Proximity of the U12 snRNA with both the 5' splice site and the branch point during early stages of spliceosome assembly

U12 snRNA is required for branch point recognition in the U12-dependent spliceosome. Using site-specific cross-linking, we have captured an unexpected interaction between the 5' end of the U12 snRNA and the -2 position upstream of the 5' splice site of P120 and SCN4a splicing substrates. The U12 snRNA nucleotides that contact the 5' exon are the same ones that form the catalytically important helix Ib with U6atac snRNA in the spliceosome catalytic core. However, the U12/5' exon interaction is transient, occurring prior to the entry of the U4atac/U6atac.U5 tri-snRNP to the spliceosome. This suggests that the helix Ib region of U12 snRNA is positioned near the 5' splice site early during spliceosome assembly and only later interacts with U6atac to form helix Ib. We also provide evidence that U12 snRNA can simultaneously interact with 5' exon sequences near 5' splice site and the branch point sequence, suggesting that the 5' splice site and branch point sequences are separated by <40 to 50 A in the complex A of the U12-dependent spliceosome. Thus, no major rearrangements are subsequently needed to position these sites for the first step of catalysis.     

snRNA interactions at 5' and 3' splice sites monitored by photoactivated crosslinking in yeast spliceosomes.

Splice site recognition and catalysis of the transesterification reactions in the spliceosome are accompanied by a dynamic series of interactions involving conserved or invariant sequences in the spliceosomal snRNAs. We have used site-specific photoactivated crosslinking in yeast spliceosomes to monitor interactions between snRNAs and exon sequences near the 5' and 3' splice sites. The last nucleotide of the 5' exon can be crosslinked to an invariant loop sequence in U5 SnRNA before and after 5' splice site cleavage. The first nucleotide of the 3' exon can also be crosslinked to the same U5 loop sequence, but this contact is only detectable after the first transesterification. These results are in close agreement with earlier data from mammalian splicing extracts, and they are consistent with a model in which U5 snRNA aligns the 5' and 3' exons for the second transesterification. After the first catalytic step of splicing, the first nucleotide of the 3' exon can also crosslink to nt U23 in U2 snRNA. This is one of a cluster of residues in U2-U6 helix I implicated by mutational analysis in the second catalytic step of splicing. The crosslinking data suggest that these residues in U2-U6 helix I are in close proximity to the scissile phosphodiester bond at the 3' splice site prior to the second transesterification. These results constitute the first biochemical evidence for a direct interaction between the 3' splice site and U2 snRNA.     

trans-splicing to spliceosomal U2 snRNA suggests disruption of branch site-U2 pairing during pre-mRNA splicing

Pairing between U2 snRNA and the branch site of spliceosomal introns is essential for spliceosome assembly and is thought to be required for the first catalytic step of splicing. We have identified an RNA comprising the 5' end of U2 snRNA and the 3' exon of the ACT1-CUP1 reporter gene, resulting from a trans-splicing reaction in which a 5' splice site-like sequence in the universally conserved branch site-binding region of U2 is used in trans as a 5' splice site for both steps of splicing in vivo. Formation of this product occurs in functional spliceosomes assembled on reporter genes whose 5' splice sites are predicted to bind poorly at the spliceosome catalytic center. Multiple spatially disparate splice sites in U2 can be used, calling into question both the fate of its pairing to the branch site and the details of its role in splicing catalysis.     

Functional interaction of a novel 15.5kD [U4/U6.U5] tri-snRNP protein with the 5' stem-loop of U4 snRNA.

Activation of the spliceosome for splicing catalysis requires the dissociation of U4 snRNA from the U4/U6 snRNA duplex prior to the first step of splicing. We characterize an evolutionarily conserved 15.5 kDa protein of the HeLa [U4/U6.U5] tri-snRNP that binds directly to the 5' stem-loop of U4 snRNA. This protein shares a novel RNA recognition motif with several RNP-associated proteins, which is essential, but not sufficient for RNA binding. The 15.5kD protein binding site on the U4 snRNA consists of an internal purine-rich loop flanked by the stem of the 5' stem-loop and a stem comprising two base pairs. Addition of an RNA oligonucleotide comprising the 5' stem-loop of U4 snRNA (U4SL) to an in vitro splicing reaction blocked the first step of pre-mRNA splicing. Interestingly, spliceosomal C complex formation was inhibited while B complexes accumulated. This indicates that the 15.5kD protein, and/or additional U4 snRNP proteins associated with it, play an important role in the late stage of spliceosome assembly, prior to step I of splicing catalysis. Our finding that the 15.5kD protein also efficiently binds to the 5' stem-loop of U4atac snRNA indicates that it may be shared by the [U4atac/U6atac.U5] tri-snRNP of the minor U12-type spliceosome.     

ATP-dependent interaction of yeast U5 snRNA loop 1 with the 5' splice site.

Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 5' exon before the first step of splicing and with the 5' and 3' exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5' and 3' exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 5' exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 5' exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5' splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 5' exon in two steps during its targeting to the 5' splice site.     

Dynamic exchanges of RNA interactions leading to catalytic core formation in the U12-dependent spliceosome

Important general insights into the mechanism of pre-mRNA splicing have emerged from studies of the U12-dependent spliceosome. Here, photochemical cross-linking analyses during U12-dependent spliceosome assembly have surprisingly revealed that an upstream 5' exon region is required for establishing two essential catalytic core interactions, U12/U6atac helix Ib and U6atac/5' splice site contacts, but not for U5/5' exon interactions or partial unwinding of U4atac/U6atac. A novel intermediate, representing an alternative pathway for catalytic core formation, is a ternary snRNA complex containing U4atac/U6atac stem II and U12/U6atac helix Ia that forms even without U6atac replacing U11 at the 5' splice site. A powerful oligonucleotide displacement method suggests that the blocked complexes analyzed to deduce the interdependence of these multiple RNA exchanges are authentic intermediates in U12-dependent spliceosome assembly.     

Extensive interactions of PRP8 protein with the 5'' and 3'' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA.

Precursor RNAs containing 4-thiouridine at specific sites were used with UV-crosslinking to map the binding sites of the yeast protein splicing factor PRP8. PRP8 protein interacts with a region of at least eight exon nucleotides at the 5' splice site and a minimum of 13 exon nucleotides and part of the polypyrimidine tract in the 3' splice site region. Crosslinking of PRP8 to mutant and duplicated 3' splice sites indicated that the interaction is not sequence specific, nor does it depend on the splice site being functional. Binding of PRP8 to the 5' exon was established before step 1 and to the 3' splice site region after step 1 of splicing. These interactions place PRP8 close to the proposed catalytic core of the spliceosome during both transesterification reactions. To date, this represents the most extensive mapping of the binding site(s) of a splicing factor on the substrate RNA. We propose that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5 snRNP.     

A ribozyme selected from variants of U6 snRNA promotes 2',5'-branch formation

In vitro selection was used to sample SnRNA-related sequences for ribozyme activities, and several 2',5'-branch-forming ribozymes were isolated. One such ribozyme is highly dependent upon an 11-nt motif that contains a conserved U6 snRNA sequence (ACAGAGA-box) known to be important for pre-mRNA splicing. The ribozyme reaction is similar to the first step of splicing in that an internal 2'-hydroxyl of an unpaired adenosine attacks at the 5'-phosphate of a guanosine. It differs in that the leaving group is diphosphate rather than a 5' exon. The finding that lariat formation can be accomplished by a small RNA with sequences related to U6 snRNA indicates that the RNA available in the spliceosome may be involved in RNA-catalyzed branch formation.     

U1 snRNA directly interacts with polypyrimidine tract-binding protein during splicing repression

Splicing of the c-src N1 exon is repressed by the polypyrimidine tract-binding protein (PTB or PTBP1). During exon repression, the U1 snRNP binds properly to the N1 exon 5' splice site but is made inactive by the presence of PTB. Examining the patterns of nuclease protection at this 5' splice site, we find that the interaction of U1 is altered by the adjacent PTB. Interestingly, UV crosslinking identifies a direct contact between the pre-mRNA-bound PTB and the U1 snRNA. EMSA, ITC, and NMR studies show that PTB RRMs 1 and 2 bind the pyrimidine-rich internal loop of U1 snRNA stem loop 4. The PTB/U1 interaction prevents further assembly of the U1 snRNP with spliceosomal components downstream. This precise interaction between a splicing regulator and?an snRNA component of the spliceosome points to a range of different mechanisms for splicing regulation.     

The conserved central domain of yeast U6 snRNA: importance of U2-U6 helix Ia in spliceosome assembly

In the pre-mRNA processing machinery of eukaryotic cells, U6 snRNA is located at or near the active site for pre-mRNA splicing catalysis, and U6 is involved in catalyzing the first chemical step of splicing. We have further defined the roles of key features of yeast U6 snRNA in the splicing process. By assaying spliceosome assembly and splicing in yeast extracts, we found that mutations of yeast U6 nt 56 and 57 are similar to previously reported deletions of U2 nt 27 or 28, all within yeast U2-U6 helix Ia. These mutations lead to the accumulation of yeast A1 spliceosomes, which form just prior to the Prp2 ATPase step and the first chemical step of splicing. These results strongly suggest that, at a late stage of spliceosome assembly, the presence of U2-U6 helix Ia is important for promoting the first chemical step of splicing, presumably by bringing together the 5' splice site region of pre-mRNA, which is base paired to U6 snRNA, and the branchsite region of the intron, which is base paired to U2 snRNA, for activation of the first chemical step of splicing, as previously proposed by Madhani and Guthrie [Cell, 1992, 71: 803-817]. In the 3' intramolecular stem-loop of U6, mutation G81C causes an allele-specific accumulation of U6 snRNP. Base pairing of the U6 3' stem-loop in yeast spliceosomes does not extend as far as to include the U6 sequence of U2-U6 helix Ib, in contrast to the human U6 3' stem-loop structure.     

Conserved domains of human U4 snRNA required for snRNP and spliceosome assembly.

U4 snRNA is phylogenetically highly conserved and organized in several domains. To determine the function of each of the domains of human U4 snRNA in the multi-step process of snRNP and spliceosome assembly, we used reconstitution procedures in combination with snRNA mutagenesis. The highly conserved 5' terminal domain of U4 snRNA consists of the stem I and stem II regions that have been proposed to base pair with U6 snRNA, and the 5' stem-loop structure. We found that each of these structural elements is essential for spliceosome assembly. However, only the stem II region is required for U4-U6 interaction, and none of these elements for Sm protein binding. In contrast, the 3' terminal domain of U4 snRNA containing the Sm binding site is dispensable for both U4-U6 interaction and spliceosome assembly. Our results support an organization of the U4 snRNP into multiple functional domains, each of which acts at distinct stages of snRNP and spliceosome assembly.     

Progression through the spliceosome cycle requires Prp38p function for U4/U6 snRNA dissociation.

The elaborate and energy-intensive spliceosome assembly pathway belies the seemingly simple chemistry of pre-mRNA splicing. Prp38p was previously identified as a protein required in vivo and in vitro for the first pre-mRNA cleavage reaction catalyzed by the spliceosome. Here we show that Prp38p is a unique component of the U4/U6.U5 tri-small nuclear ribonucleoprotein (snRNP) particle and is necessary for an essential step late in spliceosome maturation. Without Prp38p activity spliceosomes form, but arrest in a catalytically impaired state. Functional spliceosomes shed U4 snRNA before 5' splice-site cleavage. In contrast, Prp38p-defective spliceosomes retain U4 snRNA bound to its U6 snRNA base-pairing partner. Prp38p is the first tri-snRNP-specific protein shown to be dispensable for assembly, but required for conformational changes which lead to catalytic activation of the spliceosome.     

A novel approach to describe a U1 snRNA binding site

RNA duplex formation between U1 snRNA and a splice donor (SD) site can protect pre-mRNA from degradation prior to splicing and initiates formation of the spliceosome. This process was monitored, using sub-genomic HIV-1 expression vectors, by expression analysis of the glycoprotein env, whose formation critically depends on functional SD4. We systematically derived a hydrogen bond model for the complementarity between the free 5' end of U1 snRNA and 5' splice sites and numerous mutations following transient transfection of HeLa-T4+ cells with 5' splice site mutated vectors. The resulting model takes into account number, interdependence and neighborhood relationships of predicted hydrogen bond formation in a region spanning the three most 3' base pairs of the exon (-3 to -1) and the eight most 5' base pairs of the intron (+1 to +8). The model is represented by an algorithm classifying U1 snRNA binding sites which can or cannot functionally substitute SD4 with respect to Rev-mediated env expression. In a data set of 5' splice site mutations of the human ATM gene we found a significant correlation between the algorithmic classification and exon skipping (P = 0.018, chi2-test), showing that the applicability of the proposed model reaches far beyond HIV-1 splicing. However, the algorithmic classification must not be taken as an absolute measure of SD usage as it may be modified by upstream sequence elements. Upstream to SD4 we identified a fragment supporting ASF/SF2 binding. Mutating GAR nucleotide repeats within this site decreased the SD4-dependent Rev-mediated env expression, which could be balanced simply by artificially increasing the complementarity of SD4.     

Suppression of multiple substrate mutations by spliceosomal prp8 alleles suggests functional correlations with ribosomal ambiguity mutants

Conformational change within the spliceosome is required between the first catalytic step of pre-mRNA splicing, when the branch site (BS) attacks the 5' splice site, and the second step, when the 5' exon attacks the 3' splice site, yielding mRNA and lariat-intron products. A genetic screen for suppressors of BS A-to-G mutants, which stall between the two steps, identified Prp8, the highly conserved spliceosomal factor. prp8 suppressors facilitate the second step for multiple intron mutants and interact functionally with first step suppressors, alleles of PRP16 and U6 snRNA. Genetic interactions among prp8, prp16, and U6 alleles suggest that these factors control a common stage in first-to-second step transition. We propose that mutant substrates are utilized by alteration of the equilibrium between first/second step conformations, resembling tRNA miscoding caused by altered equilibrium between open/closed ribosomal conformations. This mechanistic commonality suggests that alteration of rearrangements represents an evolutionarily convenient way of modulating substrate selectivity.     

Who's on first? The U1 snRNP-5' splice site interaction and splicing

U1 small nuclear ribonucleoprotein (snRNP) is important for pre-mRNA splicing both in yeast (Saccharomyces cerevisiae) and mammalian systems. The RNA component of U1 snRNP, U1 snRNA, interacts by base pairing with pre-mRNA 5' splice sites. This article examines recent evidence suggesting that U1 snRNP is important for an early step in spliceosome assembly rather than a late step that contributes to the specificity of 5' splice-site cleavage.     

Both U2 snRNA and U12 snRNA are required for accurate splicing of exon 5 of the rat calcitonin/CGRP gene

Two classes of spliceosome are present in eukaryotic cells. Most introns in nuclear pre-mRNAs are removed by a spliceosome that requires U1, U2, U4, U5, and U6 small nuclear ribonucleoprotein particles (snRNPs). A minor class of introns are removed by a spliceosome containing U11, U12, U5, U4atac, and U6 atac snRNPs. We describe experiments that demonstrate that splicing of exon 5 of the rat calcitonin/CGRP gene requires both U2 snRNA and U12 snRNA. In vitro, splicing to calcitonin/ CGRP exon 5 RNA was dependent on U2 snRNA, as preincubation of nuclear extract with an oligonucleotide complementary to U2 snRNA abolished exon 5 splicing. Addition of an oligonucleotide complementary to U12 snRNA increased splicing at a cryptic splice site in exon 5 from <5% to 50% of total spliced RNA. Point mutations in a candidate U12 branch sequence in calcitonin/CGRP intron 4, predicted to decrease U12-pre-mRNA base-pairing, also significantly increased cryptic splicing in vitro. Calcitonin/CGRP genes containing base changes disrupting the U12 branch sequence expressed significantly decreased CGRP mRNA levels when expressed in cultured cells. Coexpression of U12 snRNAs containing base changes predicted to restore U12-pre-mRNA base pairing increased CGRP mRNA synthesis to the level of the wild-type gene. These observations indicate that accurate, efficient splicing of calcitonin/CGRP exon 5 is dependent upon both U2 and U12 snRNAs.     

Proximity of the invariant loop of U5 snRNA to the second intron residue during pre-mRNA splicing.

A photoactivatable azidophenacyl group has been introduced into seven positions in the backbone of the 11 nucleotide invariant loop of U5 snRNA. By reconstituting depleted splicing extracts with reassembled U5 snRNP particles, molecular neighbors were assessed as a function of splicing. All cross-links to the pre-mRNA mapped to the second nucleotide downstream of the 5' splice site, and formed most readily when the reactive group was at the phosphate between U5 positions 42 and 43 or 43 and 44. Both their kinetics of appearance and sensitivity to oligonucleotide inhibition suggest that these cross-links capture a late state in spliceosome assembly occurring immediately prior to the first step. A later forming, second cross-linked species is a splicing product of the first cross-link, suggesting that the U5 loop backbone maintains this position through the first step. The proximity of the U5 loop backbone to the intron's 5' end provides sufficient restrictions to develop a three-dimensional model for the arrangement of RNA components in the spliceosome during the first step of pre-mRNA splicing.     

Functional analysis of the U5 snRNA loop 1 in the second catalytic step of yeast pre-mRNA splicing.

The U5 snRNA loop 1 interacts with the 5' exon before the first step of pre-mRNA splicing and with the 5' and 3' exons following the first step. These U5-exon interactions are proposed to hold the exons in the correct orientation for the second step of splicing. Reconstitution of U5 snRNPs in vitro indicated that U5 loop 1-5' exon interactions are not necessary for the first catalytic step of splicing but are critical for the second step in yeast spliceosomes. We systematically made deletion and insertion mutations in loop 1 then monitored splicing activity and loop-exon interactions by cross-linking. Single nucleotide deletions or insertions in loop 1 permitted both steps of splicing. Larger insertions or deletions allowed the first step but progressively inhibited the second step. Analysis of selected loop 1 insertions and deletions by cross-linking revealed that inhibition of the second catalytic step resulted from misalignment of the 5' and 3' exons. These data indicate that the size of loop 1 is critical for proper alignment of the exons for the second catalytic step of splicing and that the 3' exon is positioned on loop 1 independently of the 5' exon.     

Antisense probes targeted to an internal domain in U2 snRNP specifically inhibit the second step of pre-mRNA splicing.

Functional domains within the mammalian U2 snRNP particle that are required for pre-mRNA splicing have been analysed using antisense oligonucleotides. A comparison of the melting temperatures of duplexes formed between RNA and different types of antisense oligonucleotides has demonstrated that the most stable hybrids are formed with probes made of 2'-O-allyl RNA incorporating the modified base 2-aminoadenine. We have therefore used these 2'-O-allyl probes to target sequences within the central domain of U2 snRNA. Overlapping biotinylated 2'-O-allyloligoribonucleotides complementary to the stem loop Ila region of U2 snRNA (nucleotides 54-72) specifically affinity selected U2 snRNA from HeLa nuclear extracts. These probes inhibited mRNA production in an in vitro splicing assay and caused a concomitant accumulation of splicing intermediates. Little or no inhibition of spliceosome assembly and 5' splice site cleavage was observed for all pre-mRNAs tested, indicating that the oligonucleotides were specifically inhibiting exon ligation. This effect was most striking with a 2'-O-allyloligoribonucleotide complementary to U2 snRNA nucleotides 57-68. These results provide evidence for a functional requirement for U2 snRNP in the splicing mechanism occurring after spliceosome assembly.     

Early commitment of yeast pre-mRNA to the spliceosome pathway.

Pre-mRNA splicing in vitro is preceded by complex formation (spliceosome assembly). U2 small nuclear RNA (snRNA) is found in the earliest form of the spliceosome detected by native gel electrophoresis, both in Saccharomyces cerevisiae and in metazoan extracts. To examine the requirements for the formation of this early complex (band III) in yeast extracts, we cleaved the U2 snRNA by oligonucleotide-directed RNase H digestion. U2 snRNA depletion by this means inhibits both splicing and band III formation. Using this depleted extract, we were able to design a chase experiment which shows that a pre-mRNA substrate is committed to the spliceosome assembly pathway in the absence of functional U2 snRNP. Interactions occurring during the commitment step are highly resistant to the addition of an excess of unlabeled substrate and require little or no ATP. Sequence requirements for this commitment step have been analyzed by competition experiments with deletion mutants: both the 5' splice site consensus sequence and the branch point TACTAAC box sequence are necessary. These experiments strongly suggest that the initial assembly process requires a trans-acting factor(s) (RNA and/or proteins) that recognizes and stably binds to the two consensus sequences of the pre-mRNA prior to U2 snRNP binding.