New RNA-mediated reactions by yeast mitochondrial group I introns.

The group I self-splicing reaction is initiated by attack of a guanosine nucleotide at the 5' splice site of intron-containing precursor RNA. When precursor RNA containing a yeast mitochondrial group I intron is incubated in vitro under conditions of self-splicing, guanosine nucleotide attack can also occur at other positions: (i) the 3' splice site, resulting in formation of a 3' exon carrying an extra added guanosine nucleotide at its 5' end; (ii) the first phosphodiester bond in precursor RNA synthesized from the SP6 bacteriophage promoter, leading to substitution of the first 5'-guanosine by a guanosine nucleotide from the reaction mixture; (iii) the first phosphodiester bond in already excised intron RNA, resulting in exchange of the 5' terminal guanosine nucleotide for a guanosine nucleotide from the reaction mixture. An identical sequence motif (5'-GAA-3') occurs at the 3' splice site, the 5' end of SP6 precursor RNA and at the 5' end of excised intron RNA. We propose that the aberrant reactions can be explained by base-pairing of the GAA sequence to the Internal Guide Sequence. We suggest that these reactions are mediated by the same catalytic centre of the intron RNA that governs the normal splicing reactions.     

Mutational evidence for competition between the P1 and the P10 helices of a mitochondrial group I intron.

A guanosine to cytosine transversion at position 2 of the fifth intron of the mitochondrial gene COB blocks the ligation step of splicing. This mutation prevents the formation of a base pair within the P1 helix of this group I intron--the RNA duplex formed between the 3' end of the upstream exon and the internal guide sequence. The mutation also reduces the rate of the first step of splicing (guanosine addition at the 5' splice junction) while stimulating hydrolysis at the 3' intron-exon boundary. Consequently, the ligation of exons is blocked because the 3' exon is removed prior to cleavage at the 5' splice junction. The lesion can be suppressed by second-site mutations that preserve the potential for base-pairing at this position. Because the P1 duplex and the P10 duplex (between the guide sequence and the 3' exon) overlap at the affected pairings represent alternative structures that do not, indeed cannot, form simultaneously.     

Novel RNA polymerization reaction catalyzed by a group I ribozyme.

We have converted a bacterial tRNA precursor containing a 205 nt self-splicing group I intron into a RNA enzyme that catalyzes polymerization of an external RNA substrate. The reaction involves transesterification steps analogous to both the forward and reverse exon ligation steps of group I splicing; as such it depends entirely on 3' splice site reactions. The RNA substrate is a 20 nt analogue of the ligated exons (E1.E2), whose 3' end resembles the 3' terminus of the intron RNA enzyme (IVS). The splice junction of the substrate is attacked by the 3' end of the intron, then the molecule displaces the original 3' terminal guanosine so that the new 3' terminus is brought into the active site and used as the attacking nucleophile in the next reaction. Polymerization occurs via a series of covalent enzyme-linked intermediates of the structure IVS.(E2)n, where n = 1 to > or = 18. The 5' exon accumulates during the course of the reaction and can attack the covalent intermediates to produce elongation products of structure E1.(E2)n, regenerating the intron RNA enzyme in unchanged form. In this manner, the enzyme converts 20 nt oligoribonucleotides into polyribonucleotides up to at least 180 nt by 10 nt increments. These results have significant implications for the evolution of RNA-based self-replicating systems.     

In vivo expression of the nucleolar group I intron-encoded I-dirI homing endonuclease involves the removal of a spliceosomal intron

The Didymium iridis DiSSU1 intron is located in the nuclear SSU rDNA and has an unusual twin-ribozyme organization. One of the ribozymes (DiGIR2) catalyses intron excision and exon ligation. The other ribozyme (DiGIR1), which along with the endonuclease-encoding I-DirI open reading frame (ORF) is inserted in DiGIR2, carries out hydrolysis at internal processing sites (IPS1 and IPS2) located at its 3' end. Examination of the in vivo expression of DiSSU1 shows that after excision, DiSSU1 is matured further into the I-DirI mRNA by internal DiGIR1-catalysed cleavage upstream of the ORF 5' end, as well as truncation and polyadenylation downstream of the ORF 3' end. A spliceosomal intron, the first to be reported within a group I intron and the rDNA, is removed before the I-DirI mRNA associates with the polysomes. Taken together, our results imply that DiSSU1 uses a unique combination of intron-supplied ribozyme activity and adaptation to the general RNA polymerase II pathway of mRNA expression to allow a protein to be produced from the RNA polymerase I-transcribed rDNA.     

Organization of the murine Ig-related lambda 5 gene transcribed selectively in pre-B lymphocytes.

lambda 5 is an immunoglobulin lambda light chain-related gene which is selectively transcribed in murine pre-B lymphocytes to yield a 1.2 kb poly(A)+ mRNA. Comparison of the nucleotide sequence of a 1 kb cDNA clone with the sequence of a genomic clone isolated from 70Z/3 murine pre-B lymphoma cells shows lambda 5 is composed of three exons spanning a 3.75 kb DNA segment. Conserved splice signal sequences at all exon/intron boundaries and the presence of a long open reading frame indicate that a functional mRNA molecule can be made. Exon I contains a cap-site and a potential ATG start codon as well as sequences encoding a signal peptide. This gene could encode a lambda 5 protein of 209 amino acids which has, however, not yet been identified. The 3' portion of exon II and all of exon III shows strong sequence homologies to J lambda L and C lambda L exons. Homology to the lambda L chain genes is lost in the 5' portion of exon II and throughout exon I. In exon I short homologies to leader sequences and to VH framework 1 sequences are seen.     

Short-term sequence evolution and vertical inheritance of the Naegleria twin-ribozyme group I intron

Background  

Ribosomal DNA of several species of the free-living Naegleria amoeba harbors an optional group I intron within the nuclear small subunit ribosomal RNA gene. The intron (Nae.S516) has a complex organization of two ribozyme domains (NaGIR1 and NaGIR2) and a homing endonuclease gene (NaHEG). NaGIR2 is responsible for intron excision, exon ligation, and full-length intron RNA circularization, reactions typical for nuclear group I intron ribozymes. NaGIR1, however, is essential for NaHEG expression by generating the 5' end of the homing endonuclease messenger RNA. Interestingly, this unusual class of ribozyme adds a lariat-cap at the mRNA.     

Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo.

The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailability of the usual 3' splice site for exon ligation. The thermosensitivity of this alternate splice event suggests that the sequences governing the thermodependence of MuSVts110 RNA splicing do not involve any particular 3' splice site or branch point sequence, but rather lie near the 5' end of the intron.     

Location of the 11 bp exon in the chicken pro alpha 2(I) collagen gene.

During the fine structural analysis of the 5' end of the 38 kb chicken pro alpha 2(I) collagen gene, we failed to locate an exon, only 11 bp in size, which had been predicted from the DNA sequence analysis of a cDNA clone complementary to the 5' end of the pro alpha 2(I) collagen mRNA (1). We know report the location of this 11 bp exon, exon 2, at the 5' end of a 180 bp Pst I fragment, 1900 bp 3' to exon 1 and 600 bp 5' to exon 3. Its sequence, ATGTGAGTGAG, is highly unusual in that it contains two overlapping consensus donor splice sequences. Moreover, it is flanked by two overlapping donor splice sequences but only one of the four splice sequences is actually spliced (1). The first half of intron 1 also has an unusual sequence: it is 68% GC, contains 88 CpG dinucleotides and 11 Hpa II sites. The second half is more like other intron sequences in the collagen gene with a GC content of 41%, 19 CpG, and no Hpa II sites. However it contains two sequences with 7 and 9 bp homology to the 14 bp SV40 enhancer core sequence. It is suggested that some part of intron 1 may be involved in regulation.     

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.     

DNA polymerization catalysed by a group II intron RNA in vitro.

The excised group II intron bI1 from Saccharomyces cerevisiae can act as a ribozyme catalysing various chemical reactions with different substrate RNAs in vitro . Recently, we have described an editing-like RNA polymerization reaction catalysed by the bI1 intron lariat that proceeds in the 3'-->5'direction. Here we show that the bI1 lariat RNA can also catalyse successive deoxyribonucleotide polymerization reactions on exogenous substrate molecules. The basic mechanism of the reaction involved interacting cycles between an alternative version of partial reverse splicing (lariat charging) and canonical forward splicing (lariat discharging by exon ligation). With an overall chain growth in the 3'-->5' direction, the 5' exon RNAs (IBS1dN) were elongated by successive insertion of deoxyribonucleotides derived from single deoxyribonucleotide substitutions (dA, dG, dC or dT). All four deoxyribonucleotides were used as substrates, although with different efficiencies. Our findings extend the catalytic repertoire of group II intron RNAs not only by a novel DNA polymerization activity, but also by a DNA-DNA ligation capacity, supporting the idea that ribozymes might have been part of the first primordial polymerization machinery for both RNA and DNA.     

Antisense-targeted immuno-EM localization of the pre-mRNA path in the spliceosomal C complex

A first step in understanding the architecture of the spliceosome is elucidating the positions of individual spliceosomal components and functional centers. Catalysis of the first step of pre-mRNA splicing leads to the formation of the spliceosomal C complex, which contains the pre-mRNA intermediates--the cleaved 5' exon and the intron-3' exon lariat. To topographically locate the catalytic center of the human C complex, we first determined, by DNA oligonucleotide-directed RNAse H digestions, accessible pre-mRNA regions closest to nucleotides of the cleaved 5' splice site (i.e., the 3' end of exon 1 and the 5' end of the intron) and the intron lariat branch point, which are expected to be at/near the catalytic center in complex C. For electron microscopy (EM) localization studies, C complexes were allowed to form, and biotinylated 2'-OMe RNA oligonucleotides were annealed to these accessible regions. To allow localization by EM of the bound oligonucleotide, first antibiotin antibodies and then protein A-coated colloidal gold were additionally bound. EM analyses allowed us to map the position of exon and intron nucleotides near the cleaved 5' splice site, as well as close to the anchoring site just upstream of the branch adenosine. The identified positions in the C complex EM map give first hints as to the path of the pre-mRNA splicing intermediates in an active spliceosomal C complex and further define a possible location for its catalytic center.     

Tri-partite assay for studying exon ligation by the ai5gamma group II intron

Group II introns self-splice via a two-step mechanism: cleavage at the 5' splice site followed by exon ligation at the 3' splice site. The second step has been difficult to study in vitro because it is generally faster than the first. Herein we describe development and partial kinetic characterization of a novel assay for studying the second step in isolation. In this system, a truncated linear intron (nucleotides 1-881) mediates exon ligation between two oligonucleotide substrates: a 19 nt 5' exon and a 3' substrate consisting of the last 6 nucleotides of the intron plus a 6 nucleotide 3' exon. We found that neither the exact structure of domain 6 nor the identity of nucleotides flanking the 3' splice site is critical for accurate 3' splice site choice by the ai5gamma group II intron. The multiple turnover k(cat) (0.14 min(-)(1)) is slower than the single turnover k(obs) (0.6-0.7 min(-)(1)), consistent with rate-limiting product release under steady-state conditions. Decreased single turnover rates at lower pHs were more consistent with loss of catalytic activity than with rate-limiting chemistry. Binding of the 3' substrate (K(m) = 2.6 microM) could be improved by changing a long-range A:U base pair involving the last intronic nucleotide (the gamma-gamma' interaction) to G:C (K(m(3)(')(substrate)) = 1 microM).     

Autocatalytic activities of intron 5 of the cob gene of yeast mitochondria.

The terminal intron of the mitochondrial cob gene of Saccharomyces cerevisiae can undergo autocatalytic splicing in vitro. Efficient splicing of this intron required a high concentration of monovalent ion (1 M). We found that at a high salt concentration this intron was very active and performed many of the reactions described for other group I introns. The rate of the splicing reaction was dependent on the choice of the monovalent ion; the reaction intermediate, the intron-3' exon molecule, accumulated in NH4Cl but not in KCl. In addition, the intron was more reactive in KCl, accumulating in two different circular forms: one cyclized at the 5' intron boundary and the other at 236 nucleotides from the 5' end. These circular forms were able to undergo the opening and recyclization reactions previously described for the Tetrahymena rRNA intron. Cleavage of the 5' exon-intron boundary by the addition of GTP did not require the 3' terminus of the intron and the downstream exon. An anomalous guanosine addition at the 3' exon and at the middle of the intron was also detected. Hence, this intron, which requires a functional protein to splice in vivo, demonstrated a full spectrum of characteristic reactions in the absence of proteins.     

Origin of the phosphate at the ligation junction produced by self-splicing of Tetrahymena thermophila pre-ribosomal RNA

The exons of the self-splicing pre-ribosomal RNA of Tetrahymena thermophila are joined accurately in vitro, even when only 33 nucleotides of the natural 5' exon and 38 nucleotides of the natural 3' exon remain. RNA fingerprint analysis was used to identify the unique ribonuclease T1 oligonucleotide generated by exon ligation. Secondary digests of the ligation junction oligonucleotide with ribonuclease A confirmed the identity of the fragment and demonstrated that the phosphate group that forms the phosphodiester bond at the ligation junction is derived from the 5' position of a uridine nucleotide in the RNA. This observation supports the prediction that the splice junction phosphate is derived from the 3' splice site. These results emphasize the mechanistic similarities of RNA splicing reactions of the group I introns, group II introns and nuclear pre-mRNA introns.