus sequence is YCCAC ending at the last nucleotide of the intron. An almost invariant sequence of TCCTTAAC is also found near the 3' end of the intron (the 3' upstream element). The length of the introns varies between 95 and 2940 nucleotides. The sequence organization of these introns suggests that they represent a variant class of pre-mRNA introns that might be spliced via a spliceosome mechanism employing factors distinct from those used by other pre-mRNA introns. A search of small nuclear RNA (snRNA) sequences for regions complementary to the conserved elements of this rare class of introns found a strong match between U12 snRNA and the 3' upstream element and a weaker match between U11 snRNA and the 5' splice site sequence.


Asunto(s)
Secuencia Conservada , ADN/genética , Intrones , Proteínas Nucleares/genética , Precursores del ARN/genética , Empalme del ARN/genética , Animales , Antígenos de Neoplasias/genética , Secuencia de Bases , Secuencia de Consenso , ADN/metabolismo , Humanos , Datos de Secuencia Molecular , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , ARNt Metiltransferasas
10.
Leukemia ; 29(1): 126-36, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24781015

RESUMEN

Mutations of spliceosome components are common in myeloid neoplasms. One of the affected genes, PRPF8, encodes the most evolutionarily conserved spliceosomal protein. We identified either recurrent somatic PRPF8 mutations or hemizygous deletions in 15/447 and 24/450 cases, respectively. Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis. PRPF8 defects correlated with increased myeloblasts and ring sideroblasts in cases without SF3B1 mutations. Knockdown of PRPF8 in K562 and CD34+ primary bone marrow cells increased proliferative capacity. Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects. In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions. In sum, the exploration of clinical and functional consequences suggests that PRPF8 is a novel leukemogenic gene in myeloid neoplasms with a distinct phenotype likely manifested through aberrant splicing.


Asunto(s)
Neoplasias Hematológicas/genética , Empalme del ARN , Proteínas de Unión al ARN/genética , Secuencia de Aminoácidos , Proliferación Celular , Eliminación de Gen , Técnicas de Silenciamiento del Gen , Neoplasias Hematológicas/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Datos de Secuencia Molecular , Proteínas de Unión al ARN/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Homología de Secuencia de Aminoácido
13.
Proc Natl Acad Sci U S A ; 86(20): 7795-9, 1989 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-2554290

RESUMEN

We present an adaptation of the hydroxyl radical DNA "footprinting" technique that permits high-resolution mapping of protected regions of RNA. Hydroxyl radical cleaves RNA independently of base sequence and secondary structure of the RNAs examined and allows resolution of protected regions at the single nucleotide level. By using this technique, we show that several regions of the 3' splice site of mRNA precursors are protected during the formation of splicing-specific ribonucleoprotein complexes in an in vitro splicing system. These regions include the 3' intron/exon junction and a portion of the adjacent exon, the polypyrimidine tract, and the site of branch formation. These protections appear to be due to splicing specific complexes since their formation is sensitive to point mutations at crucial residues and requires ATP and incubation. The formation of these protected regions is independent of the presence of a 5' splice site.


Asunto(s)
Hidróxidos , Precursores del ARN/genética , Empalme del ARN , Transcripción Genética , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Exones , Radicales Libres , Genes , Globinas/genética , Células HeLa/metabolismo , Humanos , Radical Hidroxilo , Intrones , Datos de Secuencia Molecular , Mapeo Nucleótido , Plásmidos , Conejos , Mapeo Restrictivo , Moldes Genéticos
14.
RNA ; 7(1): 94-105, 2001 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11214185

RESUMEN

The U6 spliceosomal snRNA forms an intramolecular stem-loop structure during spliceosome assembly that is required for splicing and is proposed to be at or near the catalytic center of the spliceosome. U6atac snRNA, the analog of U6 snRNA used in the U12-dependent splicing of the minor class of spliceosomal introns, contains a similar stem-loop whose structure but not sequence is conserved between humans and plants. To determine if the U6 and U6atac stem-loops are functionally analogous, the stem-loops from human and budding yeast U6 snRNAs were substituted for the U6atac snRNA structure and tested in an in vivo genetic suppression assay. Both chimeric U6/U6atac snRNA constructs were active for splicing in vivo. In contrast, several mutations of the native U6atac stem-loop that either delete putatively unpaired residues or disrupt the putative stem regions were inactive for splicing. Compensatory mutations that are expected to restore base pairing within the stem regions restored splicing activity. However, other mutants that retained base pairing potential were inactive, suggesting that functional groups within the stem regions may contribute to function. These results show that the U6atac snRNA stem-loop structure is required for in vivo splicing within the U12-dependent spliceosome and that its role is likely to be similar to that of the U6 snRNA intramolecular stem-loop.


Asunto(s)
ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/metabolismo , Regiones no Traducidas 5'/química , Animales , Secuencia de Bases , Células CHO , Cricetinae , Exones , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Empalme del ARN , ARN de Hongos/química , ARN Nuclear Pequeño/genética , Saccharomyces cerevisiae/genética , Transfección
15.
RNA ; 3(3): 227-33, 1997 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-9056760

RESUMEN

A notable feature of the newly described U12 snRNA-dependent class of eukaryotic nuclear pre-mRNA introns is the highly conserved 8-nt 5' splice site sequence. This sequence is virtually invariant in all known members of this class from plants to mammals. Based on sequence complementarity between this sequence and the 5' end of the U11 snRNA, we proposed that U11 snRNP may play a role in identifying and/or activating the 5' splice site for splicing. Here we show that mutations of the conserved 5' splice site sequence of a U12-dependent intron severely reduce correct splicing in vivo and that compensatory mutations in U11 snRNA can suppress the effects of the 5' splice site mutations to varying extents. This provides evidence for a required interaction between U11 snRNA and the 5' splice site sequence involving Watson-Crick base pairing. This data, in addition to a report that U11 snRNP is bound transiently to the U12-dependent spliceosome, suggests that U11 snRNP is the analogue of U1 snRNP in splicing this rare class of introns.


Asunto(s)
Intrones , Precursores del ARN/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Empalme del ARN , Transfección
16.
Nucleic Acids Res ; 21(23): 5456-62, 1993 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-8265362

RESUMEN

We have determined the effects on splicing of sulfur substitution of the non-bridging oxygens in the phosphodiester bond at the 5' splice site of a pre-mRNA intron. Pre-mRNAs containing stereochemically pure Rp and Sp phosphorothioate isomers were produced by ligation of a chemically synthesized modified RNA oligonucleotide to enzymatically synthesized RAs. When these modified pre-mRNA substrates were tested for in vitro splicing activity in a HeLa cell nuclear extract system, the RNA with the Rp diastereomeric phosphorothioate was not spliced while the Sp diastereomeric RNA spliced readily. The sulfur-containing branched trinucleotide was purified from the splicing reaction of the Sp RNA and analyzed by cleavage with a stereospecific nuclease. The results showed that the Sp phosphorothioate was inverted during the splicing reaction to the Rp configuration; a finding previously obtained for a Group I self-splicing RNA. This inversion of configuration is consistent with a transesterification mechanism for pre-mRNA splicing. The lack of splicing of the Rp modified RNA also suggests that the pro-Rp oxygen at the 5' splice site is involved in a critical chemical contact in the splicing mechanism. Additionally, we have found that the HeLa cell RNA debranching enzyme is inactive on branches containing an Rp phosphorothioate.


Asunto(s)
Empalme del ARN , ARN Mensajero/metabolismo , Adenovirus Humanos/genética , Secuencia de Bases , Técnicas In Vitro , Datos de Secuencia Molecular , Precursores de Ácido Nucleico/metabolismo , Oligodesoxirribonucleótidos/química , ARN Mensajero/ultraestructura , ARN Viral/metabolismo , Estereoisomerismo , Azufre/química
17.
RNA ; 4(6): 709-18, 1998 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-9622129

RESUMEN

The minor U12-dependent class of eukaryotic nuclear pre-mRNA introns is spliced by a distinct spliceosomal mechanism that requires the function of U11, U12, U5, U4atac, and U6atac snRNAs. Previous work has shown that U11 snRNA plays a role similar to U1 snRNA in the major class spliceosome by base pairing to the conserved 5' splice site sequence. Here we show that U6atac snRNA also base pairs to the 5' splice site in a manner analogous to that of U6 snRNA in the major class spliceosome. We show that splicing defective mutants of the 5' splice site can be activated for splicing in vivo by the coexpression of compensatory U6atac snRNA mutants. In some cases, maximal restoration of splicing required the coexpression of compensatory U11 snRNA mutants. The allelic specificity of mutant phenotype suppression is consistent with Watson-Crick base pairing between the pre-mRNA and the snRNAs. These results provide support for a model of the RNA-RNA interactions at the core of the U12-dependent spliceosome that is strikingly similar to that of the major class U2-dependent spliceosome.


Asunto(s)
Intrones , Conformación de Ácido Nucleico , Empalme del ARN , ARN Nuclear Pequeño/metabolismo , Secuencia de Bases , Células Eucariotas , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , ARN Nuclear Pequeño/química , Supresión Genética
18.
Nucleic Acids Res ; 20(8): 1949-57, 1992 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-1579497

RESUMEN

Substitution of pre-mRNA in vitro splicing substrates with alpha-phosphorothioate ribonucleotide analogs has multiple effects on the processes of spliceosome formation and splicing. A major effect of substitution is on the splicing cleavage/ligation reactions. Substitution at the 5' splice junction blocks the first cleavage/ligation reaction while substitution at the 3' splice junction blocks the second cleavage/ligation reaction. A second effect of phosphorothioate substitution is the inhibition of spliceosome formation. A substitution/interference assay was used to determine positions where substitution inhibits spliceosome formation or splicing. Substitution in the 3' splice site polypyrimidine tract was found to inhibit spliceosome formation and splicing. This effect was enhanced with multiple substitutions in the region. No sites of substitution within the exons were found which affected spliceosome formation or splicing.


Asunto(s)
Fosfatos/metabolismo , Precursores del ARN/metabolismo , Empalme del ARN/genética , ARN Mensajero/metabolismo , Tionucleótidos/metabolismo , Adenoviridae/genética , Animales , Secuencia de Bases , Globinas/genética , Datos de Secuencia Molecular , Mutación/genética , Conejos , Proteínas Virales/genética
19.
RNA ; 5(4): 525-38, 1999 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-10199569

RESUMEN

Splicing of U12-dependent introns requires the function of U11, U12, U6atac, U4atac, and U5 snRNAs. Recent studies have suggested that U6atac and U12 snRNAs interact extensively with each other, as well as with the pre-mRNA by Watson-Crick base pairing. The overall structure and many of the sequences are very similar to the highly conserved analogous regions of U6 and U2 snRNAs. We have identified the homologs of U6atac and U12 snRNAs in the plant Arabidopsis thaliana. These snRNAs are significantly diverged from human, showing overall identities of 65% for U6atac and 55% for U12 snRNA. However, there is almost complete conservation of the sequences and structures that are implicated in splicing. The sequence of plant U6atac snRNA shows complete conservation of the nucleotides that base pair to the 5' splice site sequences of U12-dependent introns in human. The immediately adjacent AGAGA sequence, which is found in human U6atac and all U6 snRNAs, is also conserved. High conservation is also observed in the sequences of U6atac and U12 that are believed to base pair with each other. The intramolecular U6atac stem-loop structure immediately adjacent to the U12 interaction region differs from the human sequence in 9 out of 21 positions. Most of these differences are in base pairing regions with compensatory changes occurring across the stem. To show that this stem-loop was functional, it was transplanted into a human suppressor U6atac snRNA expression construct. This chimeric snRNA was inactive in vivo but could be rescued by coexpression of a U4atac snRNA expression construct containing compensatory mutations that restored base pairing to the chimeric U6atac snRNA. These data show that base pairing of U4atac snRNA to U6atac snRNA has a required role in vivo and that the plant U6atac intramolecular stem-loop is the functional analog of the human sequence.


Asunto(s)
Arabidopsis/genética , ARN Nuclear Pequeño/genética , Secuencia de Bases , Clonación Molecular , Regulación de la Expresión Génica de las Plantas/genética , Humanos , Datos de Secuencia Molecular , Filogenia , Empalme del ARN , ARN de Planta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Empalmosomas/genética
20.
Mol Cell ; 1(1): 151-60, 1997 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-9659912

RESUMEN

Two types of eukaryotic nuclear introns are known: the common U2-dependent class with /GU and AG/ terminal intron dinucleotides, and the rare U12-dependent class with /AU and AC/ termini. Here we show that the U12-dependent splicing system can splice introns with /GU and AG/ termini and that such introns occur naturally. Further, U2-dependent introns with /AU and AC/termini also occur naturally and are evolutionarily conserved. Thus, the sequence of the terminal dinucleotides does not determine which spliceosomal system removes an intron. Rather, the four classes of introns described here can be sorted into two mechanistic classes (U2- or U12-dependent) by inspection of the complete set of conserved splice site sequences.


Asunto(s)
Secuencia Conservada , Intrones/genética , Empalme del ARN/genética , Ribonucleoproteína Nuclear Pequeña U2/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , Animales , Secuencia de Bases , Células CHO/fisiología , Cricetinae , Evolución Molecular , Humanos , Datos de Secuencia Molecular , Mutagénesis/genética , Nucleótidos/metabolismo
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