Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly

We recently reported that serine–arginine-rich (SR) protein-mediated pre-mRNA structural remodeling generates a pre-mRNA 3D structural scaffold that is stably recognized by the early spliceosomal components. However, the intermediate steps between the free pre-mRNA and the assembled early spliceosome are not yet characterized. By probing the early spliceosomal complexes in vitro and RNA-protein interactions in vivo, we show that the SR proteins bind the pre-mRNAs cooperatively generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some of the SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65 and U2AF35. Our work thus identifies a novel function of U1 snRNP in mammalian splicing substrate definition, explains the need for excess U1 snRNP compared to other U snRNPs in vivo, demonstrates how excess SR proteins could inhibit splicing, and provides a conceptual basis to examine if this mechanism of splicing substrate definition is employed by other splicing regulatory proteins.     

U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing

Two different experimental approaches have provided evidence that both U2 and U1 snRNPs function in pre-mRNA splicing. When the U2 snRNPs in a nuclear extract are selectively degraded using ribonuclease H and either of two deoxyoligonucleotides complementary to U2 RNA, splicing activity is abolished. Mixing an extract in which U2 has been degraded with one in which U1 has been degraded recovers activity. Use of anti-(U2)RNP autoantibodies demonstrates that U2 snRNPs associate with the precursor RNA during in vitro splicing. At 60 min, but not at 0 min, into the reaction intron fragments that include the branch-point sequence are immunoprecipitated by anti-(U2)RNP. At all times, U1 snRNPs bind the 5' splice site of the pre-mRNA. Possible interactions of the U2 snRNP with the U1 snRNP and with the pre-mRNA during splicing are considered.     

Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism.

Splicing of mRNA precursors (pre-mRNA) is preceded by assembly of the pre-mRNA with small nuclear ribonucleoprotein particles (snRNPs) and protein factors to form a splicesome. Here we show that stimulating Ser/Thr-specific protein dephosphorylation selectively inhibits an early step during mammalian spliceosome assembly. Treatment of HeLa nuclear splicing extracts with human protein phosphatase 1 (PP1) expressed in Escherichia coli, or PP1 purified from rabbit skeletal muscle, prevents pre-spliceosome E complex (early complex) formation and stable binding of U2 and U4/U6.U5 snRNPs to the pre-mRNA. PP1 does not inhibit splicing catalysis if added after spliceosome assembly has taken place. Addition of purified SR protein splicing factors restores spliceosome formation and splicing to PP1-inhibited extracts, consistent with SR proteins being targets regulated by phosphorylation. These data extend earlier observations showing that splicing catalysis, but not spliceosome assembly, is blocked by inhibiting protein phosphatases. It therefore appears that pre-mRNA splicing, in common with other biological processes, can be regulated both positively and negatively by reversible protein phosphorylation.     

Regulation of alternative 3' splice site selection by constitutive splicing factors.

We have devised an in vitro splicing assay in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. We have used this assay to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB could be partially reversed if pre-mRNAs were preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affected the joining of exons 1-3 with no dramatic increases in 1-2 splicing being detected. Preincubation of pre-mRNAs with SR proteins led to small increases in 1-2 splicing. However, if pre-mRNAs were preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there was a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus, multiple pairwise, and sometimes antagonizing, interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection.     

Functional spliceosomal A complexes can be assembled in vitro in the absence of a penta-snRNP

Two different models currently exist for the assembly pathway of the spliceosome, namely, the traditional model, in which spliceosomal snRNPs associate in a stepwise, ordered manner with the pre-mRNA, and the holospliceosome model, in which all spliceosomal snRNPs preassemble into a penta-snRNP complex. Here we have tested whether the spliceosomal A complex, which contains solely U1 and U2 snRNPs bound to pre-mRNA, is a functional, bona fide assembly intermediate. Significantly, A complexes affinity-purified from nuclear extract depleted of U4/U6 snRNPs (and thus unable to form a penta-snRNP) supported pre-mRNA splicing in nuclear extract depleted of U2 snRNPs, whereas naked pre-mRNA did not. Mixing experiments with purified A complexes and naked pre-mRNA additionally confirmed that under these conditions, A complexes do not form de novo. Thus, our studies demonstrate that holospliceosome formation is not a prerequisite for generating catalytically active spliceosomes and that, at least in vitro, the U1 and U2 snRNPs can functionally associate with the pre-mRNA, prior to and independent of the tri-snRNP. The ability to isolate functional spliceosomal A complexes paves the way to study in detail subsequent spliceosome assembly steps using purified components.     

Sequence-specific affinity selection of mammalian splicing complexes.

Antisense oligonucleotides made of 2'-OMe RNA are shown to bind specifically and efficiently to targeted sites on pre-mRNA substrates, allowing affinity selection of splicing complexes using streptavidin/biotin chromatography. The position of probe binding to the pre-mRNA influences which type of splicing complex can be selected. The accessibility of pre-mRNA sequences to antisense probes changes during the course of the splicing reaction. U1, U2, U4, U5 and U6 snRNAs are all detected in affinity-selected mammalian splicing complexes. However, antisense oligonucleotides targeted to snRNAs can block the binding of specific snRNPs to pre-mRNA. Quantitative affinity selection analyses show that only a small fraction of snRNPs in a HeLa nuclear splicing extract participate in spliceosome formation.     

Pre-mRNA splicing in vitro requires intact U4/U6 small nuclear ribonucleoprotein

Selective cleavage of U4 or U6 RNA in a HeLa cell nuclear extract inhibits splicing of pre-mRNAs containing an adenovirus or a simian virus 40 intron. RNAs in the U4/U6 small nuclear ribonucleoprotein (snRNP) were specifically degraded with RNAase H and deoxyoligonucleotides. Two oligomers complementary to U4 RNA and two complementary to U6 RNA cleave their target RNAs and inhibit the appearance of both spliced products and reaction intermediates. Splicing is reconstituted by mixing an extract containing cleaved U4 or U6 RNA with one in which splicing has been inhibited by degrading U2 RNA. All four abundant snRNPs, containing U1, U2, U5, or U4 and U6 RNAs, are now implicated in pre-mRNA splicing. Possible interactions of the U4/U6 snRNP with other components of the splicing complex are discussed.     

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.     

Deletion of the N-terminus of SF2/ASF permits RS-domain-independent pre-mRNA splicing

Serine/arginine-rich (SR) proteins are essential splicing factors with one or two RNA-recognition motifs (RRMs) and a C-terminal arginine- and serine-rich (RS) domain. SR proteins bind to exonic splicing enhancers via their RRM(s), and from this position are thought to promote splicing by antagonizing splicing silencers, recruiting other components of the splicing machinery through RS-RS domain interactions, and/or promoting RNA base-pairing through their RS domains. An RS domain tethered at an exonic splicing enhancer can function as a splicing activator, and RS domains play prominent roles in current models of SR protein functions. However, we previously reported that the RS domain of the SR protein SF2/ASF is dispensable for in vitro splicing of some pre-mRNAs. We have now extended these findings via the identification of a short inhibitory domain at the SF2/ASF N-terminus; deletion of this segment permits splicing in the absence of this SR protein's RS domain of an IgM pre-mRNA substrate previously classified as RS-domain-dependent. Deletion of the N-terminal inhibitory domain increases the splicing activity of SF2/ASF lacking its RS domain, and enhances its ability to bind pre-mRNA. Splicing of the IgM pre-mRNA in S100 complementation with SF2/ASF lacking its RS domain still requires an exonic splicing enhancer, suggesting that an SR protein RS domain is not always required for ESE-dependent splicing activation. Our data provide additional evidence that the SF2/ASF RS domain is not strictly required for constitutive splicing in vitro, contrary to prevailing models for how the domains of SR proteins function to promote splicing.     

Three-dimensional structure of the native spliceosome by cryo-electron microscopy

Splicing of pre-mRNA occurs in a multicomponent macromolecular machine--the spliceosome. The spliceosome can be assembled in vitro by a stepwise assembly of a number of snRNPs and additional proteins on exogenously added pre-mRNA. In contrast, splicing in vivo occurs in preformed particles where endogenous pre-mRNAs are packaged with all five spliceosomal U snRNPs (penta-snRNP) together with other splicing factors. Here we present a three-dimensional image reconstruction by cryo-electron microscopy of native spliceosomes, derived from cell nuclei, at a resolution of 20 angstroms. The structure revealed an elongated globular particle made up of two distinct subunits connected to each other leaving a tunnel in between. We show here that the larger subunit is a suitable candidate to accommodate the penta-snRNP, and that the tunnel could accommodate the pre-mRNA component of the spliceosome. The features this structure reveals provide new insight into the global architecture of the native splicing machine.     

The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.     

Cis-acting elements distinct from the 5' splice site promote U1-independent pre-mRNA splicing.

We have identified a class of pre-mRNAs that are spliced in HeLa extracts depleted for U1 snRNP (delta U1 extracts). Previously, we described pre-mRNAs that can be spliced in delta U1 extracts only when high concentrations of SR splicing factors are added. In contrast, the substrates characterized here are efficiently processed in delta U1 extracts without the addition of excess SR proteins. The members of this class comprise both a naturally occurring pre-mRNA, from the Drosophila fushi tarazu gene, and a chimera containing sequences from two different pre-mRNAs that individually are dependent upon U1 snRNP or excess SR proteins. Several sequence elements account for the variations in dependence on U1 snRNP and SR proteins for splicing. In one pre-mRNA, a single element was identified adjacent to the branch site. In the other, two elements flanking the 5'' splice site were found to be critical. This U1-independent splicing reaction may provide a mechanism for cells to control the extent of processing of different classes of pre-mRNAs in response to altered activities of SR proteins, and furthermore suggests that U1 snRNP-independent splicing may not be uncommon.     

Splicing factor SF4 is dispensable for the assembly of a functional splicing complex and participates in the subsequent steps of the splicing reaction.

The splicing of nuclear messenger RNA precursors (pre-mRNA) can be reconstituted in vitro with factors partially purified from HeLa cell nuclear extracts. Splicing complexes are assembled in the presence of the small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5 and U6 and the protein factors SF1, SF2, SF3 and U2AF. However, the complexes thus formed are inactive, i.e. they only contain unprocessed pre-mRNA. The intermediates and products of the splicing reaction are generated after addition of SF4. This splicing factor is a heat-labile protein which requires sulfhydryl groups for its activity. SF4 appears to participate, directly or indirectly, in the conversion of a functional but inactive splicing complex to the active spliceosome.     

U4 and U6 small nuclear ribonucleoprotein particles. 2. Evidence for their involvement in pre-mRNA splicing

The in vitro splicing of pre-mRNA of the human beta-globin gene in the presence of HeLa cell nuclear extract was investigated. Splicing was inhibited by auto-antibodies against U4 and U6 snRNP particles. No intermediates or products of the splicing reaction were evident in the presence of antibodies against U4 and U6 snRNPs which suggests their involvement in pre-mRNA splicing.     

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 SRm160/300 splicing coactivator subunits

The SRm160/300 splicing coactivator, which consists of the serine/arginine (SR)-related nuclear matrix protein of 160 kDa and a 300-kDa nuclear matrix antigen, functions in splicing by promoting critical interactions between splicing factors bound to pre-mRNA, including snRNPs and SR family proteins. In this article we report the isolation of a cDNA encoding the 300-kDa antigen and investigate the activity of it and SRm160 in splicing. Like SRm160, the 300-kDa antigen contains domains rich in alternating S and R residues but lacks an RNA recognition motif; the protein is accordingly named "SRm300." SRm300 also contains a novel and highly conserved N-terminal domain, several unique repeated motifs rich in S, R, and proline residues, and two very long polyserine tracts. Surprisingly, specific depletion of SRm300 does not prevent the splicing of pre-mRNAs shown previously to require SRm160/300. Addition of recombinant SRm160 alone to SRm160/300-depleted reactions specifically activates splicing. The results indicate that SRm160 may be the more critical component of the SRm160/300 coactivator in the splicing of SRm160/300-dependent pre-mRNAs.     

Composition and functional characterization of the yeast spliceosomal penta-snRNP.

Pre-mRNA introns are spliced in a macromolecular machine, the spliceosome. For each round of splicing, the spliceosome assembles de novo in a series of ATP-dependent steps involving numerous changes in RNA-RNA and RNA-protein interactions. As currently understood, spliceosome assembly proceeds by addition of discrete U1, U2, and U4/U6*U5 snRNPs to a pre-mRNA substrate to form functional splicing complexes. We characterized a 45S yeast penta-snRNP which contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors. The particle is functional in extracts and, when supplied with soluble factors, is capable of splicing pre-mRNA. We propose that the spliceosomal snRNPs associate prior to binding of a pre-mRNA substrate rather than with pre-mRNA via stepwise addition of discrete snRNPs.