The splicing of yeast mitochondrial group I and group II introns requires a DEAD-box protein with RNA chaperone function

Abstract

Group I and II introns self-splice in vitro, but require proteins for efficient splicing in vivo, to stabilize the catalytically active RNA structure. Recent studies showed that the splicing of some Neurospora crassa mitochondrial group I introns additionally requires a DEAD-box protein, CYT-19, which acts as an RNA chaperone to resolve nonnative structures formed during RNA folding. Here we show that, in Saccharomyces cerevisiae mitochondria, a related DEAD-box protein, Mss116p, is required for the efficient splicing of all group I and II introns, some RNA end-processing reactions, and translation of a subset of mRNAs, and that all these defects can be partially or completely suppressed by the expression of CYT-19. Results for the aI2 group II intron indicate that Mss116p is needed after binding the intron-encoded maturase, likely for the disruption of stable but inactive RNA structures. Our results suggest that both group I and II introns are prone to kinetic traps in RNA folding in vivo and that the splicing of both types of introns may require DEAD-box proteins that function as RNA chaperones.

Fig. 1.

Diagrams of the MSS116^myc, cyt-19, and chimeric MSS116/CYT-19 ^myc genes. MSS116^myc encodes the 664-aa MSS116 ORF (open box) with a 36-aa mt targeting sequence and a C-terminal myc tag. The cyt-19 cDNA (hatched box) encodes the 626-aa cyt-19 ORF with a putative 53-aa mt targeting sequence. Both Mss116p and CYT-19 contain a conserved core with nine motifs characteristic of the DEAD-box subfamily of DExH/D-box proteins (black bars, labeled for MSS116 ^myc). Percent similarity is shown for indicated regions of MSS116p and CYT-19. The chimeric MSS116/cyt-19 ^myc gene consists of the promoter and mt targeting sequence of MSS116 fused to cyt-19 codons 54–626 with a C-terminal myc tag, and the MSS116 3′ UTR. Asterisks indicate the locations of MSS116 mutations analyzed in this work.

Fig. 2.

Splicing phenotypes of yeast strains containing different MSS116 and cyt-19 alleles. (A–G) Northern hybridizations of mt RNAs from 30°C-grown strains containing the following mtDNA introns: aI1 (A), aI2 (B), aI5γ (C), bI1 (D), ω (E), aI3 and aI4 (F), and bI4 and bI5 (G). The blots were hybridized with ³²P-labeled probes complementary to COX1 exon 6 (A–C and F), COB exon 6 (D and G), and 21S rRNA exon 2 (E). (H–J) Northern hybridizations of mt RNAs from MSS116 and mss116Δ strains with ρ^- petite mtDNAs containing a 21S rRNA gene with the ω intron (H), a COX1 gene with aI5γ (I), and a COB gene with bI1 (J). Spliced mRNAs and unspliced precursor RNAs are indicated by filled and open triangles, respectively. Multiple precursor RNAs containing different combinations of introns are present in F and G. Arrows in B, C, F, and H indicate precursor RNAs with 3′ extensions. The asterisk in B indicates an aI2 splicing intermediate containing the intron and downstream exons.

Fig. 3.

Mt RNA processing and protein synthesis in yeast strains containing different MSS116 and cyt-19 alleles. (A) Northern hybridization of mt RNAs from wild-type MSS116 and mss116Δ strains with intronless (I⁰) mtDNA grown on raffinose at 30°C or 24°C. The blot was hybridized sequentially with ³²P-labeled probes complementary to COX1 exon 6 (i), ATP6 (ii), COX2 (iii), and COB exon 6 (iv). Arrow in i and upper arrow in ii indicate a polygenic precursor RNA containing COX1-ATP8-ATP6-RF3. Lower arrow in ii indicates an ATP6 precursor RNA with a 5′ extension, and arrow in iv indicates a COB precursor RNA with a 5′ extension. (B) Pulse labeling of mt translation products in strains with I⁰ mtDNA grown at 24°C. Cells were labeled with ³⁵S-SO₄ in the presence of cycloheximide, and mt proteins were analyzed in a 1% SDS/10–16% polyacrylamide gradient gel, which was scanned with a PhosphorImager. Mt translation products are identified to the left. (C) Western blots showing levels of the aI2 IEP (p62^3HA) in different strains grown at 30°C. Whole-cell proteins (≈20 μg) were analyzed in a 1% SDS/7.5% polyacrylamide gel. After blotting, the membrane was divided at the position of the 40-kDa marker, and the upper and lower halves were probed with anti-hemagglutinin and anti-porin monoclonal antibodies, respectively. Porin is a mt outer membrane protein used to confirm equal loading.

Fig. 4.

Expression of CYT-19^myc in S. cerevisiae and inability of the CYT-19 K125E mutant to support RNA splicing. (A) CYT-19^myc expression. A Western blot of whole-cell protein (≈20 μg) from the indicated strains grown at 30°C was divided at the position of 40-kDa marker, and the top and bottom halves were probed with anti-myc and anti-porin antibodies, respectively. (B) The CYT-19 K125E does not support splicing of COX1 introns. Northern hybridization was carried out with RNAs from strains grown at 30°C, which have a COX1 gene containing aI3, aI4, and aI5γ and carry CEN plasmids with the nuclear genes indicated in the figure. The blot was hybridized with a ³²P-labeled probe complementary to COX1 exon 6.

Fig. 5.

The aI2-encoded protein p62 is associated with aI2 RNA in RNP particles from cyt-19-ki. (A and B) Fractionation of p62. In A, flotation gradient-purified mitochondria from 30°C-grown MSS116 and cyt-19-ki strains, with aI2^3HA as the only mtDNA intron, were lysed with 1% Nonidet P-40 in a high-salt (500 mM KCl) buffer, and the lysates were centrifuged at 20,000 × g and 100,000 × g (18, 20). In B, flotation gradient-purified mitochondria from MSS116 and cyt-19-ki strains, with aI2^3HAΔDV as the only mtDNA intron, were lysed as above and centrifuged through a 1.85 M sucrose cushion containing 500 mM KCl buffer (18, 20). In both panels, aliquots of supernatant (S) and pellet (P) fractions were analyzed for p62^3HA by Western blotting. The band under p62^3HA in some lanes is a sporadically occurring degradation product. (C) Southern blot of ³²P-labeled cDNA synthesized in RNPs. RNP preparations from the MSS116 and cyt-19-ki strains with wild-type aI2^3HA were used in reverse transcription reactions with 20-mer DNA primer HG1, which is complementary to a 3′-exon sequence 10 nt downstream of aI2. The resulting ³²P-labeled cDNAs were hybridized to Southern blots of EcoRI-digested I⁰ (lanes 1 and 3) and aI2-containing (lanes 2 and 4) mtDNA.