Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of Ul small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of Ul snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slowmigrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact Ul and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. Ul snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by Ul-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H deavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions. Understanding the mechanism of premessenger splicing will require delineation of the pathway of assembly and disassembly of the spliceosome during the splicing reaction. Single-intron substrates of 400 to 500 nucleotides rapidly assemble into large ribonucleoprotein particle (RNP) complexes when added to in vitro splicing extracts (2, 6, 8, 12-15, 17-19, 20a, 22, 23, 26-29, 31). Fractionation of splicing reactions by sucrose gradient sedimentation reveals several different complexes (2,6,8,12,13,17,26,29; Kramer, in press). The largest complex of 50S contains precursor RNA and the reaction intermediates, exon 1 and lariat-exon 2. This complex contains small nuclear RNPs (snRNPs), including Ul snRNPs (2,8,(12)(13)(14), and undergoes rapid splicing to products without a detectable lag when added back to extract (13). Furthermore, it does not form in the absence of ATP or with substrates that lack splicing consensus sequences (2,8,12,13). On the basis of these characteristics, this complex can be considered as an active spliceosome. Smaller assemblies of 20S and 35S have also been noted which rapidly form with precursor RNA and which may be kinetic ...
Transfer RNA (tRNA) splicing is essential in Saccharomyces cerevisiae as well as in humans, and many of its features are the same in both. In yeast, the final step of this process is removal of the 2' phosphate generated at the splice junction during ligation. A nicotinamide adenine dinucleotide (NAD)-dependent phosphotransferase catalyzes removal of the 2' phosphate and produces a small molecule. It is shown here that this small molecule is an NAD derivative: adenosine diphosphate (ADP)-ribose 1"-2" cyclic phosphate. Evidence is also presented that this molecule is produced in Xenopus laevis oocytes as a result of dephosphorylation of ligated tRNA.
Ul small nuclear ribonucleoproteins (snRNPs) are required for in vitro splicing of pre-mRNA. Sequences within Ul RNA hybridize to, and thus recognize, 5' splice junctions. We have investigated the mechanism of association of Ul snRNPs with the splicesome. Ul-specific antibodies detected Ul association with precursor RNA early during assembly. Removal of the 5' terminal sequences of Ul RNA by oligo-directed cleavage or removal of Ul snRNPs by immunoprecipitation prior to the addition of precursor RNA depressed the association of all snRNPs with precursor RNA as detected by immunoprecipitation of splicing complexes by either Sm or Ul-specific antibodies. Assembly of the spliceosome as monitored by gel electrophoresis was also depressed after cleavage of Ul RNA. The dependency of Sm precipitability of precursor RNA upon the presence of Ul snRNPs suggests that Ul snRNPs participate in the early recognition of substrate RNAs by U2 to U6 snRNPs. Although removal of the 5'-terminal sequences of Ul depressed Ul snRNP association with precursor RNA, it did not eliminate it, suggesting semistable association of Ul snRNPs with the assembling spliceosome in the absence of Ul RNA hybridization. This association was not dependent upon 5' splice junction sequences but was dependent upon 3' intronic sequences, indicating that Ul snRNPs interact with factors recognizing 3' intronic sequences. Mutual dependence of 5' and 3' recognition factors suggests significant snRNP-snRNP communication during early assembly.Small nuclear ribonucleoproteins (snRNPs) recognize consensus sequence elements within pre-mRNA and target those sequences for cleavage and/or ligation by the splicing machinery (1, 3,4,6,7,9,(14)(15)(16). Ul and U2 snRNPs bind to splice junctions and branch point sequences, respectively; binding protects the consensus sequences from exogenous ribonuclease digestion (6,7,16). An Sm-reactive element, probably U5 snRNPs, recognizes 3' splice junction sequences and protects the 3' junction and adjacent polypyrimidine track against ribonuclease digestion (10, 23). The mechanism whereby individual snRNPs recognize their respective target sequences is under intensive investigation. Both splicing activity and protection of 5' splice junctions of Ul snRNPs require the sequences of Ul RNA complementary to 5' junction sequences (6,7,15,24), leading to the suggestion that hybridization of U RNAs to precursor RNA is required for association of snRNPs with the spliceosome.Studies of in vitro assembly of exogenous precursor RNA into spliceosomes (2,5,9,11,13,(19)(20)(21) indicate that assembly of exogenous precursor RNA into the active spliceosome is dependent on the presence of the consensus sequences recognized by U snRNPs. Substrates lacking all consensus sequences, however, assemble into RNP complexes, indicating that snRNPs may recognize splicing consensus sequences in precursor RNAs already complexed with heterogeneous nuclear RNP (hnRNP) RNP polypeptides (8,22). Smaller assemblies are produced by using substrates lacking 3' splic...
Aiming to facilitate the analysis of human genetic variations in the context of disease susceptibility and varied drug response, we have developed a genotyping method that entails incorporation of a chemically labile nucleotide by PCR followed by specific chemical cleavage of the resulting amplicon at the modified bases. The identity of the cleaved fragments determines the genotype of the DNA. This method, termed Incorporation and Complete Chemical Cleavage, utilizes modified nucleotides 7-deaza-7-nitro-dATP, 7-deaza-7-nitrodGTP, 5-hydroxy-dCTP, and 5-hydroxy-dUTP, which have increased chemical reactivity but are able to form standard Watson-Crick base pairs. Thus one analog is substituted for the corresponding nucleotide during PCR, generating amplicons that contain nucleotide analogs at each occurrence of the selected base throughout the target DNA except for the primer sequences. Subsequent treatment with an oxidant followed by an organic base results in chemical cleavage at each site of modification, which produces fragments of different lengths and͞or molecular weights that reflect the genotype of the original DNA sample at the site of interest. This incorporation and cleavage chemistry are widely applicable to many existing nucleic acid analysis platforms, including gel electrophoresis and mass spectrometry. By combining DNA amplification and analog incorporation into one step, this strategy eliminates preamplification, DNA-strand separation, primer extension, and purification procedures associated with traditional chain-termination chemistry and therefore presents significant advantages in terms of speed, cost, and simplicity of genotyping.
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