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.     

Yeast mRNA splicing in vitro

Synthetic actin and CYH2 pre-mRNAs containing a single intron are accurately spliced in a soluble whole cell extract of yeast. Splicing in vitro requires ATP. The excised intron is released as a lariat in which an RNA branch connects the 5' end of the molecule to the last A in the "intron conserved sequence" UACUAAC. Two other discrete RNA species produced during splicing in vitro may represent reaction intermediates: free, linear exon 1 and a form of the intron lariat extending beyond the 3' splice site to include exon 2. Both lariat forms correspond to molecules previously shown to be produced during yeast pre-mRNA splicing in vivo.     

Purification of a RNA debranching activity from HeLa cells

The splicing of messenger RNA precursors (pre-mRNA) of eukaryotic cells involves the formation of a branched RNA intermediate known as a RNA lariat. This structure is formed in the first step of the reaction when a cleavage at the 5' splice site generates the 5' exon and a RNA species containing the intron and 3' exon in which the phosphate moiety at the 5' end of the intron is forming a 2'-5' phosphodiester bond with the 2'-hydroxyl moiety of a specific adenine residue near the 3' end of the intron forming a RNA branch with the following structure: -pA2'-pX-3'-pZ-. We have purified a debranching activity approximately 700-fold from the cytosolic fraction of HeLa cells. This activity catalyzes the hydrolysis of the 2'-5' phosphodiester bond of branched RNA structures yielding a 5'-phosphate end and a 2'-hydroxyl group at the branch attachment site. The activity possessed a sedimentation coefficient of 3.5 S. The reaction catalyzed by the purified fraction requires a divalent cation and is optimal at pH 7.0. The purified activity can efficiently hydrolyze triester trinucleotide structures (pY2'-pX-3'-pZ-) prepared by digestion of RNA lariats with nuclease P1. In contrast, a 2' phosphate monoester product (-pG2'-p 3'-pC-), formed by the wheat germ RNA ligase, was not attacked.     

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.     

Spatial organization of protein-RNA interactions in the branch site-3' splice site region during pre-mRNA splicing in yeast

A series of efficiently spliced pre-mRNA substrates containing single 4-thiouridine residues were used to monitor RNA-protein interactions involving the branch site-3' splice site-3' exon region during yeast pre-mRNA splicing through cross-linking analysis. Prior to the assembly of the prespliceosome, Mud2p and the branch point bridging protein cross-link to a portion of this region in an ATP-independent fashion. Assembly of the prespliceosome leads to extensive cross-linking of the U2-associated protein Hsh155p to this region. Following the first step of splicing and in a manner independent of Prp16p, the U5 small nuclear ribonucleoprotein particle-associated protein Prp8p also associates extensively with the branch site-3' splice site-3' exon region. The subsequent cross-linking of Prp16p to the lariat intermediate is restricted to the 3' splice site and the adjacent 3' exon sequence. Using modified substrates to either mutationally or chemically block the second step, we found that the association of Prp22p with the lariat intermediate represents an authentic transient intermediate and appears to be restricted to the last eight intron nucleotides. Completion of the second step leads to the cross-linking of an unidentified approximately 80-kDa protein near the branch site sequence, suggesting a potential role for this protein in a later step in intron metabolism. Taken together, these data provide a detailed portrayal of the dynamic associations of proteins with the branch site-3' splice site region during spliceosome assembly and catalysis.     

Antisense-targeted immuno-EM localization of the pre-mRNA path in the spliceosomal C complex

A first step in understanding the architecture of the spliceosome is elucidating the positions of individual spliceosomal components and functional centers. Catalysis of the first step of pre-mRNA splicing leads to the formation of the spliceosomal C complex, which contains the pre-mRNA intermediates--the cleaved 5' exon and the intron-3' exon lariat. To topographically locate the catalytic center of the human C complex, we first determined, by DNA oligonucleotide-directed RNAse H digestions, accessible pre-mRNA regions closest to nucleotides of the cleaved 5' splice site (i.e., the 3' end of exon 1 and the 5' end of the intron) and the intron lariat branch point, which are expected to be at/near the catalytic center in complex C. For electron microscopy (EM) localization studies, C complexes were allowed to form, and biotinylated 2'-OMe RNA oligonucleotides were annealed to these accessible regions. To allow localization by EM of the bound oligonucleotide, first antibiotin antibodies and then protein A-coated colloidal gold were additionally bound. EM analyses allowed us to map the position of exon and intron nucleotides near the cleaved 5' splice site, as well as close to the anchoring site just upstream of the branch adenosine. The identified positions in the C complex EM map give first hints as to the path of the pre-mRNA splicing intermediates in an active spliceosomal C complex and further define a possible location for its catalytic center.     

Natural base-pairing interactions between 5' splice site and branch site sequences affect mammalian 5' splice site selection.

In the murine gene encoding the neuronal cell adhesion molecule (NCAM), the integrity of the 5' splice site of exon 18 (E18) is essential for regulation of alternative splicing. To further study the contribution of 5' splice site sequences, we used a simple NCAM pre-mRNA containing a portion of E18 fused to E19 and separated by a shortened intron. This RNA is spliced in vitro to produce five sets of lariat intermediates and products, the most abundant set displaying aberrant migration in acrylamide/urea gels. Base pairing interactions between positions +5 and +8 of the intron and positions -3 and -6 from the branch point were responsible for the faster migration of this set of lariat molecules. To test whether the duplex structure forms earlier and contributes to 5' splice site selection, we used NCAM substrates carrying the 5' splice sites of E17 and E18 in competition for the 3' splice site of E19. Mutations upstream of the major branch site improve E18/E19 splicing in NIH3T3 extracts, whereas compensatory mutations at positions +7 and +8 neutralize the effect of branch site mutations and curtail E18/E19 splicing. Our data suggest that duplex formation occurs early and interferes with the assembly of complexes initiated on the 5' splice site of NCAM E18. This novel type of intron interaction may exist in the introns of other mammalian pre-mRNAs.     

Cryptic branch point activation allows accurate in vitro splicing of human beta-globin intron mutants

The excised introns of pre-mRNAs and intron-containing splicing intermediates are in a lariat configuration in which the 5' end of the intron is linked by a 2'-5' phosphodiester bond (RNA branch) to a single adenosine residue near the 3' end of the intron. To determine the role of the specific sequence surrounding the RNA branch, we have mutated the branch point sequence of the human beta-globin IVS1. Pre-mRNAs lacking the authentic branch point sequence are accurately spliced in vitro; processing of the mutant pre-mRNAs generates RNA lariats due to the activation of cryptic branch points within IVS1. The cryptic branch points always occur at adenosine residues, but the sequences surrounding the branched nucleotide vary. Regardless of the type of mutation or the sequences remaining within IVS1, the cryptic branch points are 22 to 37 nucleotides upstream of the 3' splice site. These results suggest that RNA branch point selection is primarily based on a mechanism that measures the distance from the 3' splice site.     

Trans-complementation of the second step of pre-mRNA splicing by exogenous 5' exons.

During splicing of nuclear pre-mRNAs, the first step liberates the 5' exon (exon 1) and yields a lariat intron-3'exon (intron-exon 2) intermediate. The second step results in exon ligation. Previous results indicated that severe truncations of the 5' exon of the actin pre-mRNA result in a block to the second splicing step in vitro in yeast extracts, leading to an accumulation of intron-exon 2 lariat intermediates. We show that exogenous exon 1 RNA oligonucleotides can chase these stalled intermediates into lariat intron and spliced exons. This reaction requires some of the cis elements and trans-acting factors that are required for a normal second step. There is no strong sequence requirement for the exon 1 added in trans, but oligonucleotides with complementarity to the U5 snRNA conserved loop perform the chase more efficiently. Using a dominant negative mutant of the DEAH-box ATPase Prp16p and ATP depletion, we show that the stalled intermediate is blocked after the Prp16p-dependent step. These results show that exogenous RNAs with various sequences but containing no splicing signals can be incorporated into spliceosomes and undergo RNA recombination and exon shuffling during the second step of pre-mRNA splicing.     

Distribution and consensus of branch point signals in eukaryotic genes: a computerized statistical analysis.

An intermediate stage in the process of eukaryotic RNA splicing is the formation of a lariat structure. It is anchored at an adenosine residue in intron between 10 and 50 nucleotides upstream of the 3' splice site. A short conserved sequence (the branch point sequence) functions as the recognition signal for the site of lariat formation. It has been generally assumed that the branch point is recognized mainly by the presence of its unique sequence where the lariat is formed. However, the known branch point consensus sequence is found to be distributed nearly randomly throughout the gene sequence with only a slightly higher frequency in the expected lariat region. Further, the known consensus sequence is found to be clearly inadequate to specify branch points. These observations have implications for understanding the mechanism of branch point recognition in the process of splicing, and the possible evolution of the branch point signal.     

Unusual branch point selection in processing of human growth hormone pre-mRNA.

Intron A of the human growth hormone gene does not contain an A residue within 56 nucleotides preceding the 3' splice site. The analysis of the excised intron lariat revealed a C residue 28 nucleotides upstream from the 3' splice site as the major branch acceptor nucleotide. Two additional minor branched nucleotides were identified as U residues at positions -22 and -36. An adenosine substitution at position -22 results in lariat formation solely to this nucleotide. Therefore, C and U residues can function efficiently as natural branch acceptors, but an A residue is preferred if available in the proper region. In addition, the data strongly reinforce the importance of the distance constraint for lariat formation. To explain selection of the branch acceptor nucleotide, potential base-pairing interactions of branch point sequences with the U2 RNA are discussed.     

A 62,000 molecular weight spliceosome protein crosslinks to the intron polypyrimidine tract

Incubation in HeLa nuclear extract of a 32P-labeled 61 nucleotide-long RNA corresponding to the lariat branch site/polypyrimidine tract/3' splice site of the first intron of human beta-globin pre-mRNA led to the crosslinking of a single protein of approximately 62,000 mol. wt. (p62). p62 corresponds to a polypyrimidine tract-binding protein recently described by Garcia-Blanco et al. (Genes & Dev. 3: 1874-1886, 1989). Crosslinking of p62 to the 61 nt RNA was highly sequence specific. No p62 crosslinking was observed with a 60 nt pGEM vector RNA, a 63 nt RNA antisense to the 61-mer or a 72 nt U2 RNA sequence. p62 crosslinking to the 61 nt RNA was competed by unlabeled 61 nt RNA, by beta-globin pre-mRNA containing intron 1, and by poly(U) and poly(C), but was competed to a lesser extent or not at all by pGEM RNA, a beta-globin RNA lacking intron 1, or poly(A). Experiments with mutated RNAs revealed that neither the lariat branch site adenosine nor the 3' splice site were required for p62 crosslinking to polypyrimidine tract-containing RNA. Elimination of the polypyrimidine tract reduced p62 crosslinking, as did mutation of a polypyrimidine tract UU dinucleotide to GA. However, replacement of a pyrimidine-rich tract immediately adjacent (3') to the lariat branch site with a 57% A + G pGEM vector RNA sequence also significantly reduced p62 crosslinking, indicating the involvement of both this pyrimidine-rich region and the classical polypyrimidine tract adjacent to the 3' splice site. The sites of protein interaction were further defined by RNase H protection experiments, the results of which confirmed the patterns of p62 crosslinking to mutant RNAs. p62 crosslinking was efficiently competed by a DNA oligonucleotide having the same sequence as the 61 nt RNA, showing that p62 requires neither ribose 2' OH groups nor uracil bases for its interaction with the polypyrimidine tract. p62 was not crosslinked to double-stranded 61 nt RNA. Q-Sepharose chromatography of HeLa nuclear extract yielded an unbound fraction (QU) in which p62 was the only polypyrimidine tract-crosslinkable protein and a bound fraction (QB) in which, surprisingly, several non-p62 proteins were crosslinkable to the polypyrimidine tract RNA. Yet, when the two Q-Sepharose fractions were combined, p62 strongly out-competed the otherwise-crosslinkable QB proteins for polypyrimidine tract RNA crosslinking. This indicates that p62 may have the highest affinity and/or crosslinking efficiency for the intron polypyrimidine tract of any HeLa nuclear protein.     

Effect of 5'' splice site mutations on splicing of the preceding intron.

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.     

Self-splicing of group II introns in vitro: lariat formation and 3' splice site selection in mutant RNAs

Deletion or substitution of the branch A residue in group II intron bl1 significantly reduces splicing activity; yet, residual exon ligation is correct, and lariats have their branch points at the normal distance from the 3' end of the intron. Mutations in the sequence facing the branch point also allow residual lariat formation; however, free 3' exons are generated with false 5' termini, all of which are within a UCACA consensus sequence located upstream or downstream of the normal 3' splice site. These results indicate that both the conserved 3' splice site APy and the spatial arrangements in stem 6 are crucial for correct 3' splice site selection.     

Relevance of the branch point adenosine, coordination loop, and 3' exon binding site for in vivo excision of the Sinorhizobium meliloti group II intron RmInt1

Excision of the bacterial group II intron RmInt1 has been demonstrated in vivo, resulting in the formation of both intron lariat and putative intron RNA circles. We show here that the bulged adenosine in domain VI of RmInt1 is required for splicing via the branching pathway, but branch site mutants produce small numbers of RNA molecules in which the first G residue of the intron is linked to the last C residue. Mutations in the coordination loop in domain I reduced splicing efficiency, but branched templates clearly predominated among splicing products. We also found that a single substitution at the EBS3 position (G329C), preventing EBS3-IBS3 pairing, resulted in the production of 50 to 100 times more RNA molecules in which the 5' and 3' extremities were joined. We provide evidence that these intron molecules may correspond to both, intron circles linked by a 2'-5' phosphodiester bond, and tandem, head-to-tail intron copies.