General and specific functions of exonic splicing silencers in splicing control

Correct splice site recognition is critical in pre-mRNA splicing. We find that almost all of a diverse panel of exonic splicing silencer (ESS) elements alter splice site choice when placed between competing sites, consistently inhibiting use of intron-proximal 5' and 3' splice sites. Supporting a general role for ESSs in splice site definition, we found that ESSs are both abundant and highly conserved between alternative splice site pairs and that mutation of ESSs located between natural alternative splice site pairs consistently shifted splicing toward the intron-proximal site. Some exonic splicing enhancers (ESEs) promoted use of intron-proximal 5' splice sites, and tethering of hnRNP A1 and SF2/ASF proteins between competing splice sites mimicked the effects of ESS and ESE elements, respectively. Further, we observed that specific subsets of ESSs had distinct effects on a multifunctional intron retention reporter and that one of these subsets is likely preferred for regulation of endogenous intron retention events. Together, our findings provide a comprehensive picture of the functions of ESSs in the control of diverse types of splicing decisions.     

Sequestering of the 3' splice site in a theophylline-responsive riboswitch allows ligand-dependent control of alternative splicing

Despite the important role of alternative splicing in various aspects of biological processes, our ability to regulate this process at will remains a challenge. In this report, we asked whether a theophylline-responsive riboswitch could be adapted to manipulate alternative splicing. We constructed a pre-mRNA containing a single upstream 5' splice site and two 3' splice sites, of which the proximal 3' splice site is embedded in theophylline-responsive riboswitch. We show that this pre-mRNA spliced with preferential utilization of proximal 3' splice site in vitro. However, addition of theophylline to the splicing reaction promoted splicing at distal 3' splice site thereby changing the ratio of distal-to-proximal 3' splice site usage by more than twofold. Our data suggest that theophylline influenced 3' splice site choice without affecting the kinetics of the splicing reaction. We conclude that an in vitro selected riboswitch can be adapted to control alternative splicing, which may find many applications in basic, biotechnological, and biomedical research.     

Specific and stable intron-factor interactions are established early during in vitro pre-mRNA splicing

Biochemical components (splicing factors) interact with specific intron regions during pre-mRNA splicing in vitro. The pre-mRNA specifically associates with factors at both the branch point and the 5' splice site and these RNA-factor interactions are maintained in the intron-containing RNA processing products. The first detectable event, the ATP-dependent association of a factor (or factors) with the branch point, is mediated by at least one factor containing an essential nucleic acid component. Mutant RNA substrates that lack either the 5' splice site or the vast majority of exon sequences can still associate with the branch point binding factor(s). However, this branch point-factor interaction does not occur with a mutant RNA substrate that contains the branch point but that lacks the 3' splice site consensus sequence. These results suggest that selection of the 3' splice site accompanied by the association of a factor with the branch point may be the initial step in mammalian pre-mRNA splicing.     

Selection of the bovine papillomavirus type 1 nucleotide 3225 3' splice site is regulated through an exonic splicing enhancer and its juxtaposed exonic splicing suppressor.

Alternative splicing is an important mechanism for the regulation of bovine papillomavirus type 1 (BPV-1) gene expression during the virus life cycle. However, one 3' splice site, located at nucleotide (nt) 3225, is used for the processing of most BPV-1 pre-mRNAs in BPV-1-transformed C127 cells and at early to intermediate times in productively infected warts. At late stages of the viral life cycle, an alternative 3' splice site at nt 3605 is used for the processing of the late pre-mRNA. In this study, we used in vitro splicing in HeLa cell nuclear extracts to identify cis elements which regulate BPV-1 3' splice site selection. Two purine-rich exonic splicing enhancers were identified downstream of nt 3225. These sequences, designated SE1 (nt 3256 to 3305) and SE2 (nt 3477 to 3526), were shown to strongly stimulate the splicing of a chimeric Drosophila doublesex pre-mRNA, which contains a weak 3' splice site. A BPV-1 late pre-mRNA containing the nt 3225 3' splice site but lacking both SE1 and SE2 was spliced poorly, indicating that this 3' splice site is inherently weak. Analysis of the 3' splice site suggested that this feature is due to both a nonconsensus branch point sequence and a suboptimal polypyrimidine tract. Addition of SE1 to the late pre-mRNA dramatically stimulated splicing, indicating that SE1 also functions as an exonic splicing enhancer in its normal context. However, a late pre-mRNA containing both SE1 and SE2 as well as the sequence in between was spliced inefficiently. Further mapping studies demonstrated that a 48-nt pyrimidine-rich region immediately downstream of SE1 was responsible for this suppression of splicing. Thus, these data suggest that selection of the BPV-1 nt 3225 3' splice site is regulated by both positive and negative exonic sequences.     

A purine-rich intronic element enhances alternative splicing of thyroid hormone receptor mRNA.

The mammalian thyroid hormone receptor gene c-erbAalpha gives rise to two mRNAs that code for distinct isoforms, TRalpha1 and TRalpha2, with antagonistic functions. Alternative processing of these mRNAs involves the mutually exclusive use of a TRalpha1-specific polyadenylation site or TRalpha2-specific 5' splice site. A previous investigation of TRalpha minigene expression defined a critical role for the TRalpha2 5' splice site in directing alternative processing. Mutational analysis reported here shows that purine residues within a highly conserved intronic element, SEa2, enhance splicing of TRalpha2 in vitro as well as in vivo. Although SEalpha2 is located within the intron of TRalpha2 mRNA, it activates splicing of a heterologous dsx pre-mRNA when located in the downstream exon. Competition with wild-type and mutant RNAs indicates that SEalpha2 functions by binding trans-acting factors in HeLa nuclear extract. Protein-RNA crosslinking identifies several proteins, including SF2/ASF and hnRNP H, that bind specifically to SEalpha2. SEalpha2 also includes an element resembling a 5' splice site consensus sequence that is critical for splicing enhancer activity. Mutations within this pseudo-5' splice site sequence have a dramatic effect on splicing and protein binding. Thus SEa2 and its associated factors are required for splicing of TRalpha2 pre-mRNA.     

Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences

We have investigated the early events of pre-mRNA splicing in vitro by sucrose gradient sedimentation analysis. Time course experiments revealed the assembly, in two steps, of a large (50S) pre-mRNA splicing complex, preceded by formation of two other complexes that sediment at approximately 22S and 35S. Pre-mRNA and the intermediates and products of the in vitro splicing reaction cosediment with the 50S complex, while only pre-mRNA is associated with the 22S and 35S complexes. No splicing is observed in the absence of a 50S complex. Formation of the 50S complex requires ATP, whereas formation of the 22S and 35S complexes does not. U-snRNPs are necessary for assembly of the 35S and the 50S complexes but not for assembly of the 22S complex. Analysis with mutant substrate RNAs demonstrated that a polypyrimidine stretch near the 3' splice site and an intact 5' splice site are absolutely required for splicing complex formation.     

Identification of a novel sequence that governs both polyadenylation and alternative splicing in region E3 of adenovirus.

Region E3 encodes four major overlapping mRNAs with different splicing patterns. There are two poly(A) sites, an upstream site called E3A and a downstream site called E3B. We have analyzed virus mutants with deletions or insertions in E3 in order to identify sequences that function in the alternative processing of E3 pre-mRNAs, and to understand what determines which poly(A) sites and which splice sites are used. In previous studies we established that the 5' boundary of the E3A poly(A) signal is at an ATTAAA sequence. We now show, using viable virus mutants, that the 3' boundary of the E3A signal is located within 47-62 nucleotides (nt) downstream of the ATTAAA (17-32 nt downstream of the last microheterogenous poly(A) addition site). Our data further suggest that the spacing between the ATTAAA, the cleavage sites, and the essential downstream sequences may be important in E3A 3' end formation. Of particular interest, these mutants suggest a novel mechanism for the control of alternative pre-mRNA processing. Mutants which are almost completely defective in E3A 3' end formation display greatly increased use of a 3' splice site located 4 nt upstream of the ATTAAA. The mRNA that uses this 3' splice site is polyadenylated at the E3B poly(A) site. We suggest, for this particular case, that alternative pre-mRNA processing could be determined by a competition between trans-acting factors that function in E3A 3' end formation or in splicing. These factors could compete for overlapping sequences in pre-mRNA.     

The role of the polypyrimidine stretch at the SV40 early pre-mRNA 3'' splice site in alternative splicing.

We have studied the role in pre-mRNA splicing of the nucleotide sequence preceding the SV40 early region 3' splice site. Somewhat surprisingly, neither the pyrimidine at the highly conserved -3 position, nor the polypyrimidine stretch that extends from -5 to -15, relative to the 3' splice site, were found to be required for efficient splicing. Mutations that delete this region or create polypurine insertions at position -2 had no significant effects on the efficiency of SV40 early pre-mRNA splicing in vivo or in vitro. Interestingly, however, the pyrimidine content of this region had substantial effects on the alternative splicing pattern of this pre-mRNA in vivo. Mutations that increased the number of pyrimidine residues resulted in more efficient utilization of the large T antigen mRNA 5' splice site relative to the small t 5' splice site, while mutations that increased the purine content enhanced small t mRNA splicing. A possible molecular mechanism for these findings, as well as a model that proposes a role for the polypyrimidine stretch in alternative splicing, are discussed.     

Use of RNase H and primer extension to analyze RNA splicing.

A new method for the characterization of pre-mRNA splicing products is presented. In this method RNA molecules are hybridized to an oligodeoxynucleotide complementary to exon sequences upstream of a given 5' splice site, and the RNA strands of the resulting RNA:DNA hybrids are cleaved by RNase H. The cleaved RNAs are then subjected to primer extension using a 32P-labelled primer complementary to exon sequences downstream of an appropriate 3' splice site. Since the primer extension products all terminate at the site of RNase H cleavage, their lengths are indicative of the splice sites utilized. The method simplifies the study of the processing of complex pre-mRNAs by allowing the splicing events between any two exons to be analyzed. We have used this approach to characterize the RNAs generated by expression of the rat tropomyosin 1 (Tm 1) gene in various rat tissues and in cultured cells after transient transfection. The results demonstrate that this method is suitable for the analysis of alternative RNA processing in vivo.     

A novel protein factor is required for use of distal alternative 5'' splice sites in vitro.

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.     

Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing.

PRP8 protein of Saccharomyces cerevisiae interacts directly with pre-mRNA in spliceosomes, shown previously by UV-crosslinking. To analyse at which steps of splicing and with which precursor-derived RNA species the interaction(s) take place, UV-crosslinking was combined with PRP8-specific immunoprecipitation and the coprecipitated RNA species were analysed. Specific precipitation of intron-exon 2 and excised intron species was observed. PRP8 protein could be UV-crosslinked to pre-mRNA in PRP2-depleted spliceosomes stalled before initiation of the splicing reaction. Thus, the interaction of PRP8 protein with substrate RNA is established prior to the first transesterification reaction, is maintained during both steps of splicing and continues with the excised intron after completion of the splicing reaction. RNase T1 treatment of spliceosomes revealed that substrate RNA fragments of the 5' splice site region and the branchpoint-3' splice site region could be coimmunoprecipitated with PRP8 specific antibodies, indicating that these are potential sites of interaction for PRP8 protein with substrate RNA. Protection of the branch-point-3' splice site region was detected only after step 1 of splicing. The results allow a first glimpse at the pattern of PRP8 protein-RNA interactions during splicing and provide a fundamental basis for future analysis of these interactions.