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.     

Promoter choice determines splice site selection in protocadherin alpha and gamma pre-mRNA splicing

A family of mammalian protocadherin (Pcdh) proteins is encoded by three closely linked gene clusters (alpha, beta, and gamma). Multiple alpha and gamma Pcdh mRNAs are expressed in distinct patterns in the nervous system and are generated by alternative pre-mRNA splicing between different "variable" exons and three "constant" exons within each cluster. We show that each Pcdh variable exon is preceded by a promoter and that promoter choice determines which variable exon is included in a Pcdh mRNA. In addition, we provide evidence that alternative splicing of variable exons within a gene cluster occurs via a cis-splicing mechanism. However, virtually every variable exon can engage in trans-splicing with constant exons from another cluster, albeit at a far lower level.     

Novel splicing factor RBM25 modulates Bcl-x pre-mRNA 5' splice site selection

RBM25 has been shown to associate with splicing cofactors SRm160/300 and assembled splicing complexes, but little is known about its splicing regulation. Here, we characterize the functional role of RBM25 in alternative pre-mRNA splicing. Increased RBM25 expression correlated with increased apoptosis and specifically affected the expression of Bcl-x isoforms. RBM25 stimulated proapoptotic Bcl-x_S 5′ splice site (5′ ss) selection in a dose-dependent manner, whereas its depletion caused the accumulation of antiapoptotic Bcl-x_L. Furthermore, RBM25 specifically bound to Bcl-x RNA through a CGGGCA sequence located within exon 2. Mutation in this element abolished the ability of RBM25 to enhance Bcl-x_S 5′ ss selection, leading to decreased Bcl-x_S isoform expression. Binding of RBM25 was shown to promote the recruitment of the U1 small nuclear ribonucleoprotein particle (snRNP) to the weak 5′ ss; however, it was not required when a strong consensus 5′ ss was present. In support of a role for RBM25 in modulating the selection of a 5′ ss, we demonstrated that RBM25 associated selectively with the human homolog of yeast U1 snRNP-associated factor hLuc7A. These data suggest a novel mode for Bcl-x_S 5′ ss activation in which binding of RBM25 with exonic element CGGGCA may stabilize the pre-mRNA-U1 snRNP through interactions with hLuc7A.     

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

Background  

We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins.     

Systematic study of sequence motifs for RNA trans splicing in Trypanosoma brucei

mRNA maturation in Trypanosoma brucei depends upon trans splicing, and variations in trans-splicing efficiency could be an important step in controlling the levels of individual mRNAs. RNA splicing requires specific sequence elements, including conserved 5' splice sites, branch points, pyrimidine-rich regions [poly(Y) tracts], 3' splice sites (3'SS), and sometimes enhancer elements. To analyze sequence requirements for efficient trans splicing in the poly(Y) tract and around the 3'SS, we constructed a luciferase-beta-galactosidase double-reporter system. By testing approximately 90 sequences, we demonstrated that the optimum poly(Y) tract length is approximately 25 nucleotides. Interspersing a purely uridine-containing poly(Y) tract with cytidine resulted in increased trans-splicing efficiency, whereas purines led to a large decrease. The position of the poly(Y) tract relative to the 3'SS is important, and an AC dinucleotide at positions -3 and -4 can lead to a 20-fold decrease in trans splicing. However, efficient trans splicing can be restored by inserting a second AG dinucleotide downstream, which does not function as a splice site but may aid in recruitment of the splicing machinery. These findings should assist in the development of improved algorithms for computationally identifying a 3'SS and help to discriminate noncoding open reading frames from true genes in current efforts to annotate the T. brucei genome.     

Cloning and characterization of a variant surface glycoprotein expression site from Trypanosoma equiperdum.

Variant surface glycoprotein (VSG) genes of African trypanosomes are expressed when they are inserted into one of several telomere-linked expression sites. We cloned and characterized an 11-kilobase (kb) DNA fragment located upstream of an expressed VSG gene. A DNA sequence of 1.8 kb that is located immediately upstream of the inserted VSG gene contains sequences homologous to the 76-base-pair repeats described as being upstream of VSG genes in Trypanosoma brucei (D. A. Campbell, M. P. Van Bree, and J. C. Boothroyd, Nucleic Acids Res. 12:2759-2774). There are no such sequences elsewhere in the 11-kb cloned region. Southern blot analysis using probes from the cloned region revealed multiple unlinked copies of the same or very similar regions. At least three of these are located near telomeres, and two have been shown to be used for the expression of known Trypanosoma equiperdum VSG genes. Like VSG genes, the upstream sequences themselves can be duplicated and deleted. The choice of expression site to be used by a duplicated VSG gene is nonrandom; the site used for expression of the parental VSG gene is strongly favored for use in the daughter variant. Furthermore, even when the parental expression site is not used, the VSG gene occupying it is replaced. Thus, an active expression site is a preferential target for gene conversion in the next variation event.     

Substrate recognition and splice site determination in yeast tRNA splicing

S. cerevisae tRNA introns interrupt the gene at a constant position in the anticodon loop. Pre-tRNAs are matured by an endonuclease and a ligase. The endonuclease alone can accurately release the intron from the pre-tRNA. Here, we investigate the mechanism of splice site selection by the endonuclease. We propose that it initially recognizes features in the mature domain common to all tRNAs. Once positioned on the enzyme, the splice sites are recognizable because they are a fixed distance from the mature domain. To test this hypothesis, we developed a system for synthesizing pre-tRNA by bacteriophage T7 RNA polymerase. To search for recognition sites, we made several mutations. Mutations of C56 and U8 strongly affect endonuclease recognition of pre-tRNA. With insertion and deletion mutations, we show that the anticodon stem determines splicing specificity. The sequence and structure of the intron are not strong determinants of splice site selection.     

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.     

RNA splicing: group I intron crystal structures reveal the basis of splice site selection and metal ion catalysis

The group I intron has served as a model for RNA catalysis since its discovery 25 years ago. Four recently determined high-resolution crystal structures complement extensive biochemical studies on this system. Structures of the Azoarcus, Tetrahymena and bacteriophage Twort group I introns mimic different states of the splicing or ribozyme reaction pathway and provide information on splice site selection and metal ion catalysis. The 5'-splice site is selected by formation of a conserved G.U wobble pair between the 5'-exon terminus and the intron. The 3'-splice site is identified through stacking of three base triples, in which the middle triple contains the conserved terminal nucleotide of the intron, OmegaG. The structures support a two-metal-ion mechanism for group I intron splicing that might have corollaries to group II intron and pre-mRNA splicing by the spliceosome.     

Principles of 3' splice site selection and alternative splicing for an unusual group II intron from Bacillus anthracis

We investigated the self-splicing properties of two introns from the bacterium Bacillus anthracis. One intron (B.a.I1) splices poorly in vitro despite having typical structural motifs, while the second (B.a.I2) splices well while having apparently degenerated features. The spliced exons of B.a.I2 were sequenced, and splicing was found to occur at a 3' site shifted one nucleotide from the expected position, thus restoring missing gamma-gamma' and IBS3-EBS3 pairings, but leaving the two conserved exonic ORFs out of frame. Because of the unexpected splice site, the principles for 3' intron definition were examined, which showed that the 3' splice site is flexible but contingent on gamma-gamma' and IBS3-EBS3 pairings, and can be as far away as four nucleotides from the wild-type site. Surprisingly, alternative splicing occurs at position +4 for wild-type B.a.I2 intron, both in vitro and in vivo, and the alternative event fuses the two conserved exon ORFs, presumably leading to translation of the downstream ORF. The finding suggests that the structural irregularities of B.a.I2 may be an adaptation to facilitate gene expression in vivo.     

The essential pre-mRNA splicing factor SF2 influences 5' splice site selection by activating proximal sites

SF2 is a 33 kd protein factor required for 5' splice site cleavage and lariat formation during pre-mRNA splicing in HeLa cell extracts. In addition to its essential role in constitutive splicing, SF2 can strongly influence 5' splice site selection. When pre-mRNAs containing multiple cis-competing 5' splice sites are spliced in vitro, high concentrations of purified SF2 promote the use of the 5' splice site closest to the 3' splice site. However, SF2 discriminates properly between authentic and cryptic splice sites. These effects of SF2 on splice site selection may reflect the cellular mechanisms that prevent exon skipping and ensure the accuracy of splicing. In addition, alterations in the concentration or activity of SF2, and of other general splicing factors, may serve to regulate alternative splicing in vivo.     

Wobble splicing reveals the role of the branch point sequence-to-NAGNAG region in 3' tandem splice site selection

Alternative splicing involving the 3' tandem splice site NAGNAG sequence may play a role in the structure-function diversity of proteins. However, how 3' tandem splice site utilization is determined is not well understood. We previously demonstrated that 3' NAGNAG-based wobble splicing occurs mostly in a tissue- and developmental stage-independent manner. Bioinformatic analysis reveals that the nucleotide preceding the AG dinucleotide may influence 3' splice site utilization; this is also supported by an in vivo splicing assay. Moreover, we found that the intron sequence plays an important role in 3' splice site selection for NAGNAG wobble splicing. Mutations of the region between the branch site and the NAGNAG 3' splice site, indeed, affected the ratio of the distal/proximal AG selection. Finally, we found that single nucleotide polymorphisms around the NAGNAG motif could affect the splice site choice, which may lead to a change in mRNA patterns and influence protein function. We conclude that the NAGNAG motif and its upstream region to the branch point sequence are required for 3' tandem splice site selection.     

Controlled turnover and 3'' trimming of the trans splicing precursor of Trypanosoma brucei.

The maturation of mRNAs in Trypanosoma brucei involves a novel step, in which a short capped sequence is spliced in trans onto the 5' end of nascent mRNAs from a 140-nucleotide precursor. This precursor is called the mini-exon-derived RNA or medRNA. We have used drugs and ultraviolet irradiation as inhibitors to probe the synthesis and processing of medRNA in vivo. Inhibition of RNA synthesis by chloroquine shows that the half-life of medRNA is about 4 minutes. Despite this high turnover, only limited accumulation of medRNA could be achieved following a block in the synthesis of high molecular weight splice acceptor substrates by UV irradiation. This implies that there is a constraint on the steady-state levels of medRNA and that excess medRNA is degraded in the cell. A 3' shortened version of medRNA accumulates upon a block in normal medRNA processing by UV irradiation or upon treatment of the cells with actinomycin D or novobiocin but was shown not to participate in trans splicing, making it a likely candidate for an in vivo degradation intermediate.     

Half pint regulates alternative splice site selection in Drosophila

Alternative splicing is used by metazoans to increase protein diversity and to alter gene expression during development. However, few factors that control splice site choice in vivo have been identified. Here we describe a factor, Half pint (Hfp), that regulates RNA splicing in Drosophila. Females harboring hypomorphic mutations in hfp lay short eggs and show defects in germline mitosis, nuclear morphology, and RNA localization during oogenesis. We find that hfp encodes the Drosophila ortholog of human PUF60 and functions in both constitutive and alternative splicing in vivo. In particular, hfp mutants display striking defects in the developmentally regulated splicing of ovarian tumor (otu). Furthermore, transgenic expression of the missing otu splice form can rescue the ovarian phenotypes of hfp.     

Alternative 5'' splice site selection induced by heat shock.

The mouse HSP47 gene consists of six exons separated by five introns. Three HSP47 cDNAs differing only in their 5' noncoding regions have been reported. One of these alternatively spliced mRNAs was detected only after heat shock, which caused an alternative 5' splice donor site selection. Other stress inducers, including an amino acid analog and sodium arsenite, had no effect on the alternative splicing. The alternatively spliced mRNA, which was 169 nucleotides longer in the 5' noncoding region compared to mRNA transcribed in non-heat shock conditions, was efficiently translated under heat shock conditions. This novel finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression.