Phosphorothioate substitution identifies phosphate groups important for pre-mRNA splicing.

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.     

A downstream splicing enhancer is essential for in vitro pre-mRNA splicing.

Splicing enhancers have previously been shown to promote processing of introns containing weak splicing signals. Here, we extend these studies by showing that also 'strong' constitutively active introns are absolutely dependent on a downstream splicing enhancer for activity in vitro. SR protein binding to exonic enhancer elements or U1 snRNP binding to a downstream 5' splice site serve redundant functions as activators of splicing. We further show that a 5' splice site is most effective as an enhancer of splicing. Thus, a 5' splice site is functional in S100 extracts, under conditions where a SR enhancer is nonfunctional. Also, splice site pairing occurs efficiently in the absence of exonic SR enhancers, emphasizing the significance of a downstream 5' splice site as the enhancer element in vertebrate splicing.     

Stabilization of the tau exon 10 stem loop alters pre-mRNA splicing

Neurofibrillary tangles containing filaments of the microtubule-associated protein tau are found in a variety of neurodegenerative diseases. Mutations in the tau gene itself cause frontotemporal dementia with parkinsonism, demonstrating the critical role of tau in pathogenesis. Many of these mutations in tau are silent, are found at the 5'-splice site of exon 10, and lead to increased inclusion of exon 10. These silent mutations are predicted to destabilize a stem loop structure at the exon 10 5'-splice site; however, the existence of this stem loop under physiological conditions and its role in splice regulation are controversial. Here we show that base changes that stabilize this stem loop in vitro substantially decrease exon 10 inclusion in a wild type tau minigene and rescue the increase in exon 10 splicing caused by a dementia-causing point mutation. Moreover, we probed the intracellular structure of the tau stem loop with antisense RNA and demonstrate that the stability of the stem loop dictates antisense effectiveness. Together these results validate the stem loop as a bona fide structure regulating tau exon 10 splicing.     

Ribonucleoprotein complex formation during pre-mRNA splicing in vitro.

The ribonucleoprotein (RNP) structures of the pre-mRNA and RNA processing products generated during in vitro splicing of an SP6/beta-globin pre-mRNA were characterized by sucrose gradient sedimentation analysis. Early, during the initial lag phase of the splicing reaction, the pre-mRNA sedimented heterogeneously but was detected in both 40S and 60S RNP complexes. An RNA substrate lacking a 3' splice site consensus sequence was not assembled into the 60S RNP complex. The two splicing intermediates, the first exon RNA species and an RNA species containing the intron and the second exon in a lariat configuration (IVS1-exon 2 RNA species), were found exclusively in a 60S RNP complex. These two splicing intermediates cosedimented under a variety of conditions, indicating that they are contained in the same RNP complex. The products of the splicing reaction, accurately spliced RNA and the excised IVS1 lariat RNA species, are released from the 60S RNP complex and detected in smaller RNP complexes. Sequence-specific RNA-factor interactions within these RNP complexes were evidenced by the preferential protection of the pre-mRNA branch point from RNase A digestion and protection of the 2'-5' phosphodiester bond of the lariat RNA species from enzymatic debranching. The various RNP complexes were further characterized and could be distinguished by immunoprecipitation with anti-Sm and anti-(U1)RNP antibodies.     

Antisense RNA inhibits splicing of pre-mRNA in vitro.

Antisense RNAs complementary to human beta-globin pre-mRNA or to a chimeric globin/adenovirus E2a pre-mRNA specifically and efficiently inhibit pre-mRNA splicing in vitro. The level of inhibition depends on the length, position and concentration of the antisense RNA relative to the pre-mRNA substrate. Antisense RNAs complementary to sequences greater than 80 nucleotides downstream of the globin 3' splice site inhibit at least as efficiently as those extending across the splice sites. Thus splicing is sensitive to perturbations involving exon sequences some distance from the splice sites. Inhibition is mediated by factors which affect the annealing of antisense and substrate RNAs. Direct analysis of RNA duplex formation demonstrates the presence of an activity in HeLa cell nuclear extract which promotes the rapid annealing of complementary RNAs in an ATP-independent manner. Both annealing and inhibition are greatly reduced when antisense RNA is added to the splicing reaction greater than or equal to 5 min after substrate. This result may reflect a transition between an open structure, in which annealing of antisense RNA with pre-mRNA is facilitated, and a closed complex in which pre-mRNA is sequestered at an early stage of spliceosome assembly.     

The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro.

We have shown previously that truncation of the human beta-globin pre-mRNA in the second exon, 14 nucleotides downstream from the 3' splice site, leads to inhibition of splicing but not cleavage at the 5' splice site. We now show that several nonglobin sequences substituted at this site can restore splicing and that the efficiency of splicing depends on the length of the second (downstream) exon and not a specific sequence. Deletions in the first exon have no effect on the efficiency of in vitro splicing. Surprisingly, an intron fragment from the 5' region of the human or rabbit beta-globin intron 2, when placed 14 nucleotides downstream from the 3' splice site, inhibited all the steps in splicing beginning with cleavage at the 5' splice site. This result suggests that the intron 2 fragment carries a "poison" sequence that can inhibit the splicing of an upstream intron.     

Inhibition of nuclear pre-mRNA splicing by antibiotics in vitro.

A number of antibiotics have been reported to disturb the decoding process in prokaryotic translation and to inhibit the function of various natural ribozymes. We investigated the effect of several antibiotics on in vitro splicing of a eukaryotic nuclear pre-mRNA (beta-globin). Of the eight antibiotics studied, erythromycin, Cl-tetracycline and streptomycin were identified as splicing inhibitors in nuclear HeLa cell extract. The K(i) values were 160, 180 and 230 microm, respectively. Cl-tetracycline-mediated and streptomycin-mediated splicing inhibition were in the molar inhibition range for hammerhead and human hepatitis delta virus ribozyme self-cleavage (tetracycline), of group-I intron self-splicing (streptomycin) and inhibition of RNase P cleavage by some aminoglycosides. Cl-tetracycline and the aminocyclitol glycoside streptomycin were found to have an indirect effect on splicing by unspecific binding to the pre-mRNA, suggesting that the inhibition is the result of disturbance of the correct folding of the pre-mRNA into the splicing-compatible tertiary structure by the charged groups of these antibiotics. The macrolide, erythromycin, the strongest inhibitor, had only a slight effect on formation of the presplicing complexes A and B, but almost completely inhibited formation of the splicing-active C complex by binding to nuclear extract component(s). This results in direct inhibition of the second step of pre-mRNA splicing. To our knowledge, this is the first report on specific inhibition of nuclear splicing by an antibiotic. The functional groups involved in the interaction of erythromycin with snRNAs and/or splicing factors require further investigation.     

Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing.

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.     

The role of U5 snRNP in pre-mRNA splicing.

The current model for the function of the U5 small nuclear ribonucleoprotein particle (snRNP) in the spliceosome proposes that U5 carries binding sites for the 5' and 3' exons, allowing the spliceosome to 'tether' the 5' exon intermediate produced by the first catalytic step and align it with the 3' exon for the second step. Functional analysis of U5 snRNA in cis-spliceosomes has provided support for this model, and data from nematode and trypanosome splicing systems suggest that U5 or a U5-like snRNA performs a similar role in trans-splicing.     

Detection of pre-mRNA splicing in vitro by an RNA-templated fluorogenic reaction

RNA splicing is an important target for basic research of disease mechanisms and for drug discovery. Here, we report a new method for analysis of the in vitro RNA splicing process that produces fluorescence using a reduction-triggered fluorescence (RETF) probe. The fluorescence signal is produced only when the two probes bind side-by-side with a specific RNA target. Precursor messenger RNA and mature messenger RNA originating from the chicken δ-crystallin (CDC) gene were successfully discriminated in solution using an RETF probe with the assistance of helper oligonucleotide strands. Also, we successfully applied RETF probes to the detection of emerging mature mRNA in an in vitro splicing process.     

Epigenetics in alternative pre-mRNA splicing

Alternative splicing plays critical roles in differentiation, development, and disease and is a major source for protein diversity in higher eukaryotes. Analysis of alternative splicing regulation has traditionally focused on RNA sequence elements and their associated splicing factors, but recent provocative studies point to a key function of chromatin structure and histone modifications in alternative splicing regulation. These insights suggest that epigenetic regulation determines not only what parts of the genome are expressed but also how they are spliced.     

RNA helicase dynamics in pre-mRNA splicing

The DExH-box NTPase/helicase Prp22p plays two important roles in pre-mRNA splicing. It promotes the second transesterification reaction and then catalyzes the ATP-dependent release of mature mRNA from the spliceosome. Evidence that helicase activity is important emerged from the analysis of Prp22p motif III (SAT) mutations that uncouple the NTPase and helicase activities. We find that S635A and T637A hydrolyse ATP, but are defective in unwinding duplex RNA and releasing mRNA from the spliceosome. The S635A mutation is lethal in vivo at 相似文献