BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing

Here, we show that dBCAS2 (CG4980, human Breast Carcinoma Amplified Sequence 2 ortholog) is essential for the viability of Drosophila melanogaster. We find that ubiquitous or tissue-specific depletion of dBCAS2 leads to larval lethality, wing deformities, impaired splicing, and apoptosis. More importantly, overexpression of hBCAS2 rescues these defects. Furthermore, the C-terminal coiled-coil domain of hBCAS2 binds directly to CDC5L and recruits hPrp19/PLRG1 to form a core complex for splicing in mammalian cells and can partially restore wing damage induced by knocking down dBCAS2 in flies. In summary, Drosophila and human BCAS2 share a similar function in RNA splicing, which affects cell viability.     

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.     

Specific binding of an exonic splicing enhancer by the pre-mRNA splicing factor SRp55.

Purine-rich exonic splicing enhancers (ESEs) have been identified in many alternatively spliced exons. Alternative splicing of several ESE-containing exons has been shown to depend on subsets of the SR protein family of pre-mRNA splicing factors. In this report, we show that purified SR protein family member SRp55 by itself binds a 30-nt ESE-containing exon, the alternatively spliced exon 5 of avian cardiac troponin T. We show that purified SRp55 binds specifically to this RNA sequence with an apparent Kd of 60 nM as assayed by gel mobility retardation experiments. Mutations in the exon 5 sequence that increase or decrease exon 5 inclusion in vivo and in vitro have correspondingly different affinities for SRp55 in our assays. The exon 5 sequence contains two purine-rich motifs, common to many ESEs, and both are required for SRp55 binding. Hill plot analysis of binding titration reactions indicates that there is a cooperative binding of at least two SRp55 proteins to the exon sequence. Chemical modification interference studies using kethoxal show that SRp55 binding to exon 5 requires the N1 and/or the N2 of almost every G residue in the exon. Dimethylsulfate modification interference studies indicate that none of the N1 positions of A residues in the exon are important for binding. We postulate that SRp55 may recognize both primary sequence and RNA secondary structural elements within pre-mRNA.     

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.     

Human splicing factor SF3a, but not SF1, is essential for pre-mRNA splicing in vivo

The three subunits of human splicing factor SF3a are essential for the formation of the functional 17S U2 snRNP and prespliceosome assembly in vitro. RNAi-mediated depletion indicates that each subunit is essential for viability of human cells. Knockdown of single subunits results in a general block in splicing strongly suggesting that SF3a is a constitutive splicing factor in vivo. In contrast, splicing of several endogenous and reporter pre-mRNAs is not affected after knockdown of SF1, which functions at the onset of spliceosome assembly in vitro and is essential for cell viability. Thus, SF1 may only be required for the splicing of a subset of pre-mRNAs. We also observe a reorganization of U2 snRNP components in SF3a-depleted cells, where U2 snRNA and U2-B' are significantly reduced in nuclear speckles and the nucleoplasm, but still present in Cajal bodies. Together with the observation that the 17S U2 snRNP cannot be detected in extracts from SF3a-depleted cells, our results provide further evidence for a function of Cajal bodies in U2 snRNP biogenesis.     

Ubiquitin-like protein Hub1 is required for pre-mRNA splicing and localization of an essential splicing factor in fission yeast

Hub1/Ubl5 is a member of the family of ubiquitin-like proteins (UBLs). The tertiary structure of Hub1 is similar to that of ubiquitin; however, it differs from known modifiers in that there is no conserved glycine residue near the C terminus which, in ubiquitin and UBLs, is required for covalent modification of target proteins. Instead, there is a conserved dityrosine motif proximal to the terminal nonconserved amino acid. In S. cerevisiae, high molecular weight adducts can be formed in vivo from Hub1, but the structure of these adducts is not known, and they could be either covalent or noncovalent. The budding yeast HUB1 gene is not essential, but Delta hub1 mutants display defects in mating. Here, we report that fission yeast hub1 is an essential gene, whose loss results in cell cycle defects and inefficient pre-mRNA splicing. A screen for Hub1 interactors identified Snu66, a component of the U4/U6.U5 tri-snRNP splicing complex. Furthermore, overexpression of Snu66 suppresses the lethality of a hub1ts mutant. In cells lacking functional hub1, the nuclear localization of Snu66 is disrupted, suggesting that an important role for Hub1 is the correct subcellular targeting of Snu66, although our data suggest that Hub1 is likely to perform other roles in splicing as well.     

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.     

SMNrp is an essential pre-mRNA splicing factor required for the formation of the mature spliceosome

SMNrp, also termed SPF30, has recently been identified in spliceosomes assembled in vitro. We have functionally characterized this protein and show that it is an essential splicing factor. We show that SMNrp is a 17S U2 snRNP-associated protein that appears in the pre-spliceosome (complex A) and the mature spliceosome (complex B) during splicing. Immunodepletion of SMNrp from nuclear extract inhibits the first step of pre-mRNA splicing by preventing the formation of complex B. Re-addition of recombinant SMNrp to immunodepleted extract reconstitutes both spliceosome formation and splicing. Mutations in two domains of SMNrp, although similarly deleterious for splicing, differed in their consequences on U2 snRNP binding, suggesting that SMNrp may also engage in interactions with splicing factors other than the U2 snRNP. In agreement with this, we present evidence for an additional interaction between SMNrp and the [U4/U6.U5] tri-snRNP. A candidate that may mediate this interaction, namely the U4/U6-90 kDa protein, has been identified. We suggest that SMNrp, as a U2 snRNP-associated protein, facilitates the recruitment of the [U4/U6.U5] tri-snRNP to the pre-spliceosome.     

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.     

A complex intronic splicing enhancer from the c-src pre-mRNA activates inclusion of a heterologous exon.

The mouse c-src gene contains a short neuron-specific exon, N1. To characterize the sequences that regulate N1 splicing, we used a heterologous gene, derived from the human beta-globin gene, containing a short internal exon that is usually skipped by the splicing machinery. Various fragments from the src gene were inserted into the globin substrate to measure their effects on the splicing of the test exon. These clones were transiently expressed in neuronal and nonneuronal cell lines, and the level of exon inclusion was measured by primer extension. Several sequences from the N1 exon region induced the splicing of the heterologous exon. The most powerful effect was seen with a sequence from the intron downstream of the N1 exon. This sequence acted as a strong splicing enhancer, activating splicing of the test exon when placed in the intron downstream. The enhancer was strongest in neuronal LA-N-5 cells but also activated splicing in nonneuronal HEK293 cells. Deletion and linker scanning mutagenesis indicate that the enhancer is made up of multiple smaller elements that must act in combination. One of these elements was identified as the sequence UGCAUG. Three copies of this element can strongly activate splicing of the test exon in LA-N-5 neuroblastoma cells. These component elements of the src splicing enhancer are also apparently involved in the splicing of other short cassette exons.     

Assembly of pre-mRNA splicing complex is cap dependent.

To study the influence of the ubiquitous cap structure of nuclear pre-mRNAs on the assembly of a functional splicing complex, the in vitro splicing of a truncated human metallothionein pre-mRNA was examined in the presence of the cap analogue m7GTP. Significant inhibition of splicing was observed at a concentration as low as 5 microM m7GTP. Analysis of the splicing reaction on glycerol density gradients showed two complexes sedimenting at 45S and 22S. When the reaction was carried out in presence of m7GTP a marked decrease of the material sedimenting at 45S, representing the active splicing complex, was observed. When capped pre-mRNA was replaced by uncapped pre-mRNA, complex formation was significantly reduced. These data indicate that the cap structure plays an important yet unknown role in the assembly of spliceosomes.