Reprogramming alternative pre-messenger RNA splicing through the use of protein-binding antisense oligonucleotides

Alternative pre-messenger RNA splicing is a major contributor to proteomic diversity in higher eukaryotes and represents a key step in the control of protein function in a large variety of biological systems. As a means of artificially altering splice site choice, we have investigated the impact of positioning proteins in the vicinity of 5' splice sites. We find that a recombinant GST-MS2 protein interferes with 5' splice site use, most efficiently when it binds upstream of that site. To broaden the use of proteins as steric inhibitors of splicing, we have tested the activity of antisense oligonucleotides carrying binding sites for the heterogeneous nuclear ribonucleoprotein A1/A2 proteins. In a HeLa cell extract, tailed oligonucleotides complementary to exonic sequences elicit strong shifts in 5' splice site selection. In four different human cell lines, an interfering oligonucleotide carrying A1/A2 binding sites also shifted the alternative splicing of the Bcl-x pre-mRNA more efficiently than oligonucleotides acting through duplex formation only. The use of protein-binding oligonucleotides that interfere with U1 small nuclear ribonucleoprotein binding therefore represents a novel and powerful approach to control splice site selection in cells.     

The nuclear matrix phosphoprotein p255 associates with splicing complexes as part of the [U4/U6.U5] tri-snRNP particle.

The monoclonal antibody CC3 recognizes a phosphorylated epitope present on an interphase protein of 255 kDa. Previous work has shown that p255 is localized mainly to nuclear speckles and remains associated with the nuclear matrix scaffold following extraction with non-ionic detergents, nucleases and high salt. The association of p255 with splicing complexes is suggested by the finding that mAb CC3 can inhibit in vitro splicing and immunoprecipitate pre-messenger RNA and splicing products. Small nuclear RNA immunoprecipitation assays show that p255 is a component of the U5 small nuclear ribonucleoprotein (snRNP) and the [U4/U6.U5] tri-snRNP complex. In RNase protection assays, mAb CC3 immunoprecipitates fragments containing branch site and 3' splice site sequences. As predicted for a [U4/U6.U5]-associated component, the recovery of the branch site-protected fragment requires binding of U2 snRNP and is inhibited by EDTA. p255 may correspond to the previously identified p220 protein, the mammalian analogue of the yeast PRP8 protein. Our results suggest that changes in the phosphorylation of p255 may be part of control mechanisms that interface splicing activity with nuclear organization.     

A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly

Pre-mRNA splicing complex assembly is mediated by two specific pre-mRNA-snRNP interactions: U1 snRNP binds to the 5' splice site and U2 snRNP binds to the branch point. Here we show that unlike a purified U1 snRNP, which can bind to a 5' splice site, a partially purified U2 snRNP cannot interact with its target pre-mRNA sequence. We identify a previously uncharacterized activity, U2AF, that is required for the U2 snRNP-branch point interaction and splicing complex formation. Using RNA substrate exclusion and competition assays, we demonstrate that U2AF binds to the 3' splice site region prior to the U2 snRNP-branch point interaction. This provides an explanation for the necessity of the 3' splice site region in U2 snRNP binding and, hence, the first step of splicing.     

Molecular consequences of specific intron mutations on yeast mRNA splicing in vivo and in vitro

We have altered the TACTAAC sequence in the yeast CYH2m gene intron to TACTACC. This mutation changes the nucleotide at the normal position of the branch in intron RNA lariats produced during pre-mRNA splicing, and it prevents splicing in vivo. In a yeast pre-mRNA splicing system, CYH2m pre-mRNA carrying the TACTACC mutation is not specifically cut or rearranged in any way. Substitution of an A for the first G of the CYH2m intron, converting the highly conserved GTATGT 5' splice site sequence to ATATGT, also blocks intron excision in vivo and in vitro: pre-mRNA carrying this mutation was still cut normally at the mutant 5' splice site in vitro, to give authentic exon 1 and an intron-exon 2 lariat RNA with an A-A 2'-5' phosphodiester linkage at the branch point. This lariat RNA is a dead-end product. The subsequent cleavage at the 3' splice site is therefore sensitive to the sequence of the 5' end of the intron attached at the branch point.     

Short artificial hairpins sequester splicing signals and inhibit yeast pre-mRNA splicing.

To examine the stability of yeast (Saccharomyces cerevisiae) pre-mRNA structures, we inserted a series of small sequence elements that generated potential RNA hairpins at the 5' splice site and branch point regions. We analyzed spliceosome assembly and splicing in vitro as well as splicing and nuclear pre-mRNA retention in vivo. Surprisingly, the inhibition of in vivo splicing approximately paralleled that of in vitro splicing. Even a 6-nucleotide hairpin could be shown to inhibit splicing, and a 15-nucleotide hairpin gave rise to almost complete inhibition. The in vitro results indicate that hairpins that sequester the 5' splice site have a major effect on the early steps of spliceosome assembly, including U1 small nuclear ribonucleoprotein binding. The in vivo experiments lead to comparable conclusions as the sequestering hairpins apparently result in the transport of pre-mRNA to the cytoplasm. The observations are compared with previous data from both yeast and mammalian systems and suggest an important effect of pre-mRNA structure on in vivo splicing.     

A novel protein factor is required for use of distal alternative 5'' splice sites in vitro.

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.     

Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles.

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 U1 small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of U1 snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slow-migrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact U1 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. U1 snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by U1-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 cleavage 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.     

Characterization of a U2AF-independent commitment complex (E') in the mammalian spliceosome assembly pathway

Early recognition of pre-mRNA during spliceosome assembly in mammals proceeds through the association of U1 small nuclear ribonucleoprotein particle (snRNP) with the 5' splice site as well as the interactions of the branch binding protein SF1 with the branch region and the U2 snRNP auxiliary factor U2AF with the polypyrimidine tract and 3' splice site. These factors, along with members of the SR protein family, direct the ATP-independent formation of the early (E) complex that commits the pre-mRNA to splicing. We report here the observation in U2AF-depleted HeLa nuclear extract of a distinct, ATP-independent complex designated E' which can be chased into E complex and itself commits a pre-mRNA to the splicing pathway. The E' complex is characterized by a U1 snRNA-5' splice site base pairing, which follows the actual commitment step, an interaction of SF1 with the branch region, and a close association of the 5' splice site with the branch region. These results demonstrate that both commitment to splicing and the early proximity of conserved sequences within pre-mRNA substrates can occur in a minimal complex lacking U2AF, which may function as a precursor to E complex in spliceosome assembly.     

Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle.

A two-site model for the binding of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was tested in order to understand how exon partners are selected in complex pre-mRNAs containing alternative exons. In this model, it is proposed that two U1 snRNPs define a functional unit of splicing by base pairing to the 3' boundary of the downstream exon as well as the 5' boundary of the intron to be spliced. Three-exon substrates contained the alternatively spliced exon 4 (E4) region of the preprotachykinin gene. Combined 5' splice site mutations at neighboring exons demonstrate that weakened binding of U1 snRNP at the downstream site and improved U1 snRNP binding at the upstream site result in the failure to rescue splicing of the intron between the mutations. These results indicate the stringency of the requirement for binding a second U1 snRNP to the downstream 5' splice site for these substrates as opposed to an alternative model in which a certain threshold level of U1 snRNP can be provided at either site. Further support for the two-site model is provided by single-site mutations in the 5' splice site of the third exon, E5, that weaken base complementarity to U1 RNA. These mutations block E5 branchpoint formation and, surprisingly, generate novel branchpoints that are specified chiefly by their proximity to a cryptic 5' splice site located at the 3' terminus of the pre-mRNA. The experiments shown here demonstrate a true stimulation of 3' splice site activity by the downstream binding of U1 snRNP and suggest a possible mechanism by which combinatorial patterns of exon selection are achieved for alternatively spliced pre-mRNAs.     

A specific 31-nucleotide domain of U1 RNA directly interacts with the 70K small nuclear ribonucleoprotein component.

We have defined the nucleotide sequence of a protein-binding domain within U1 RNA that specifically recognizes and binds both to a U1 small nuclear ribonucleoprotein component (the 70K protein) and to the previously defined RNA-binding domain of the 70K protein. We have investigated direct interactions between purified U1 RNA and 70K protein by reconstitution in vitro. Thirty-one nucleotides of U1 RNA, corresponding to stem-loop I, were required for this interaction. Nucleotides at the 5' end of U1 RNA that are involved in base pairing with the 5' splice site of pre-mRNA were not required for binding. In contrast to other reports, these findings demonstrate that a specific domain of U1 RNA can bind directly to the 70K protein independently of any other snRNP-associated proteins.     

A catalogue of splice junction and putative branch point sequences from plant introns.

Splice junction and possible branch point sequences have been collected from 177 plant introns. Consensus sequences for the 5' and 3' splice junctions and for possible branch points have been derived. The splice junction consensus sequences were virtually identical to those of animal introns except that the polypyrimidine stretch at the 3' splice junction was less pronounced in the plant introns. A search for possible branch points with sequences related to the yeast, vertebrate and fungal consensus sequences revealed a similar sequence in plant introns.     

Specific and stable intron-factor interactions are established early during in vitro pre-mRNA splicing

Biochemical components (splicing factors) interact with specific intron regions during pre-mRNA splicing in vitro. The pre-mRNA specifically associates with factors at both the branch point and the 5' splice site and these RNA-factor interactions are maintained in the intron-containing RNA processing products. The first detectable event, the ATP-dependent association of a factor (or factors) with the branch point, is mediated by at least one factor containing an essential nucleic acid component. Mutant RNA substrates that lack either the 5' splice site or the vast majority of exon sequences can still associate with the branch point binding factor(s). However, this branch point-factor interaction does not occur with a mutant RNA substrate that contains the branch point but that lacks the 3' splice site consensus sequence. These results suggest that selection of the 3' splice site accompanied by the association of a factor with the branch point may be the initial step in mammalian pre-mRNA splicing.     

Evidence for nuclear factors involved in recognition of 5'' splice sites.

To investigate soluble factors involved in pre-messenger RNA splicing we have fractionated nuclear extract by simple centrifugation to produce a supernatant pellet pair. Factors larger than 15S including U2, U4, U5, and U6 snRNPs fractionate with the pellet; U1 snRNPs distribute equally in pellet and supernatant. Each fraction is individually incompetent for splicing and spliceosome assembly; mixing restores wild type activity and assembly. The pellet fraction directs an aberrant assembly pathway in which proper 3', but improper 5' splice site recognition occurs. Complexes formed with the pellet fraction are distinguishable from wild-type complexes using native gel electrophoresis. Pellet complexes contain U1 snRNP antigens and their formation requires ATP, U1 snRNPs, U2 snRNPs, and sequences at the 3' end of the intron - properties shared with the initial steps of normal assembly and directed by sequences at the 3' end of the intron. In contrast, pellet complex assembly shows no dependence on the presence of a 5' splice junction within precursor RNA. Furthermore, binding of factors to the 5' splice junction is deficient in pellet assemblies. Thus, the pellet lacks a factor required for proper recognition of 5' splice sites. This factor can be supplied by the supernatant. Complementation occurs when supernatant U1 RNA is destroyed, suggesting that the supernatant factor recognizing 5' splice sites is not U1 snRNPs.     

The splicing regulator TIA-1 interacts with U1-C to promote U1 snRNP recruitment to 5' splice sites

The U1 small nuclear ribonucleoprotein (U1 snRNP) binds to the pre-mRNA 5' splice site (ss) at early stages of spliceosome assembly. Recruitment of U1 to a class of weak 5' ss is promoted by binding of the protein TIA-1 to uridine-rich sequences immediately downstream from the 5' ss. Here we describe a molecular dissection of the activities of TIA-1. RNA recognition motifs (RRMs) 2 and 3 are necessary and sufficient for binding to the pre-mRNA. The non- consensus RRM1 and the C-terminal glutamine-rich (Q) domain are required for association with U1 snRNP and to facilitate its recruitment to 5' ss. Co-precipitation experiments revealed a specific and direct interaction involving the N-terminal region of the U1 protein U1-C and the Q-rich domain of TIA-1, an interaction enhanced by RRM1. The results argue that binding of TIA-1 in the vicinity of a 5' ss helps to stabilize U1 snRNP recruitment, at least in part, via a direct interaction with U1-C, thus providing one molecular mechanism for the function of this splicing regulator.     

Reconstituted U1 small nuclear ribonucleoprotein complex restores 5' splice site cleavage activity

Functional reconstitution of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was performed using in vitro transcribed U1 snRNA. Hela cell nuclear extract was depleted of its constituent snRNPs by centrifugation at 100,000 X g. The supernatant was devoid of snRNAs and lacked cleavage activity in splicing reactions using in vitro transcribed beta-globin pre-mRNA as substrate. The resulting pellet which contained the snRNAs, retained 5' splice site cleavage activity in a similar splicing reaction. Supplementation of the inactive supernatant fraction with in vitro transcribed U1 snRNA, partially restored 5' splice site cleavage activity thereby demonstrating the specific requirement of U1 snRNP in the initial stage of pre-mRNA splicing.     

The Prp19-associated complex is required for specifying interactions of U5 and U6 with pre-mRNA during spliceosome activation

Activation of the spliceosome involves a major structural change in the spliceosome, including release of U1 and U4 small nuclear ribonucleoprotein particles and the addition of a large protein complex, the Prp19-associated complex. We previously showed that the Prp19-associated complex is required for stable association of U5 and U6 with the spliceosome after U4 is released. Changes within the spliceosome upon binding of the Prp19-associated complex include remodeling of the U6/5' splice site interaction and destabilization of Lsm proteins to allow further interaction of U6 with the intron sequence. Here, we further analyzed interactions of U5 and U6 with pre-mRNA at various stages of spliceosome assembly from initial binding of tri-small nuclear ribonucleoprotein complex to the activated spliceosome to reveal stepwise changes of interactions. We demonstrate that both U5 and U6 interacted with pre-mRNA in dynamic manners spanning over a large region of U6 and the 5' exon sequences prior to the activation of the spliceosome. During spliceosome activation, interactions were locked down to small regions, and the Prp19-associated complex was required for defining the specificity of interaction of U5 and U6 with the 5' splice site to stabilize their association with the spliceosome after U4 is dissociated.