Uridine branch acceptor is a cis-acting element involved in regulation of the alternative processing of calcitonin/CGRP-l pre-mRNA.

The human calcitonin/CGRP-I (CALC-I) gene contains 6 exons and encodes two polypeptide precursors. In thyroid C-cells, calcitonin (CT) mRNA is produced by splicing of exons 1-2-3 to exon 4 (CT-encoding) and polyadenylation at exon 4. CGRP-I mRNA is produced in particular neural cells by splicing of exons 1-2-3 to exon 5 (CGRP-I-encoding) and the polyadenylated exon 6. We previously reported that model precursor RNAs containing the exon 3 to exon 5 region of the CALC-I gene are processed predominantly into CGRP-I mRNA in vitro, in nuclear extracts of several cell types (neural and non-neural). Using truncated precursor RNAs containing only the exon 3 to exon 4 region of the CALC-I gene it was shown that CT splicing is an inefficient reaction in which a uridine residue serves as the major site of lariat formation. Here we report that the low CT splicing efficiency and the dominance of CGRP-I splicing over CT splicing in vitro are primarily due to the usage of the CT-specific uridine branch acceptor. Mutation of this uridine residue into an adenosine residue resulted in a strong increase in CT splicing efficiency causing a reversal of the splicing pattern. In addition, it was shown that this point mutation also increased CT splicing efficiency in vivo. These results and data obtained from other experiments involving mutation of the CT splice acceptor site suggest that the uridine branch acceptor is a cis-acting element involved in regulation of the alternative processing of the CALC-I pre-mRNA.     

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.     

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.     

Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons.

Circularly permuted group I intron precursor RNAs, containing end-to-end fused exons which interrupt half-intron sequences, were generated and tested for self-splicing activity. An autocatalytic RNA can form when the primary order of essential intron sequence elements, splice sites, and exons are permuted in this manner. Covalent attachment of guanosine to the 5' half-intron product, and accurate exon ligation indicated that the mechanism and specificity of splicing were not altered. However, because the exons were fused and the order of the splice sites reversed, splicing released the fused-exon as a circle. With this arrangement of splice sites, circular exon production was a prediction of the group I splicing mechanism. Circular RNAs have properties that would make them attractive for certain studies of RNA structure and function. Reversal of splice site sequences in a context that allows splicing, such as those generated by circularly permuted group I introns, could be used to generate short defined sequences of circular RNA in vitro and perhaps in vivo.     

Self-splicing of a group II intron in yeast mitochondria: dependence on 5'' exon sequences.

It has been previously suggested that self-splicing of group II introns starts with a nucleophilic attack of the 2' OH group from the branchpoint adenosine on the 5' splice junction. To investigate the sequences governing the specificity of this attack, a series of Bal31 nuclease deletion mutants was constructed in which progressively larger amounts of 5' exon have been removed starting from its 5' end. The ability of mutant RNAs to carry out self-splicing in vitro was studied. Involvement of 5' exon sequences in self-splicing activity is indicated by the fact that a mutant in which as many as 18 nucleotides of 5' exon remain is seriously disturbed in splicing, while larger deletions eliminate splicing entirely. Mutants containing a truncated 5' exon form aberrant RNAs. One of these is a 425-nucleotide RNA containing the 5' exon as well as sequences of the 5' part of the intron. Its 3' end maps at position 374 of the 887-nucleotide intron. The other is a less abundant lariat RNA probably originating from the remainder of the intron linked to the 3' exon. We interpret this large dependence of reactivity of the intron on 5' exon and adjoining intron sequences as evidence for base-pairing interactions between the exon and parts of the intron, leading to an RNA folding necessary for splicing. Possible folding models are discussed.     

In vitro splicing analysis of mini-gene constructs of the alternatively processed human calcitonin/CGRP-I pre-mRNA

The human calcitonin/CGRP-I (CALC-I) gene can be alternatively expressed into calcitonin mRNA in thyroid C-cells and into CGRP-I mRNA in particular nerve cells. Formation of calcitonin mRNA requires splicing of exons 1, 2, 3 and 4 and addition of poly(A) at exon 4, whereas splicing of exons 1, 2, 3, 5 and 6 and addition of poly(A) at exon 6 yields CGRP-I mRNA. The calcitonin and CGRP-I mRNA-specific splicing reactions were investigated in vitro, in nuclear extracts of HeLa cells, using model precursor RNAs containing the exon 3 to exon 5 region of the gene. A precursor RNA containing the full-length exon 3 to exon 5 region was only poorly spliced in vitro. Therefore, a systematic analysis was performed of the effect of deletions introduced in the intron 3, exon 4 and intron 4 of this precursor RNA on calcitonin/CGRP mRNA-specific splicing. The deletions increased the efficiency of splicing considerably. In all cases CGRP mRNA-specific splicing is strongly favoured over calcitonin mRNA-specific splicing. In addition, splicing reactions using cryptic 5' splice sites were detected which interfered with the usage of processing signals for calcitonin and CGRP mRNA-specific splicing. The results imply a major regulatory role for the exon 4 poly(A) addition reaction in the generation of calcitonin mRNA.     

Minimal exon sequence requirements for efficient in vitro splicing of mono-intronic nuclear pre-mRNA

Measurements of the in vitro splicing efficiency of deletion mutant RNA precursors containing the small intron of the rabbit beta-globin gene, which are truncated in the first or in the second exon, revealed that no more than approximately 20 nucleotides of either exon are necessary for efficient splicing. At least for the second exon, this minimal length requirement is globin sequence-independent. Reduction of the exon-2 length to 14 nucleotides resulted in very inefficient splicing, whereas further reduction to 5 nucleotides apparently abolished the second splicing step (3' cutting and ligation), whereas the first step (5' cutting and branching) still occurred. The splicing efficiency of a double-mutant substrate retaining approximately 20 nucleotides of each exon was reduced to 50%. A kinetic study indicated that in the reaction of this double-mutant substrate the second, but not the first, splicing step was delayed, in contrast to the reaction of the wild-type precursor. Duplication or triplication of the entire sequence of exon-1 did not affect the splicing efficiency, whereas elongation of this exon with approximately 100 nucleotides of 5'-flanking (nontranscribed) beta-globin sequence diminished the level of correct splicing with the simultaneous appearance of aberrant lariat forms. We conclude that for mono-intronic precursors in which there is only one choice of splice sites, most of the exon sequences are not mechanistically involved in the splicing process.     

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.     

Unusual branch point selection involved in splicing of the alternatively processed Calcitonin/CGRP-I pre-mRNA.

To study splice site selection in alternative RNA processing we used the human Calcitonin/CGRP-I (CALC-I) gene. Expression of the CALC-I gene in thyroid C-cells results predominantly in calcitonin (CT) mRNA (containing exons 1 to 4) whereas CGRP-I mRNA (containing exons 1,2,3,5 and 6) is the exclusive product in particular nerve cells. We previously reported that a model precursor RNA containing the exon 3 to exon 5 region is predominantly processed into CGRP-I mRNA in vitro using nuclear extracts of three different cell types. To study CT specific processing in Hela cell nuclear extracts we have used precursor RNAs corresponding to the exon 3 to exon 4 region containing only CT specific processing signals. The results revealed the usage of a uridine residue 23 nucleotides upstream of the 3' splice site as the major site of lariat formation in CT specific splicing. The implications of this finding for the alternative, tissue specific processing of the CALC-I pre-mRNA and for branch point selection in general are discussed.     

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.     

An alternative helix in the 26S rRNA promotes excision and integration of the Tetrahymena intervening sequence.

A highly conserved ribosomal stem-loop immediately upstream of the Tetrahymena splice junction can inhibit both forward and reverse self-splicing by competing with base pairing between the 5' exon and the guide sequence of the intervening sequence. Formation of this unproductive hairpin is preferred in precursor RNAs with short exons and results in a lower rate of splicing. Inhibition of self-splicing is not observed in longer precursors, suggesting that additional interactions in the extended exons can influence the equilibrium between the productive and unproductive hairpins at the 5' splice site. An alternative pairing upstream of the 5' splice site has been identified and is proposed to stabilize the active conformer of the pre-rRNA. Nucleotide changes that alter the ability to form this additional helix were made, and the self-splicing rates were compared. Precursors in which the proposed stem is stabilized splice more rapidly than the wild type, whereas RNAs that contain a base mismatch splice more slowly. The ability of DNA oligomers to bind the RNA, as detected by RNase H digestion, correlates with the predicted secondary structure of the RNA. We also show that a 236-nucleotide RNA containing the natural splice junction is a substrate for intervening sequence integration. As in the forward reaction, reverse splicing is enhanced in ligated exon substrates in which the alternative rRNA pairing is more stable.     

A multicomponent complex is involved in the splicing of messenger RNA precursors

A multicomponent complex termed spliceosome (splicing body) is unique to the splicing of messenger RNA precursors in vitro. This 60S RNA-protein complex contains RNAs from the previously characterized bipartite splicing intermediate, the 5' exon RNA, and the lariat intervening sequence-3' exon RNA, as well as some intact 455 nucleotide precursor RNA. This complex contains snRNPs, particularly U1 RNP, as shown by immunoprecipitation with specific antisera. Formation of the 60S complex appears to be an early and essential step in splicing, because the 60S complex forms during the early stage, or lag time, of the reaction before the first covalent modification, cleavage at the 5' splice site of precursor RNA. The 60S complex forms only under conditions that permit splicing; both ATP and a precursor RNA containing authentic 5' and 3' splice sites are required for formation, while antiserum specific for U1 RNP inhibits its formation. RNA within the 60S complex, predominantly precursor RNA, was chased into products with accelerated kinetics and more complete conversion than purified precursor RNA.