Figure 1.
Secondary structure of the spliceozyme, based on the group I intron ribozyme from Tetrahymena.
(A) Before the reaction, the substrate consists of two exons (red) and an intervening intron (blue). The spliceozyme (black) uses its 5′-terminus to form the P1 duplex, the P1 extension (P1ex), and the 5′-duplex to position the substrate 5′-splice site (filled triangle). The ribozyme 3′-terminus forms the P9.2 duplex and the P9.0 duplex to position the substrate 3′-splice site (empty triangle). (B) After two transphosphorylation reactions the 5′-exon and the 3′-exon of the substrate are joined. The 5′-terminus of the intron is capped by an exogenous G (black) and the 3′-terminus of the intron is liberated. The sequences correspond to the removal of an intron from splice site 258 of the CAT pre-mRNA used in this study. The internal sequence of the intron is drawn as a bold blue line. Note that a hairpin terminator is added to the 3′-terminus for all reactions in cells. The secondary structure of the ribozyme is based on [58] with the alteration that the P4-P6 domain was positioned on the right side for clarity.
Figure 2.
Influence of the spliceozyme 5′-terminus design on the reaction in vitro.
The ribozyme 5′-terminus ends in (A) a P1 duplex, (B) a P1 extension, or (C) a 5′-duplex (see Figure 1). The corresponding constructs are labeled as 5′ P1, 5′ P1ex, and 5′ EGS, respectively. The top panels show autoradiograms of internally radiolabeled splicing products after separation by denaturing polyacrylamide gel electrophoresis. The marker (M) shows the position of pre-mRNA (778 nt), mRNA (678 nt), and 5′-exon (278 nt). The 3′-exon and the intron had a length of 400 nt and 100 nt, respectively. For each spliceozyme construct, two splicing reactions are analyzed with spliceozyme concentrations of 100 nM and 1 µM. Samples were taken at reaction times between 0 and 60 minutes. A schematic of the substrates and reaction products is shown to the right of the image, with exons in red and the intron in blue. Bottom panels show the quantitation of the disappearance of the CAT pre-mRNA (triangles) and the appearance of the CAT mRNA (squares). Empty symbols correspond to 100 nM spliceozyme, while filled symbols correspond to 1 µM spliceozyme concentration. Grey lines show single-exponential curve fits to the products and double-exponential curve fits to the reaction products. Error bars are standard deviations from three experiments.
Figure 3.
Effect of substrate recognition sequences on product formation.
(A) Kinetics of spliceozyme splicing in vitro, measured as the molar percentage of the initital substrate concentration (CAT pre-mRNA) converted to product (CAT mRNA). The reactions were analyzed as in Figure 2. Data from spliceozymes with a 3′-terminal P9.2 helix of 9 base pairs are shown with filled symbols, including spliceozyme 5′-termini of a 5′-duplex (triangles), P1 extension helix (circles), and P1 helix (squares). Empty symbols denote results from spliceozymes terminating in a P1 helix at the 5′-terminus, and the 3′-terminal P9.2 helix truncated to 8 base pairs (diamonds), 7 base pairs (squares), 6 base pairs (triangles), and 5 base pairs (circles). Error bars correspond to standard deviations from three experiments. (B) Sequences of splicing junctions resulting from RT-PCR, cloning and sequencing of trans-splicing products from the reaction with a P9.2 helix of 5 base pairs and a P1 helix at the 5′-terminus. Note that the wild type sequence (WT) differs from the splicing products in three silent mutations (empty triangles), which confirmed that the sequences were splicing products and not a contamination by the wild type gene. Nine out of ten sequences showed the sequence expected from correct splicing at the 3′-terminal G, whereas one sequence indicated that the guanosine four nucleotides downstream of the intended 3′-splice site was used instead. The sequence participating in the P1 helix is underlined, containing the 5′-splice site (filled triangle).
Figure 4.
Spliceozyme activity in E. coli cells.
(A) Schematic of the plasmid used for the expression of spliceozyme (Sz; black) and CAT pre-mRNA (CAT; red) containing an intron (blue) in E. coli cells. Restriction sites used in cloning of the plasmid are indicated. (B) Quantitation of E. coli cell growth on LB-agar plates containing 8 µg/mL chloramphenicol, by measuring the A600 of cell suspensions from washing the plates after incubation. The black column on the left shows the A600 from cells that express the wild type CAT gene without intron. Grey columns denote the A600 resulting from plasmids containing a CAT gene with an intron and wild-type spliceozymes. White columns denote the A600 from the same constructs as in the grey columns but with six mutations in the catalytic core of the spliceozymes. The black column on the right denotes the A600 resulting from cells containing the pUC19 plasmid without spliceozyme or CAT gene. Two different intron sequences with a length of 64 nucleotides were inserted into plasmid pUCSz2 for this assay (64-CiC2 and 64-SiC3; note that both of these introns were selected for their efficient removal by the spliceozyme; see materials and methods). Note the logarithmic scale for the A600. Error bars are standard deviations from three biological samples.
Figure 5.
Effect of the internal intron sequence on spliceozyme activity in E. coli cells.
(A) Secondary structure of the spliceozyme (black), with the substrate exons in red and the position of the randomized N64 sequence indicated in the internal region of the intron (blue). (B) Quantitation of E. coli cell growth on LB-agar plates containing 10 µg/mL chloramphenicol. The resulting A600 is given for 20 clones, ten of which were chosen before selection on LB-chloramphenicol plates (white columns), and ten of which were chosen after this selection step (grey columns). The name of each clone is given below, with ‘L’ indicating clones from the unselected library and ‘S’ indicating selected clones, 100 denoting the length of the intron, the letter i indicating that the internal region of the intron was randomized, and the number after ‘C’ denoting the clone number. Error bars are standard deviations from three experiments. (C) Sequences of 20 cloned intron sequences, sorted according to their activity in cells. The left column lists the clone names, using the same nomenclature as in (B). The middle column shows the internal sequence of that clone that resulted from the N64 library, which was inserted between the constant regions of the intron (see (A)). The right column shows the growth activity, measured as A600 of cell suspensions that resulted from washing LB-growth plates containing chloramphenicol, relative to growth on medium without chloramphenicol.
Figure 6.
Effect of the 3′-terminal intron sequence on spliceozyme activity in E. coli cells.
(A) Secondary structure of the spliceozyme (black), with the substrate exons in red, and the position of the randomized N10 sequence in the 3′-terminal region of the intron (blue). The helices P9.0 and P9.2 are labeled. (B) List of 20 analyzed clones, sorted according to the resulting activity in cells. The clone name is given on the left, the growth on LB-agar plates containing chloramphenicol is shown as horizontal columns, and the N10 sequence is given on the right. Clone names containing ‘L’ indicate the unselected library, while ‘S’ indicates a selection step with chloramphenicol. The letter ‘j’ denotes that the randomized region is located near the 3′-terminus of the CAT mRNA intron. The growth was measured on plates containing 10 µg/mL chloramphenicol and normalized to growth on medium without chloramphenicol. The sequence at the top (WT) corresponds to the wild type sequence of the Tetrahymena ribozyme. Nucleotides that are identical with the wild type sequence are colored in blue. The position 10 tolerates a U, which is consistent with its base-pairing role, and is colored in red. Errors are standard deviations from three experiments.
Figure 7.
Quantitation of CAT enzyme activity, CAT RNA level, and plasmid level in E. coli cells.
(A) Units of CAT activity detected in E. coli cell extract, containing different plasmid constructs, as indicated below the column graphs. No detectable CAT activity was found when E. coli cells contained the plasmid pUC19 (left black column; -0.00045±0.00053 units per OD600). Grey columns show the CAT activity of constructs expressing a spliceozyme and a CAT pre-mRNA containing an intron that mediated efficient bacterial resistance to chloramphenicol. White columns show the CAT activity mediated by introns that led to poor or no bacterial resistance to chloramphenicol. Construct names starting with 64 or 100 label the length of the intron in the CAT pre-mRNA gene, ‘S’ denotes that the clone was selected for activity on medium with chloramphenicol, ‘L’ denotes that the clone was chosen from an un-selected library, ‘I’ denotes that the library for this selection was randomized in the internal region of the intron, and the number after C is the clone number. CAT WT denotes a construct without spliceozyme, and in which the CAT gene did not contain an intron, resulting in 0.13±0.01 units of CAT activity per OD600. The percent of CAT activity relative to the CAT WT construct is noted above each column. (B) Level of CAT RNAs estimated by RT-qPCR of the CAT RNA 3′-exon. This assay measures the sum of CAT pre-mRNA and CAT mRNA. (C) Molar amount of plasmid isolated from logarithmically growing E. coli cells. Note that the unit OD600 is used as an absolute value, corresponding to the cells in a volume of 1 mL cell suspension with OD600 = 1. For all graphs shown in Figure 7, error bars denote standard deviations from three biological replicates.