HPV18 Utilizes Two Alternative Branch Sites for E6~*I Splicing to Produce E7 Protein

Human papillomavirus 18(HPV18) E6 and E7 oncogenes are transcribed as a single bicistronic E6 E7 pre-mRNA. The E6 ORF region in the bicistronic E6 E7 pre-mRNA contains an intron. Splicing of this intron disrupts the E6 ORF integrity and produces a spliced E6~*I RNA for efficient E7 translation. Here we report that the E6 intron has two overlapped branch point sequences(BPS) upstream of its 30 splice site, with an identical heptamer AACUA■C, for E6~*I splicing. One heptamer has a branch site adenosine(underlined) at nt 384 and the other at nt 388. E6~*I splicing efficiency correlates to the expression level of E6 and E7 proteins and depends on the selection of which branch site. In general, E6~*I splicing prefers the 30 ss-proximal branch site at nt 388 over the distal branch site at nt 384. Inactivation of the nt 388 branch site was found to activate a cryptic acceptor site at nt 636 for aberrant RNA splicing. Together, these data suggest that HPV18 modulates its production ratio of E6 and E7 proteins by alternative selection of the two mapped branch sites for the E6~*I splicing, which could be beneficial in its productive or oncogenic infection according to the host cell environment.     

Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5′ splice sites (5′ ss) and three 3′ splice sites (3′ ss) normally used in HPV16⁺ cervical cancer and its derived cell lines. The choice of two novel alternative 5′ ss (nt 221 5′ ss and nt 191 5′ ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5′ ss and nt 409 3′ ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3′ ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3′ ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of ⁹¹QYNK⁹⁴ to ⁹¹PSFW⁹⁴ displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.     

A highly stable duplex structure sequesters the 5' splice site region of hnRNP A1 alternative exon 7B.

Exon 7B in the hnRNP A1 pre-mRNA is alternatively spliced to yield A1 and A1(B), two proteins that differ in their ability to modulate 5' splice site selection. Sequencing the murine intron downstream of exon 7B revealed the existence of several regions of similarity to the corresponding human intron. In vitro splicing assays indicate that an 84-nt region (CE6IO) decreases splicing to the proximal 5' splice site in a pre-mRNA carrying the 5' splice sites of exon 7 and 7B. In vivo, the CE6IO element promotes exon 7B skipping in pre-mRNAs expressed from a mini-gene containing the hnRNP A1 alternative splicing unit. Using oligonucleotide-targeted RNase H cleavage assays, we provide support for the existence of highly stable base pairing interactions between CE6IO and the 5' splice site region of exon 7B. Duplex formation occurs in naked pre-mRNA, resists incubation in splicing extracts, and is associated with a reduction in the assembly of U1 snRNP-dependent complexes to the 5' splice site of exon 7B. Our results demonstrate that pre-mRNA secondary structure plays an important role in promoting exon 7B skipping in the A1 pre-mRNA.     

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.     

Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo.

The aminoglycoside antibiotic neomycin B inhibits translation in prokaryotes and interferes with RNA-protein interactions in HIV both in vivo and in vitro. Hitherto, inhibition of ribozyme catalysis has only been observed in vitro. We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (td) intron in vivo. All antibiotics tested inhibited splicing, even chloramphenicol, which does not inhibit splicing in vitro. Splicing of the td intron in vivo requires translation for proper folding of the pre-mRNA. In the absence of translation, two interactions between sequences in the upstream exon and the 5' and 3' splice sites trap the pre-mRNA in splicing-incompetent conformations. Their disruption by mutations rendered splicing less dependent on translation and also less sensitive to neomycin B. Intron splicing was affected by neither neomycin B nor gentamicin in Escherichia coli strains carrying antibiotic-resistance genes that modify the ribosomal RNA. Taken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their interference with translation. This was further confirmed by assaying splicing of the Tetrahymena group I intron, which is inserted in the E. coli 23 S rRNA and, thus, not translated. Furthermore, neomycin B, paromomycin, and streptomycin enhanced missplicing in antibiotic-sensitive strains. Missplicing is caused by an alternative structural element containing a cryptic 5' splice site, which serves as a substrate for the ribozyme. Our results demonstrate that aminoglycoside antibiotics display different effects on ribozymes in vivo and in vitro.     

The cap and the 3' splice site similarly affect polyadenylation efficiency.

The 5' cap of a mammalian pre-mRNA has been shown to interact with splicing components at the adjacent 5' splice site for processing of the first exon and the removal of the first intron (E. Izaurralde, J. Lewis, C. McGuigan, M. Jankowska, E. Darzynkiewicz, and I.W. Mattaj, Cell 78:657-668, 1994). Likewise, it has been shown that processing of the last exon and removal of the last intron involve interaction between splicing components at the 3' splice site and the polyadenylation complex at the polyadenylation signal (M. Niwa, S. D. Rose, and S.M. Berget, Genes Dev. 4:1552-1559, 1990; M. Niwa and S. M. Berget, Genes Dev. 5:2086-2095, 1991). These findings suggest that the cap provides a function in first exon processing which is similar to the function of the 3' splice site at last exon processing. To determine whether caps and 3' splice sites function similarly, we compared the effects of the cap and the 3' splice site on the in vitro utilization of the simian virus 40 late polyadenylation signal. We show that the presence of a m7GpppG cap, but not a cap analog, can positively affect the efficiency of polyadenylation of a polyadenylation-only substrate. Cap analogs do not stimulate polyadenylation because they fail to bind titratable cap-binding factors. The failure of cap analogs to stimulate polyadenylation can be overcome if a 3' splice site is present upstream of the polyadenylation signal. These data indicate that factors interacting with the cap or the 3' splice site function similarly to affect polyadenylation signal, along with m7GpppG cap, is inhibitory to polyadenylation. This finding suggests that the interaction between the cap-binding complexes and splicing components at the 5' splice site may form a complex which is inhibitory to further processing if splicing of an adjacent intron is not achieved.     

Genetic interaction between U6 snRNA and the first intron nucleotide in Saccharomyces cerevisiae.

Nuclear pre-mRNA splicing necessitates specific recognition of the pre-mRNA splice sites. It is known that 5' splice site selection requires base pairing of U6 snRNA with intron positions 4-6. However, no factor recognizing the highly conserved 5' splice site GU has yet been identified. We have tested if the known U6 snRNA-pre-mRNA interaction could be extended to include the first intron nucleotides and the conserved 50GAG52 sequence of U6 snRNA. We observe that some combinations of 5' splice site and U6 snRNA mutations produce a specific synthetic block to the first splicing step. In addition, the U6-G52U allele can switch between two competing 5' splice sites harboring different nucleotides following the cleavage site. These results indicate that U6 snRNA position 52 interacts with the first nucleotide of the intron before 5' splice site cleavage. Some combinations of U6 snRNA and pre-mRNA mutations also blocked the second splicing step, suggesting a role for the corresponding nucleotides in a proofreading step before exon ligation. From studies in diverse organisms, various functions have been ascribed to the conserved U6 snRNA 47ACAGAG52 sequence. Our results suggest that these discrepancies might reflect variations between different experimental systems and point to an important conserved role of this sequence in the splicing reaction.     

Exon and intron sequences, respectively, repress and activate splicing of a fibroblast growth factor receptor 2 alternative exon.

Two alternative exons, BEK and K-SAM, code for part of the ligand binding site of fibroblast growth factor receptor 2. Splicing of these exons is mutually exclusive, and the choice between them is made in a tissue-specific manner. We identify here pre-mRNA sequences involved in controlling splicing of the K-SAM exon. The short K-SAM exon sequence 5'-TAGGGCAGGC-3' inhibits splicing of the exon. This inhibition can be overcome by mutating either the exon's 5' or 3' splice site to make it correspond more closely to the relevant consensus sequence. Two separate sequence elements in the intron immediately downstream of the K-SAM exon, one of which is a sequence rich in pyrimidines, are both needed for efficient K-SAM exon splicing. This is no longer the case if either the exon's 5' or 3' splice site is reinforced. Furthermore, if the exon inhibitory sequence is removed, the intron sequences are not required for splicing of the K-SAM exon in a cell line which normally splices this exon. At least three elements are thus involved in controlling splicing of the K-SAM exon: suboptimal 5' and 3' splice sites, an exon inhibitory sequence, and intron activating sequences.     

tau Exon 10 expression involves a bipartite intron 10 regulatory sequence and weak 5' and 3' splice sites

tau mutations that deregulate alternative exon 10 (E10) splicing cause frontotemporal dementia with parkinsonism chromosome 17-type by several mechanisms. Previously we showed that E10 splicing involved exon splicing enhancer sequences at the 5' and 3' ends of E10, an exon splicing silencer, a weak 5' splice site, and an intron splicing silencer (ISS) within intron 10 (I10). Here, we identify additional regulatory sequences in I10 using both non-neuronal and neuronal cells. The ISS sequence extends from I10 nucleotides 11-18, which is sufficient to inhibit use of a weakened 5' splice site of a heterologous exon. Furthermore, ISS function is location-independent but requires proximity to a weak 5' splice site. Thus, the ISS functions as a linear sequence. A new cis-acting element, the intron splicing modulator (ISM), was identified immediately downstream of the ISS at I10 positions 19-26. The ISM and ISS form a bipartite regulatory element, within which the ISM functions when the ISS is present, mitigating E10 repression by the ISS. Additionally, the 3' splice site of E10 is weak and requires exon splicing enhancer elements for efficient E10 inclusion. Thus far, tau FTDP-17 splicing mutations affect six predicted cis-regulatory sequences.     

Modulation of exon skipping by high-affinity hnRNP A1-binding sites and by intron elements that repress splice site utilization

The RNA-binding protein hnRNP A1 is a splicing regulator produced by exclusion of alternative exon 7B from the A1 pre-mRNA. Each intron flanking exon 7B contains a high-affinity A1-binding site. The A1-binding elements promote exon skipping in vivo, activate distal 5' splice site selection in vitro and improve the responsiveness of pre-mRNAs to increases in the concentration of A1. Whereas the glycine-rich C-terminal domain of A1 is not required for binding, it is essential to activate the distal 5' splice site. Because A1 complexes can interact simultaneously with two A1-binding sites, we propose that an interaction between bound A1 proteins facilitates the pairing of distant splice sites. Based on the distribution of putative A1-binding sites in various pre-mRNAs, an A1-mediated change in pre-mRNA conformation may help define the borders of mammalian introns. We also identify an intron element which represses the 3' splice site of exon 7B. The activity of this element is mediated by a factor distinct from A1. Our results suggest that exon 7B skipping results from the concerted action of several intron elements that modulate splice site recognition and pairing.     

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.     

Requirement for SLU7 in yeast pre-mRNA splicing is dictated by the distance between the branchpoint and the 3' splice site.

Yeast pre-mRNA splicing factors SLU7 and PRP16 are required for cleavage of the 3' splice site and exon ligation in vitro. Using natural and model precursor RNAs, we found that SLU7 is dispensable for splicing of RNAs in which the 3' splice site is in close proximity to the branchpoint. SLU7 is only required when the interval between the branchpoint and the 3' splice site is greater than 7 nt. In contrast, PRP16 is essential for splicing of all pre-mRNAs tested. Immunoprecipitation of the products of step 1 by anti-SLU7 antibodies demonstrates that SLU7 is a component of the spliceosome. Recruitment of SLU7 to the spliceosome is greatly enhanced by prior addition of PRP16. PRP16 is liberated from the spliceosome after completion of step 2, whereas SLU7 remains bound to the excised intron and spliced mature RNA until the spliceosome disassembles, in a reaction that requires ATP.     

Donor site competition is involved in the regulation of alternative splicing of the rat beta-tropomyosin pre-mRNA

The rat beta-tropomyosin (beta-TM) gene encodes both skeletal muscle beta-TM mRNA and nonmuscle TM-1 mRNA via alternative RNA splicing. This gene contains eleven exons: exons 1-5, 8, and 9 are common to both mRNAs; exons 6 and 11 are used in fibroblasts as well as in smooth muscle, whereas exons 7 and 10 are used in skeletal muscle. Previously we demonstrated that utilization of the 3' splice site of exon 7 is blocked in nonmuscle cells. In this study, we use both in vitro and in vivo methods to investigate the regulation of the 5' splice site of exon 7 in nonmuscle cells. The 5' splice site of exon 7 is used efficiently in the absence of flanking sequences, but its utilization is suppressed almost completely when the upstream exon 6 and intron 6 are present. The suppression of the 5' splice site of exon 7 does not result from the sequences at the 3' end of intron 6 that block the use of the 3' splice site of exon 7. However, mutating two conserved nucleotides GU at the 5' splice site of exon 6 results in the efficient use of the 5' splice site of exon 7. In addition, a mutation that changes the 5' splice site of exon 7 to the consensus U1 snRNA binding site strongly stimulates the splicing of exon 7 to the downstream common exon 8. Collectively, these studies demonstrate that 5' splice site competition is responsible, in part, for the suppression of exon 7 usage in nonmuscle cells.     

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.     

Inhibition of pre-mRNA splicing by antisense RNA in vitro: effect of RNA containing sequences complementary to exons.

The objective of the experiments described in this paper was to determine the feasibility of inhibition of pre-mRNA splicing by antisense RNA in vitro. Three different types of antisense RNA were utilized: antisense RNA complementary to the spliced RNA molecule; antisense RNA complementary to the unprocessed mRNA precursor molecule; and antisense RNA complementary to the 5' and 3' splice junctions. Whereas antisense RNA complementary to mRNA had little effect on splicing, antisense RNAs complementary to mRNA precursor or to splice junctions strongly inhibited splicing of pre-mRNA molecule. The results obtained indicate that the inhibitory effect is most likely due to hybrid formation between pre-mRNA and antisense RNA molecules and that antisense RNA complementary to the exon portion but not to the intron portion of splice junction exhibit an inhibitory effect. This inhibition can be overcome by bringing together 5' and 3' splice junctions via hybrid formation with antisense RNA complementary to the spliced RNA molecule.     

Exon skipping in purine nucleoside phosphorylase mRNA processing leading to severe immunodeficiency.

We report a defect in splicing of precursor messenger RNA (pre-mRNA) resulting from a naturally occurring mutation of the gene encoding purine nucleoside phosphorylase (PNP) in a patient with PNP-deficient severe combined immunodeficiency. This defects results from a G to T transversion at the terminal nucleotide of exon 2 within the 5' splice site of intron 2 and causes skipping of exon 2 during processing of PNP pre-mRNA. Translation of the misspliced mRNA results in a reading frameshift at the exon 1-exon 3 junction. The predicted polypeptide encoded by the aberrant mRNA is severely truncated, terminating at 31 amino acids. Only 4 residues at the NH2 terminus of the polypeptide correspond to PNP amino acids. Otherwise the translation product of the misspliced mRNA differs completely from PNP in amino acid sequence and has no PNP activity. The finding of exon skipping in PNP is the first report of a splicing defect resulting in PNP-deficient severe combined immunodeficiency. Analysis of the genomic context of the G-1 to T mutation of the 5' splice site lends support for the exon definition model of pre-mRNA splicing and contributes to the understanding of splice site selection.