The role of branchpoint and 3'-exon sequences in the control of balanced splicing of avian retrovirus RNA.

We previously described an avian sarcoma-leukosis virus (ASLV) insertion mutation that causes a decrease in the ratio of unspliced to spliced RNA in vivo, resulting in a replication defect. Pseudorevertant viruses containing cis-acting suppressor mutations that restored the normal ratio were isolated. One class of the suppressor mutations consists of single-base changes or small deletions near the 3' splice site, while another consists of deletions in the 3' exon. In this paper we report results from an in vitro analysis of wild-type, mutant, and pseudorevertant pre-mRNA splicing. We find that wild-type RNA is spliced inefficiently in vitro, and that the insertion mutation and suppressors act directly at the level of splicing. Characterization of splicing intermediates reveals that the insertion mutation and suppressor mutations located within the intron alter the pattern of lariat formation. In contrast, suppressor mutations consisting of 3' exon deletions act at an earlier step in the splicing pathway. Thus, the efficiency of splicing at the env 3' splice site can be affected at the level of spliceosome assembly, lariat formation, or cleavage at the 3' splice site and exon ligation.     

A chemical modification/interference study of yeast pre-mRNA spliceosome assembly and splicing

A chemical modification/interference assay was used to determine the yeast pre-mRNA sequence requirements for in vitro spliceosome assembly and splicing. Modifications of any of the nucleotides within the 5' splice site and branch point (TACTAAC box) consensus sequences as well as less conserved intron and exon positions were found to inhibit assembly and/or splicing. The interference pattern of the 5' splice site and TACTAAC box lesions increased as spliceosome assembly proceeded (complex III----complex I----complex II) and as splicing proceeded, suggesting that these sequence elements play multiple roles in the assembly of yeast spliceosomes and in the removal of intervening sequences. Furthermore, modification (or mutation) of a TACTAAC-like sequence upstream of the branch point was found to inhibit the rate of spliceosome assembly, implying a possible role for degenerate branch point sequences in modulating the efficiency of spliceosome assembly.     

A novel role for the 3' region of introns in pre-mRNA splicing of Saccharomyces cerevisiae

To investigate the importance of sequences between the yeast (Saccharomyces cerevisiae) branch point (TACTAAC box) and 3' splice site (AG), we generated a series of pre-mRNA substrates that differed in the length of RNA retained on the 3' side of the TACTAAC box. These pre-mRNAs were compared as substrates for the first step of in vitro splicing (5' cleavage and lariat formation) and in vitro spliceosome assembly (complex formation) in a whole-cell yeast extract. The results indicate that for rp51A pre-mRNA at least 29 nucleotides of RNA on the 3' side of the TACTAAC box are required for 5' cleavage and lariat formation, as smaller substrates fail to manifest any detectable cleavage or ligation events. Analysis of splicing complex assembly indicates that these smaller substrates undergo efficient yet incomplete complex formation; they are blocked at a late stage of spliceosome assembly, the complex I to complex II transition (Pikielny et al. 1986), a result which suggests that the failure to form lariats is due to a specific assembly defect. The lariat formation block (and assembly defect) can be relieved by the addition of ribohomopolymer "tails" to the 3' end of the shortened rp51A pre-mRNAs, and similar results were obtained with shortened actin pre-mRNAs. The results of this study indicate that this region of the pre-mRNA serves a specific function late in in vitro spliceosome assembly.     

Interaction of the U1 snRNP with nonconserved intronic sequences affects 5' splice site selection

Intron definition and splice site selection occur at an early stage during assembly of the spliceosome, the complex mediating pre-mRNA splicing. Association of U1 snRNP with the pre-mRNA is required for these early steps. We report here that the yeast U1 snRNP-specific protein Nam8p is a component of the commitment complexes, the first stable complexes assembled on pre-mRNA. In vitro and in vivo, Nam8p becomes indispensable for efficient 5' splice site recognition when this process is impaired as a result of the presence of noncanonical 5' splice sites or the absence of a cap structure. Nam8p stabilizes commitment complexes in the latter conditions. Consistent with this, Nam8p interacts with the pre-mRNA downstream of the 5' splice site, in a region of nonconserved sequence. Substitutions in this region affect splicing efficiency and alternative splice site choice in a Nam8p-dependent manner. Therefore, Nam8p is involved in a novel mechanism by which a snRNP component can affect splice site choice and regulate intron removal through its interaction with a nonconserved sequence. This supports a model where early 5' splice recognition results from a network of interactions established by the splicing machinery with various regions of the pre-mRNA.     

Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly

The pairing of 5′ and 3′ splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs and make particular alternative splicing choices. Here, we report a new interaction between stem–loop 4 (SL4) of the U1 snRNA, which recognizes the 5′ splice site, and a component of the U2 small nuclear ribonucleoprotein particle (snRNP) complex, which assembles across the intron at the 3′ splice site. Using a U1 snRNP complementation assay, we found that SL4 is essential for splicing in vivo. The addition of free U1-SL4 to a splicing reaction in vitro inhibits splicing and blocks complex assembly prior to formation of the prespliceosomal A complex, indicating a requirement for a SL4 contact in spliceosome assembly. To characterize the interactions of this RNA structure, we used a combination of stable isotope labeling by amino acids in cell culture (SILAC), biotin/Neutravidin affinity pull-down, and mass spectrometry. We show that U1-SL4 interacts with the SF3A1 protein of the U2 snRNP. We found that this interaction between the U1 snRNA and SF3A1 occurs within prespliceosomal complexes assembled on the pre-mRNA. Thus, SL4 of the U1 snRNA is important for splicing, and its interaction with SF3A1 mediates contact between the 5′ and 3′ splice site complexes within the assembling spliceosome.     

Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing

To study the determinants of splice site selection, we have inserted synthetic 5' and 3' splice sites at different positions within beta-globin genes and analyzed the resultant RNA substrates for in vitro splicing, factor binding, and complex assembly. We show that consensus 5' and 3' splice site sequences are insufficient to determine splice site utilization; in the presence or absence of the authentic site, the synthetic sites are variably active in a position-dependent manner. However, regardless of position or utilization, the synthetic 5' and 3' splice sites are bound by the appropriate splicing factors. Thus, binding of splicing factors is necessary but not sufficient for splice site utilization. Finally, we demonstrate that a block to efficient splicing can occur at multiple steps in the pathway of normal splicing complex assembly.     

Analysis of the LAMB3 gene in a junctional epidermolysis bullosa patient reveals exonic splicing and allele-specific nonsense-mediated mRNA decay

How splicing, the process of intron removal in pre-messenger RNA (mRNA), is carried out with such fidelity in human cells is still not understood, although some general rules are being proposed mainly by in vitro experiments. These rules are currently being redefined by analysis of splicing mechanisms in patients presenting splicing defects. We analysed material of a patient suffering from junctional epidermolysis bullosa, a heritable blistering skin disease. Absence of laminin-5 protein together with hypoplastic hemidesmosomes at the dermo-epidermal junction in the patient's skin was shown by immunohistochemical analysis and immunoelectron microscopy. Subsequent DNA analysis revealed heterozygosity for the mutations R635X and 3009C-->T in the LAMB3 gene. The latter did not alter codon translation, but introduced an exonic splice site in exon 20. Interestingly, this exonic splice site, which presented a splice score of only 68.6, was preferentially used by the spliceosome over the wild-type splice site at the exon 20-intron 20 border, which showed a splice score of 92.2. LAMB3 mRNA was still detectable in RT-PCR analysis although the aberrantly spliced mRNA leads to a stop codon in exon 21, 5' of the commonly assumed 3' border for nonsense-mediated mRNA decay. These results describe an exception to the proposed rules of pre-mRNA splicing and RNA degradation.     

5' exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

A general consequence of pre-mRNA splicing is the stable deposition of several proteins 20-24 nucleotides (nt) upstream of exon-exon junctions on spliced mRNAs. This exon junction complex (EJC) contains factors involved in mRNA export, cytoplasmic localization, and nonsense-mediated mRNA decay. Here we probed the mechanism and timing of EJC assembly. Over the course of splicing, the 5' exon is subject to numerous dynamic protein-RNA interactions involving at least nine distinct polypeptides. Within the fully assembled spliceosome, these interactions afford protection to the last 25-27 nt of the 5' exon intermediate. Coincident with exon ligation, interactions at the 3' end of the 5' exon disappear, and new species associate with position -24. Mass spectrometry and Western blotting of purified H, C, and mRNP complexes revealed that at least one EJC component, REF/Aly, can interact with pre-mRNA prior to spliceosome assembly, whereas Y14, Magoh, RNPS1, UAP56, and SRm160 are found in intermediate-containing spliceosomes. Upon exon ligation, association of RNPS1, UAP56, and SRm160 is destabilized. In contrast, REF/Aly, Y14, and Magoh remain stably bound to spliced mRNA, indicating that these three proteins are components of the EJC core.     

Identification of two distinct intron elements involved in alternative splicing of beta-tropomyosin pre-mRNA

The rat beta-tropomyosin gene encodes two isoforms, termed skeletal muscle beta-tropomyosin and fibroblast last tropomyosim 1 (TM-1), via an alternative RNA processing mechanism. The gene contains 11 exons. Exons 1-5 and exons 8 and 9 are common to all mRNAs expressed from the gene. Exons 6 and 11 are used in fibroblasts, as well as smooth muscle, whereas exons 7 and 10 are used only in skeletal muscle. In the present studies we focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). We have identified two distinct elements in the intron, upstream of exon 7, involved in splice site selection. The first element is comprised of a polypyrimidine tract located 89-143 nucleotides upstream of the 3' splice site, which specifies the location of the lariat branchpoints used, 144-153 nucleotides upstream of exon 7. The 3' splice site AG dinucleotide has no role in selection of these branchpoints. The second element is comprised of intron sequences located between the polypyrimidine tract and the 3' splice site of exon 7. It contains an important determinant in alternative splice site selection, because deletion of these sequences results in the use of the skeletal muscle-specific exon in nonmuscle cells. We propose that the use of lariat branchpoints located far upstream from a 3' splice site may be a general feature of some alternatively excised introns, reflecting the presence of regulatory sequences located between the lariat branch site and the 3' splice site. The data also indicate that alternative splicing of the rat beta-tropomyosin gene is regulated by a somewhat different mechanism from that described for rat alpha-tropomyosin gene and the transformer-2 gene of Drosophila melanogaster.     

Human step II splicing factor hSlu7 functions in restructuring the spliceosome between the catalytic steps of splicing

The spliceosome catalyzes pre-mRNA splicing in two steps. After catalytic step I, a major remodeling of the spliceosome occurs to establish the active site for step II. Here, we report the isolation of a cDNA encoding hSlu7, the human homolog of the yeast second step splicing factor Slu7. We show that hSlu7 associates with the spliceosome late in the splicing pathway, but at a stage prior to recognition of the 3' splice site for step II. In the absence of hSlu7, splicing is stalled between the catalytic steps in a novel complex, the CDeltahSlu7 complex. We provide evidence that this complex differs significantly in structure from the known spliceosomal complexes, yet is a functional intermediate between the catalytic steps of splicing. Together, our observations indicate that hSlu7 is required for a structural alteration of the spliceosome prior to the establishment of the catalytically active spliceosome for step II.     

Association of U6 snRNA with the 5'-splice site region of pre-mRNA in the spliceosome.

U6 snRNA is one of the five RNA species required for splicing of nuclear pre-mRNAs. High conservation of its sequence has led to the hypothesis that U6 snRNA plays a catalytic role in splicing. If this is the case, U6 snRNA should be localized close to sites where the splicing reaction occurs. However, this has never been demonstrated. Here, we have shown that U6 snRNA is cross-linked to the 5'-splice site region of pre-mRNA by UV irradiation during the in vitro splicing reaction. We have also detected the cross-link of U6 snRNA and the region around the branchpoint of the intron lariat. The results show that U6 snRNA is present near the splice sites in the splicing reaction and support the idea that U6 snRNA is a catalytic element in the spliceosome.     

Protein components specifically associated with prespliceosome and spliceosome complexes.

We have carried out a systematic analysis of the protein composition of highly purified mammalian spliceosomes. We show that > 30 distinct proteins, including 20 previously unidentified components [designated spliceosome-associated proteins (SAPs)], are specifically associated with the spliceosome in a salt-resistant complex. In contrast to these spliceosome-specific proteins, we show that hnRNP proteins are not tightly associated with purified prespliceosome and spliceosome complexes. The splicing factor U2AF65, U1 snRNP-specific proteins, and several SAPs are present in the earliest prespliceosome complex (E). A set of 10 proteins is then added to the first ATP-dependent prespliceosome complex (A), and concomitantly, a significant decrease in the level of U2AF65 is observed. The fully assembled spliceosome is formed by the addition of 12 proteins in a reaction that requires ATP and both the 5' and 3' splice sites.     

Identification of eight proteins that cross-link to pre-mRNA in the yeast commitment complex

In the yeast commitment complex and the mammalian E complex, there is an important base-pairing interaction between the 5' end of U1 snRNA and the conserved 5' splice site region of pre-mRNA. But no protein contacts between splicing proteins and the pre-mRNA substrate have been defined in or near this region of early splicing complexes. To address this issue, we used 4-thiouridine-substituted 5' splice site-containing RNAs as substrates and identified eight cross-linked proteins, all of which were identified previously as commitment complex components. The proteins were localized to three domains: the exon, the six nucleotides of the 5' ss region, and the downstream intron. The results indicate that the 5' splice site region and environs are dense with protein contacts in the commitment complex and suggest that some of them make important contributions to formation or stability of the U1 snRNP-pre-mRNA complex.     

Metal ion catalysis during group II intron self-splicing: parallels with the spliceosome

The identical reaction pathway executed by the spliceosome and self-splicing group II intron ribozymes has prompted the idea that both may be derived from a common molecular ancestor. The minimal sequence and structural similarities between group II introns and the spliceosomal small nuclear RNAs, however, have left this proposal in question. Mechanistic comparisons between group II self-splicing introns and the spliceosome are therefore important in determining whether these two splicing machineries may be related. Here we show that 3'-sulfur substitution at the 5' splice site of a group II intron causes a metal specificity switch during the first step of splicing. In contrast, 3'-sulfur substitution has no significant effect on the metal specificity of the second step of cis-splicing. Isolation of the second step uncovers a metal specificity switch that is masked during the cis-splicing reaction. These results demonstrate that group II intron ribozymes are metalloenzymes that use a catalytic metal ion for leaving group stabilization during both steps of self-splicing. Furthermore, because 3'-sulfur substitution of a spliceosomal intron has precisely the same effects as were observed during cis-splicing of the group II intron, these results provide striking parallels between the catalytic mechanisms employed by these two systems.     

U2 toggles iteratively between the stem IIa and stem IIc conformations to promote pre-mRNA splicing

To ligate exons in pre-messenger RNA (pre-mRNA) splicing, the spliceosome must reposition the substrate after cleaving the 5' splice site. Because spliceosomal small nuclear RNAs (snRNAs) bind the substrate, snRNA structures may rearrange to reposition the substrate. However, such rearrangements have remained undefined. Although U2 stem IIc inhibits binding of U2 snRNP to pre-mRNA during assembly, we found that weakening U2 stem IIc suppressed a mutation in prp16, a DExD/H box ATPase that promotes splicing after 5' splice site cleavage. The prp16 mutation was also suppressed by mutations flanking stem IIc, suggesting that Prp16p facilitates a switch from stem IIc to the mutually exclusive U2 stem IIa, which activates binding of U2 to pre-mRNA during assembly. Providing evidence that stem IIa switches back to stem IIc before exon ligation, disrupting stem IIa suppressed 3' splice site mutations, and disrupting stem IIc impaired exon ligation. Disrupting stem IIc also exacerbated the 5' splice site cleavage defects of certain substrate mutations, suggesting a parallel role for stem IIc at both catalytic stages. We propose that U2, much like the ribosome, toggles between two conformations--a closed stem IIc conformation that promotes catalysis and an open stem IIa conformation that promotes substrate binding and release.     

Alpha-tropomyosin mutually exclusive exon selection: competition between branchpoint/polypyrimidine tracts determines default exon choice

We have used exons 2 and 3 of the rat alpha-tropomyosin gene to analyze the basis of mutually exclusive exon selection. The basis of the strict mutually exclusive behavior of this exon pair is enforced by the proximity of the exon 3 branchpoint to the 5' splice site of exon 2. With the exception of smooth muscle cells, exon 3 rather than exon 2 is incorporated into mRNA in all cell types. We show here, using both in vivo and in vitro cell-free systems, that this alternative exon selection is a consequence of general principles that govern 3' splice site selection. In the absence of exon 3, exon 2 is utilized efficiently in all cells. Selection of exon 3 is therefore the default result of a competition between exons 2 and 3 for the flanking constitutive splice sites. The basis of this competition is the relative strength of the polypyrimidine tract/branchpoint elements of the two exons. The major determinant of this splice site strength is the pyrimidine content adjacent to the branchpoint, and this involves no other sequence specificity. The branchpoint elements play an important but secondary role. The functional strengths of the different polypyrimidine tract/branchpoint combinations, as determined in cis competition assays, showed a perfect correlation with their binding affinities to a spliceosome component that interacts with the pre-mRNA in an ATP-independent manner. Selection of exon 3 in most cell types therefore reflects the preferential interaction of these splice site elements with constitutive splicing factors early in spliceosome assembly. The aspects of splice site selection analyzed here are likely to be of general applicability to constitutive and alternative pre-mRNA splicing.