Length changes in the joining segment between domains 5 and 6 of a group II intron inhibit self-splicing and alter 3' splice site selection.

Domain 5 (D5) and domain 6 (D6) are adjacent folded hairpin substructures of self-splicing group II introns that appear to interact within the active ribozyme. Here we describe the effects of changing the length of the 3-nucleotide segment joining D5 to D6 [called J(56)3] on the splicing reactions of intron 5 gamma of the COXI gene of yeast mitochondrial DNA. Shortened variants J(56)0 and J(56)1 were defective in vitro for branching, and the second splicing step was performed inefficiently and inaccurately. The lengthened variant J(56)5 had a milder defect-splicing occurred at a reduced rate but with correct branching and a mostly accurate 3' splice junction choice. Yeast mitochondria were transformed with the J(56)5 allele, and the resulting yeast strain was respiration deficient because of ineffective aI5 gamma splicing. Respiration-competent revertants were recovered, and in one type a single joiner nucleotide was deleted while in the other type a nucleotide of D6 was deleted. Although these revertants still showed partial splicing blocks in vivo and in vitro, including a substantial defect in the second step of splicing, both spliced accurately in vivo. These results establish that a 3-nucleotide J(56) is optimal for this intron, especially for the accuracy of 3' splice junction selection, and indicate that D5 and D6 are probably not coaxially stacked.     

Identification of splicing signals in introns of yeast mitochondrial split genes: mutational alterations in intron bI1 and secondary structures in related introns.

Four mitochondrial mutations are known to block excision of intron I1 of the cob gene in S.cerevisiae. The nucleotide sequence alteration of one of them, M4873, has been determined. It is a deletion of 1 bp in a run of five G's at a distance of 30 to 34 bp upstream to the 3' splice point. Reversion is found to occur by restoration of the run of five G's either by insertion of 1 G (wild type reversion) or by transition A leads to G next to this run of G's (pseudo-wild type reversion). The effect of mutation and reversion on RNA splicing indicates that the run of five G's is of critical importance for intron I1 excision, possibly in participating in the formation of a splice signal with a helical structure. This presumption is confirmed by the observation that this sequence is part of a larger sequence of some 80 bp next to the 3' splice point which is conserved to some extend in the four mitochondrial introns (bI1, aI1, aI2, aI5) that survive after excision as circular RNAs. Most striking is the conservation of this sequence at the level of secondary structure.     

Role of the 5' hairpin structure in the splicing accuracy of the fourth intron of the yeast cob-box gene

The splicing mechanism of the maturase-coding introns is poorly understood. We have systematically examined the phenotypes of a large number of revertants from the mitochondrial mutation G2457. This mutation results from a single base change near the 5' splicing site. We show here that this base change does not completely block the splicing of the intron but rather affects the specificity of the splicing process. We examine four classes of revertants which allow us to characterize the crucial role of a stem and loop structure in the accuracy of the intron excision process. An unexpected class of revertant suggests that other elements are involved in this mechanism. Reversion of G2457 can also occur via the excision in the mitochondrial genome of the intron coding sequence. These results are discussed in relation to the possible role fulfilled by the maturase in the control of intron splicing.     

Splice site selection by intron aI3 of the COX1 gene from Saccharomyces cerevisiae.

Interactions of the 5' and 3' splice sites with intron internal sequences of the yeast mitochondrial group I intron aI3 were studied using mutation analysis. The results can be fully explained by the splice guide model in which the splice sites are defined by the Internal Guide Sequence. No evidence was found for an alternative interaction between intron nucleotides preceding the 3' splice site and nucleotides in the vicinity of the core region as was found for the Tetrahymena intron. Our results also suggest that binding of the 5' and 3' splice site nucleotides to the IGS can not take place simultaneously. The intron must therefore undergo conformational changes as the reaction proceeds from the first step of self splicing, GTP attack at the 5' splice site, to exon ligation, the second step.     

Determination of functional domains in intron bI1 of yeast mitochondrial RNA by studies of mitochondrial mutations and a nuclear suppressor.

The sequence of intron 1 in the cob gene in mtDNA (bI1) of the yeast strain 777-3A has been determined. Furthermore, we have performed a systematic search for complementary sequence stretches within this intron RNA, and within the RNA of intron 5 gamma of the oxi3 gene (aI5 gamma) which shares distinctive sequences with bI1. Possible secondary structure models derived from this analysis show nearly identical core structures for bI1 and aI5 gamma RNA with conserved sequence stretches in prominent positions. These core structures are similar to those previously reported for RNAs of introns having very limited sequence homology with bI1 and aI5 gamma. In two mutants which are defective in bI1 excision from cob pre-mRNA, nucleotide sequence alterations in bI1 have been determined. One mutation (G5049) apparently affects the stability of a hybrid stretch in the proposed secondary structure of bI1 RNA whereas the other one (M1301), a deletion of one A in a run of five As, affects a sequence which is conserved in bI1 and aI5 gamma and is involved in the formation of a distinct secondary structure. Out of seven revertants of M1301, three were found to have restored the wild-type bI1 sequence AAAAA, three others had the related sequence AAAAG which is functionally indistinguishable from wild-type, whereas one revertant had a nuclear mutation which suppresses the splicing defect exerted by the mitochondrial mutation M1301. This nuclear suppressor (SUP-101) is allele specific and dominant. The possible role of the sequence affected by M1301 in terms of a recognition site for a nuclear gene product will be discussed.     

Mne1 is a novel component of the mitochondrial splicing apparatus responsible for processing of a COX1 group I intron in yeast

Saccharomyces cerevisiae cells lacking Mne1 are deficient in intron splicing in the gene encoding the Cox1 subunit of cytochrome oxidase but contain wild-type levels of the bc(1) complex. Thus, Mne1 has no role in splicing of COB introns or expression of the COB gene. Northern experiments suggest that splicing of the COX1 aI5β intron is dependent on Mne1 in addition to the previously known Mrs1, Mss116, Pet54, and Suv3 factors. Processing of the aI5β intron is similarly impaired in mne1Δ and mrs1Δ cells and overexpression of Mrs1 partially restores the respiratory function of mne1Δ cells. Mrs1 is known to function in the initial transesterification reaction of splicing. Mne1 is a mitochondrial matrix protein loosely associated with the inner membrane and is found in a high mass ribonucleoprotein complex specifically associated with the COX1 mRNA even within an intronless strain. Mne1 does not appear to have a secondary function in COX1 processing or translation, because disruption of MNE1 in cells containing intronless mtDNA does not lead to a respiratory growth defect. Thus, the primary defect in mne1Δ cells is splicing of the aI5β intron in COX1.     

A maturase-encoding group IIA intron of yeast mitochondria self-splices in vitro.

Intron 1 of the coxI gene of yeast mitochondrial DNA (aI1) is a group IIA intron that encodes a maturase function required for its splicing in vivo. It is shown here to self-splice in vitro under some reaction conditions reported earlier to yield efficient self-splicing of group IIB introns of yeast mtDNA that do not encode maturase functions. Unlike the group IIB introns, aI1 is inactive in 10 mM Mg2+ (including spermidine) and requires much higher levels of Mg2+ and added salts (1M NH4Cl or KCl or 2M (NH4)2SO4) for ready detection of splicing activity. In KCl-stimulated reactions, splicing occurs with little normal branch formation; a post-splicing reaction of linear excised intron RNA that forms shorter lariat RNAs with branches at cryptic sites was evident in those samples. At low levels of added NH4Cl or KCl, the precursor RNA carries out the first reaction step but appears blocked in the splicing step. AI1 RNA is most reactive at 37-42 degrees C, as compared with 45 degrees C for the group IIB introns; and it lacks the KCl- or NH4Cl-dependent spliced-exon reopening reaction that is evident for the self-splicing group IIB introns of yeast mitochondria. Like the group IIB intron aI5 gamma, the domain 4 of aI1 can be largely deleted in cis, without blocking splicing; also, trans-splicing of half molecules interrupted in domain 4 occurs. This is the first report of a maturase-encoding intron of either group I or group II that self-splices in vitro.     

Structural analysis of the Neurospora mitochondrial large rRNA intron and construction of a mini-intron that shows protein-dependent splicing.

The gene encoding the Neurospora mitochondrial large rRNA contains a single group I intron of 2.3 kilobases that is not self-splicing in vitro. We showed previously that the splicing of this intron in vivo and in vitro is dependent on the Neurospora cyt-18 protein, mitochondrial tyrosyl-tRNA synthetase. In the present work, we carried out further structural analysis of the intron and constructed mutant derivatives of it in order to identify features that are either required for splicing or prevent it from self-splicing. Previous studies showed that the intron contains a large hairpin structure near the 5' splice site. By mapping RNase III cleavage sites, we identified this hairpin structure as an extended P2 stem. We construct a mini-intron of 388 nucleotides by deleting the 426-amino acid intron open reading frame, most of the 5' intron hairpin, and all of L8. This mini-intron shows the same protein-dependent splicing as the full length intron, but is still not self-splicing. Further deletions, which remove all of P2 or all or part of P4, P6, P7, or P9, inactivate splicing, suggesting that an intact group I intron core structure is required. Strengthening the P1, P10, or P9.0 pairings did not enable the mini-intron to self-splice. Our findings indicate that the inability of the mitochondrial large rRNA intron to self-splice reflects deficiency of a structure or activity required for cleavage at the 5' splice site, either in the intron core itself or in the interaction between the core and the P1 stem.     

Maturase and endonuclease functions depend on separate conserved domains of the bifunctional protein encoded by the group I intron aI4 alpha of yeast mitochondrial DNA.

Intron 4 alpha (aI4 alpha) of the yeast mitochondrial COXI gene is a mobile group I intron that contains a reading frame encoding both the homing endonuclease I-SceII and a latent maturase capable of splicing both aI4 alpha and the fourth intron of the cytochrome b (COB) gene (bI4). The aI4 alpha reading frame is a member of a large gene family recognized by the presence of related dodecapeptide sequence motifs called P1 and P2. In this study, missense mutations of P1 and P2 were placed in mitochondrial DNA by biolistic transformation. The effects of the mutations on intron mobility, endonuclease I-SceII activity and maturase function were tested. The mutations of P1 strongly affected mobility and endonuclease I-SceII activity, but had little or no effect on maturase function; mutations of P2 affected splicing but not mobility or endonuclease I-SceII activity. Surprisingly, the conditional (temperature-sensitive) mutations at P1 and P2 block one or the other function of the protein but not both. This study indicates that the two functions depend on separate domains of the intron-encoded protein.     

A novel mitochondrial DEAD box protein (Mrh4) required for maintenance of mtDNA in Saccharomyces cerevisiae

In a screen of nuclear genes that assist splicing of mitochondrial localized group II introns in yeast we isolated low-copy number suppressors of splicing and respiratory-deficient point mutants of intron aI5gamma, the last intron of the gene encoding cytochrome c oxidase subunit I. One of the genes found contains the open reading frame (ORF) YGL064c that has previously been proposed to encode a putative RNA helicase of the DEAD box family. Deletion of the ORF gives rise to 100% cytoplasmic petites, indicating that the protein plays an essential role in the mitochondrial RNA metabolism. Overexpression of YGL064c-GFP fusions clearly revealed a mitochondrial localization of the protein. The gene encodes the fourth putative RNA helicase of Saccharomyces cerevisiae implicated in a mitochondrial function and was therefore termed MRH4 (for mitochondrial RNA helicase).     

Imprecise excision of the Caenorhabditis elegans transposon Tc1 creates functional 5' splice sites.

Imprecise excision of the Caenorhabditis elegans transposon Tc1 from a specific site of insertion within the unc-54 myosin heavy chain gene generates either wild-type or partial phenotypic revertants. Wild-type revertants and one class of partial revertants contain insertions of four nucleotides in the unc-54 third exon (Tc1 "footprints"). Such revertants express large amounts of functional unc-54 myosin despite having what would appear to be frameshifting insertions in the unc-54 third exon. We demonstrate that these Tc1 footprints act as efficient 5' splice sites for removal of the unc-54 third intron. Splicing of these new 5' splice sites to the normal third intron splice acceptor removes the Tc1 footprint from the mature mRNA and restores the normal translational reading frame. Partial revertant unc-54(r661), which contains a single nucleotide substitution relative to the wild-type gene, is spliced similarly, except that the use of its new 5' splice site creates a frameshift in the mature mRNA rather than removing one. In all of these revertants, two alternative 5' splice sites are available to remove intron 3. We determined the relative efficiency with which each alternative 5' splice site is used by stabilizing frameshifted mRNAs with smg(-) genetic backgrounds. In all cases, the upstream member of the two alternative sites is used preferentially (> 75% utilization). This may reflect an inherent preference of the splicing machinery for the upstream member of two closely spaced 5' splice sites. Creation of new 5' splice sites may be a general characteristic of Tc1 insertion and excision events.