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Rnase Mitochondrial RNA Processing Correctly Cleaves a Novel R Loop at the Mitochondrial DNA Leading-Strand Origin of Replication

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Rnase Mitochondrial RNA Processing Correctly Cleaves a Novel R Loop at the Mitochondrial DNA Leading-Strand Origin of Replication Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press RNase mitochondrial RNA processing correctly cleaves a novel R loop at the mitochondrial DNA leading-strand origin of replication Daniel Y. Lee and David A. Clayton 1 Department of Developmental Biology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, California 94305-5427 USA The precursor primer RNA for mammalian mitochondrial DNA leading-strand replication remains as a persistent R loop formed during transcription through the mitochondrial DNA control region. We have examined model R loops, which exist in a novel and physiologically accurate preprimer conformation, as potential substrates for mammalian RNase mitochondrial RNA processing (MRP). Mouse RNase MRP accurately cleaves an R loop containing the mouse mitochondrial DNA origin. The multiple cleavage sites on the R-loop substrate match the priming sites observed in vivo, suggesting that RNase MRP alone is capable of generating virtually all of the leading-strand replication primers. [Key Words: Replication priming; mitochondrial RNA processing; R loop; RNA-DNA hybrid; RNase MRP] Received December 19, 1996; revised version accepted January 30, 1997. Mammalian mitochondrial DNA (mtDNA) replicates by excellent opportunity for investigating the RNA process- a mechanism of unidirectional displacement synthesis ing mechanism in vitro. initiating from two distinct origins, one for each strand RNase mitochondrial RNA processing (MRP), a ribo- of the duplex circular genome (Clayton 1982). The nucleoprotein (RNP) endoribonuclease, was originally mtDNA heavy (H)-strand is synthesized first, giving rise described as an RNA processing activity purified from to the so-called displacement-loop (D-loop) DNA strand. mouse mitochondrial extracts that cleaved single- Examination of the nucleic acid species from the stranded L-strand RNA in vitro at only one of the mul- H-strand origin (OH) of human and mouse mtDNAs re- tiple in vivo priming sites (Chang and Clayton 1987a, bl. vealed discrete groups of light (L)-strand transcripts (so Despite the precision of the RNA cleavage reaction, an named because the L-strand served as template) that ter- issue relating to the substrate structure remained unre- minated precisely where H-strand synthesis initiated solved. Although primer RNAs were known to be asso- (Gillum and Clayton 1979; Chang and Clayton 1985; ciated stably with mtDNA when isolated from mito- Chang et al. 1985). In mouse mitochondria, some of the chondria, simple linear RNA-DNA heteroduplex sub- newly synthesized DNA strands were found to contain strates constructed with the primer RNA and its cDNA RNA chains covalently attached at their 5' termini were not cleaved by RNase MRP (Chang and Clayton (Chang et al. 1985). These RNA species were shown to 1985, 1987b). However, it has become clear recently that have the same 5' termini, which mapped exactly to the such RNA-DNA heteroduplexes do not represent the L-strand transcriptional promoter (LSP). Therefore, prod- physiological state of the preprimer complex, prompting ucts of transcription were proposed to prime DNA syn- us to re-evaluate the substrate specificity in the RNase thesis at the mtDNA O H. Because the L-strand tran- MRP processing model. scripts extended beyond the replication origin, the action Xu and Clayton (1995, 1996) reported that transcrip- of a site-specific endoribonuclease was postulated to gen- tion by mitochondrial RNA polymerase through the rep- erate the mature primer RNA 3' termini. The in vivo lication control regions of yeast and human mtDNA re- mapping data, which document the RNA to DNA tran- sulted in persistent RNA-DNA hybrids also known as R sition sites with nucleotide-level precision, provided an loops. We have shown recently that model R loops like those formed via transcription can be reconstituted by hybridizing the L-strand RNA and its corresponding su- percoiled DNA template in the presence of formamide XCorresponding author. Present address: Howard Hughes Medical Insti- tute, Chevy Chase, Maryland 20815-6789 USA. (Lee and Clayton 1996). Distinct from simple RNA- E-MAIL [email protected]; FAX (301) 215-8828. cDNA heteroduplexes, three-stranded R loops thus 582 GENES & DEVELOPMENT 11:582-592 © 1997 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/97 $5.00 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Mitochondrial R-loop processing by RNase MRP formed were remarkably stable and exhibited unusual A structural properties that were proposed to be important sPo/ in primer RNA metabolism. These findings provided IIIIIIIIlllLIIIIIIIRNA crucial insight into defining a physiologically accurate 5[-~/~,,,~, preprimer complex as a potential substrate for an RNA processing enzyme. We now report the surprising finding pMR718B DNA that mouse RNase MRP cleaves R-loop substrates in < vitro at virtually all of the priming sites observed in vivo, >9 including the major sites of RNA to DNA synthesis that collectively constitute the leading-strand replication ori- B gin. 5' GAAUACACGG AAUUCGAGCU CGCCCACUAA AAUAUAAGUC Results AUAUUUUGGG AACUACUAGA AUUGAUCAGG ACAUAGGGUU The mouse R-loop substrate is processed by RNase UGAUAGUUAA UAUUAUAUGU CUUUCAAGUU CUUAGUGUUU MRP W VW TT ~ WT WV UUGGGGUUUG GCAUUAAGAG GAGGGGGUGG GGGGUUUGGA RNase MRP was purified from mouse LA9 cells by meth- --CSB III -j I--CSB II-J ods described previously (Chang and Clayton 1987a, b) q V V GAGUUAAAAU UUGGUAUUGA GUAGCAUUUA UGUCUAACAA with additional chromatographic steps (see Materials i .... V T ~ T and Methods). Enzymatic activity was followed by RNA GCAUGAAUAA UUAGCCUUAG GUGAUUGGGU UUUGCGGACU cleavage with the standard single-stranded L-strand -CSB I RNA substrate and by direct quantitation of the RNA AAUGAUUCUU CACCGUAGGU GCGUCUAGAC UGUGUGCUGU subunit of RNase MRP. From the final ion exchange V V V VW W VV (Mono Q) chromatography, a single fraction correspond- CCUUUCAUGC CUUGACGGCU AUGUUGAUGA AAGUAGGCCA ing to the peak of activity was used, unless stated oth- erwise. These highly purified enzyme fractions con- AAAUAAAAAG AUACCAAA 3' tained no detectable levels of nonspecific exonuclease, Figure 1. Structure of the R-loop substrate. (A) Diagram of the ribonuclease, and deoxyribonuclease activities under our reconstituted R loop containing the mouse mtDNA control re- standard reaction conditions. The model R-loop sub- gion. The double-stranded supercoiled plasmid (pMR718B) and strate containing the mouse mtDNA OH region (Fig. the single-stranded RNA derived from the same template by in 1A, B) was prepared by annealing a transcript synthesized vitro transcription (SP6 RNA polymerase) is base-paired with in vitro from pMR718B (Chang and Clayton 1987a) and the complementary DNA strand in a complex configuration. (B) the same supercoiled template DNA in the presence of Nucleotide sequence of the RNA in the R-loop substrate. Con- served sequence blocks (CSB) are designated with brackets and formamide as described elsewhere (Lee and Clayton sites of E. coli RNase H cleavage are denoted with arrowheads 1996). (Lee and Clayton 1996). Filled, hatched, and open arrowheads A characteristic feature of persistent R loops generated represent, respectively, strong, intermediate, and weak RNase either by transcription or reconstituted in formamide is H cleavage at that site, indicating the nonuniform RNA-DNA the discrete and limited position of the RNA-DNA base- base-pairing throughout the length of the RNA. The 5' region of paired region (Lee and Clayton 1996; Xu and Clayton the RNA strand that is largely unstructured is designated by 1996). When the final Mono Q fractions were assayed italic text. with the R-loop substrate in which the RNA was uniquely radiolabeled at the 3' terminus, multiple cleav- age products were observed (Fig. 2C). This R-loop pro- cessing activity coeluted with the standard single- with the gross similarity of the free and R-loop forms of stranded RNA cleavage activity (Fig. 2B) and with the the primer RNA as determined by structural probing as- RNA subunit of the RNase MRP holoenzyme (Fig. 2A). says (Lee and Clayton 1996). Comparison of the relatively simple RNase MRP cleav- To confirm that these cleavage reactions were cata- age pattern on the free RNA substrate to the more com- lyzed by RNase MRP, we carried out specific inhibition plex pattern on the R loop revealed similar overall reac- experiments by exploiting the known ribonucleoprotein tion kinetics (Fig. 2D). However, cleavage at the major composition of the holoenzyme. The requirement of an site on the free RNA, located between conserved se- RNA component of RNase MRP was shown previously quence block (CSB)II and CSBIII, was less efficient in the by loss of cleavage activity upon digestion with micro- R loop, and what was observed initially as inefficient coccal nuclease (Chang and Clayton 1987a). Consistent cleavage at other discrete sites on the free RNA was with these previous results, pretreatment of the Mono Q highly enhanced in the R-loop substrate. This similarity fraction with micrococcal nuclease (with Ca 2÷, an essen- in the cleavage pattern was accentuated with extended tial metal cofactor) abolished cleavage at all sites on the digestion times, suggesting that the RNA strand exhibits R-loop substrate (Fig. 3A, lane 7); micrococcal nuclease certain structural features of the R loop even in the ab- with EGTA-chelated Ca 2÷ had no effect (Fig. 3A, lane 8). sence of DNA (Fig. 2D). This observation is consistent Predigestion of the Mono Q fraction with proteinase K GENES & DEVELOPMENT 583 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Lee and Clayton Figure 2. R-loop processing activity of RNase A 46 48 50 52 54 56 D MRP. (A) Purification profile of RNase MRP holo- i!ii ~i! i~ii ~i~ ,~~ free RNA R-loop ~~ ~,___~ , , i , enzyme. The RNA subunit of RNase MRP was ex- "--MRP L° o °. Loo o°~. ~°°~oo ° tracted from Mono Q column fractions (numbers • ~,~ :ErOo "LO,-~O,--~UO,--L6,--~ ,- at top of lanes).
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