Biochimica et Biophysica Acta, 1089 (1991) 33-39 ~ 1991 Elsevier Science Publishers B.V. 0167-4781/91/$03.50 ADONIS 0167478191001255 BBAEXP 92236
5' Flanking sequences of human MRP/7-2 RNA gene are required and sufficient for the transcription by RNA polymerase Ill Yan Yuan and Ram Reddy Department of Pharmacololo; Baylor College of Medicine. Houston. TX (U S.A.I (Received 17 September 19901 (Revised manuscript received 30 November ! 990)
Key words: Mitochondrial RNA processing RNA; Transcription: RNA polymerase i11: Cloning: (Human)
Human mitochondrial RNA processing (MRP) RNA is a 270 nucicotide-long small RNA found as ribonucleoprotein particles. In this study, we isolated four human genomic clones with homology to human MRP RNA. Two of these doues contained one copy each of the real gene coding for human MRP RNA; the other two clones represented a processed pseudogene. The Southern blot with the genumic DNA showed that the haploid human genome contains o n e copy of real gene and a few pseudogenes for M R P / 7 - 2 RNA. The human MRP RNA is synthesized by R N A polymerase !!i and the 5 ' flanking sequences - 8 4 to 1 of MRP RNA gene, containing "rATA and PSE-like elements, a r e required and sufficient for transeription in vitro.
Introduction In eukaryotic cells, nucleoli contain many small RNAs in the form of small ribonucleoprotein particles; these include U3, U8, U13, U14 and 7-2 RNAs. U snRNAs in animal nucleoli contain trimethylguanosine cap structures, and are bound to a common 34 kDa protein termed "Fibrillarin' [1-3]. In contrast, 7-2 RNA, as well as RNaseP RNA in mammalian cells, are bound to a 40 kDa To antigen and contain pppG or pppA on their 5' ends [1,4-6]. The U3 RNP particles are required components for a sequence-specific cleavage of preribosomal RNA in the nucleolus [7]. The functions of U8, UI3, and U14 RNPs are not known. 7-2 RNA was initially found as a nucleolar RNA [8]. Using immunoclectron microscopy, Reimer et al. [5] showed that the 7-2 RNA is associated with the To antigen and is localized to the granular compartment of the nucleolus. The nucleolar function of 7-2 RNPs is
Abbreviations: bp, base pair(s), MRP, mitochondrial RNA processing; nt, nucleotide(s); ~ IlL RNA polymerase i11: RNP(s), ribonucleoprotein(s); snRNA(s), small nuclear RNA(s). Correspondence: R. Reddy, Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, U.S.A.
not known, even though it is presumed to be involved in the processing of preribosomal RNA [9]. Pioneering studies carried out by Clayton and his associates with the MRP (mitochondrial RNA processing) RNase showed that the MRP RNase in mitochondria is responsible for a sequence-specific cleavage of the mitochondrial RNA; this cleavage generates an RNA primer that is used as a primer for mitochondriai DNA replication [10,11]. Results obtained in our laboratory showed that rat nucleolar 7-2 RNA is homologous to mouse MRP RNA [9], and it appears that nucleolar 7-2 RNA and MRP RNA are identical [6]. These data show that MRP RNase is functional in mitochondria as well as in nucleofi. MRP RNPs share several features with RNaseP [6]; RNaseP is an endoribonudease that processes precursor tRNAs to generate tRNAs with mature 5' termini [12]. Both RNPs are immunoprecipitabic with anti-To/Th antibodies [1,4,6,13] and are synthesized by RNA polymerase ill (this study; Ref. 14). Both RNAs show primary sequence homology to each other in four conserved blocks [6] and these conserved sequence elements appear to reside in similar stems in the proposed secondary structures of MRP and human RNaseP RNAs [15,16]. All the above properties show that MRP RNase is analogous to RNaseP in structure and in function 116,17]. In the present study, human MRP/7-2 RNA gene
34 was isolated and characterized. The synthesis of MRP/7-2 RNA in vitro was investigated. The results show that the human genome contains one copy of MRP gene and is transcribed by RNA polymerase ill in vitro. Upstream regulatory elements are necessary and sufficient for transcription of this gene in vitro.
Human MRP/7-2 RNA was isolated from HeLa cell nuclei and purified as described previously [9]. Doublestranded MRP cDNA was synthesized using the following deoxyoligonucleotides: 5' ccgaattcgtgctgaaaggcctg 3' corresponding to nueleotides 2 to 18 and 5' gaatgagccccgtgtggt 3' complementary to 237 to 254 nueleotides of MRP RNA. ccgaa sequence in one of the above oligos is an extra linker sequence. The cDNA was cloned into the SmaI site of the Bluescript plasmid vector. MRP cDNA insert was purified on polyacrylamide gels and 32p-labeled by either multiprimer procedure [18] or Polymerase Chain Reaction (PCR) with [a-S2pkICTP as the precursor. The PCR mixture (50 pl) contained 1 pM each of unlabeled dATE dGTP, dTTP, 100/tCi [a-32P]dCTP (3000 Ci/mmol), 300 rig each of deoxyoligonueleotide and 5 ng of DNA template. The labeled probe was purified on a Sephadex G-25 column and used for hybridization.
M R P ( - 84/226) were prepared by PCR using deoxyoligonueleotides corresponding to nucleotides - 8 4 to - 6 2 and deoxyoligonucleotides complementary to nueleotides 2 to - 1 9 , 114-101 or 226-209 of MRP RNA, respectively. The fragments were cloned into Bluescript vector at Smal site. 3' deletion construct M R P ( - 7 3 1 / 1 - 1 4 ) was made by using a convenient Stul restriction site. The 745 bp-long Pstl/Stul fragment was subcloned into Pstl/Smal digested Bluescript plasmid. Analysis of DNA by Southern blotting. 10 pg of HeLa nuclear DNA or 0.1-0.5 /tg of cloned DNA was digested with the appropriate restriction endonucleases, fractionated on agarose gels, blots were prepared and probed by the method of Southern [20]. The hybridization conditions were the same as those employed for screening the genomic library. In vitro transcriptions. Whole HeLa cell extract was prepared by the method of Weil et al. [22]. 3 /tg of supercoiled plasmid DNA was incubated at 30°C for 1-3 h in a 50/tl reaction mixture containing 50% (v/v) HeLa cell extracts, 0.6 mM each of unlabeled CTP, UTP, ATP, 0.025 mM GTP, 5-50/tCi of [a-32p]GTP, 6 mM creatine phosphate, 20 mM KCI, 1 mM DTT and 10 mM Tris-HCi (pH 8.1). The transcribed RNAs were subjected to SDS/phenol extraction and precipitated with ethanol and fractionated by electrophoresis on 10% polyacrylamide/7 M urea gels.
Isolation of genomic clones containing human MRP / 7-2 RNA sequences. A human genomic library (ACTT
Results
Materials and Methods
Preparation of cDNA probes for MRP/7-2 RNA.
57760), prepared from lymph node DNA by partial Alul/HaeIll digestion and cloning into charon 4A phage DNA, was screened by the method of Benton and Davis [19]. Hybridization was done in 5 x SSC, 0.05% sodium pyrophosphate, 2 × Denhardt's solution, 0.1% SDS and 50 p g / m l of Escherichia coil tRNA. The nitrocellulose filters were prehybridized for 6 h, hybridized for 16 h and washed in 2 x SSC, 1 × SSC and 0.5 × SSC containing 0.1% SDS each for 15 min at 42°C. Recombinant A phage DNAs, containing sequences complementary to MRP cDNA, were mapped by Southern hybridization [20] and appropriate DNA restriction fragments were subcloned into Bluescript plasmid vector. DNA sequences were determined by the chain-termination method of Sanger et al. [21] using MI3 universal, reverse or synthetic deoxyoligonueleotide primers.
Isolation of human MRP / 7-2 RNA gene MRP RNA cDNA insert was isolated, labeled by PCR in the presence of [a-32p]dCTP and 400000 phage plaques from a human genomic library were screened. After the first screening, three strong positives and four weak positives were observed. A total of four clones, representing two strong (hMRP-1, hMRP-2) and hMRP-2 -
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Construction of MRP / 7-2 RNA gene deletion mutants. 5' deletion DNAs M R P ( - 8 4 / + 28), M R P ( - 6 / + 28) and M R P ( 1 - 2 7 0 / + 28) were prepared by amplifying plasmid DNA by PCR using deoxyoligonucleotides corresponding to nueleotides - 8 4 to - 6 2 , - 6 to 15 or 2 to 18 of MRP RNA and the M13 universal primer, digesting the amplified DNA with Pstl and inserting into Bluescript vector digested with Sinai and Pstl. 3" deletion constructs M R P ( - 84/2), M R P ( - 84/114) and
hMRP-4
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Fig. 1. Restficti,~n maps of human MRP RNA gene and pseudogene.
The data obtained from the Southern blots of cloned phase MRP-2 and MRP.4 DNA as well as from the genomicblots were used to construct this restrictionpattern surroundingthe human MRP gene and a pseudogene,hMRP, humanMRP.
35 MRP-2
-160 -140 -120 -100 A G C T A T T C T G C T A G C C A C A A T G C C T C T G A A A G C C T A T A G T CTTAGAAAGTTATGCCCGAA AACGGTTTTTTTAATCTCAC
MRP-2
-80 -60 -40 -20 -I G C C A C C A A C T T T C T C A C C C T A A T C A T A A A A C A C A A T T T C T TTAGGGCTATAAAATACTAC T C T G T G A A G C T G A G G A C G ~
[qRP-2
1 20 40 60 80 G T T C G T G C T G A A G G C C T G T A T C C T A G G C T A C A C A C T G A G G ACTCTGTTCCTCCCCTTTCC GCCTAGGGGAAAGTCCCCGG
MRP-2 MRP-4
100 120 140 160 A C C T C G G G C A G A G A G T G C C A C G T G C A T A C G C A C G T A G A C A TTCCCCGCTTCCCACTCCAA AGTCCGCCAAGAAGCGTATC G G A C A G A G G C C T T A A G T T T A G T A A T A G T A G G G A G A G T A G G G G G G C C T G G C G G T G C A C A T G ATATGAGGAAATGGCATCCA
MRP-2 MRP-4
180 200 22O 240 C C G C T G A G C G G C G T G G C G C G G G G G C G T C A T C C G T C A G C T C C C T C T A G T T A C G C A G G C A G T GCGTGTCCGCGCACCAACCA CAGGATCTGGCAGCTAGTTA AATGATGGAATTCAGAGAAC
MRP-2 P,H P - 4
260 +1 C A C G G G G C T C A T T C T C A G C G CGG~r(.~'I-FI-t-FtGT'rCC~A *G****AACG********** * * * * * * e * * l * ~
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GACCACGGCTCACTGCAG AAG~GAAJU~GAAAA
÷5O AGAAGAAGAAGAAAAGAAAA
Fig. 2. Nucleotide sequences of human MRP gene and a pseudogene. The top line shows the sequence of the human MRP gene (clone hMRP-2); the sequence of MRP pseudogene (clone MRP-4) is shown below the real gene sequence. Stars indicate identical bases and dots indicate deletions. The 5" flanking sequences are numbered from - 1 to - 1 6 0 , the transcribed portion of MRP RNA is numbered from I to 270 and the 3' flanking region from + 1 to + 28.
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Fig. 3. Southern blot analysis of human genomic D N A and ~timation of the gene copy number. (A) l0 ~g each of HeLa cell nuclear D N A was digested with restriction endonucleases indicated above each lane, fractionated on a 0.8~ agarose gel, transferred to a nitrocellulose filter and probed with labeled MRP eDNA. (B) Each lane contained 10/tg of HeLa nuclear D N A digested with Pstl, and mixed with 13 pg. 40 pg, or 80 Pig of cloned hMRP-2 plasmid D N A digested with Pstl and BstN! corresponding to 1, 3, or 6 copy equivalents, respectively, per haploid human genome.
36 two weak hybridization signals (hMRP-3, hMRP-4), were plaque-purified and characterized. The restriction map of the phage DNAs indicated that both the clones with strong hybridization signal are identical to each other; similarly, the two clones with weak homology to MRP DNA probe appeared to be from the same genomic locus. The restriction maps surrounding the MRP related sequences are shown in Fig. 1. A 1 kb Pstl fragment from hMRP-2 DNA, and 2.8 kb Pstl fragment from hMRP-4 were suhcloned into the Pstl site of Biuescript vector. The nucleotide sequence of MRP / 7-2 gene and pseudogene The nucleotide sequence of a portion of the I kb Pstl fragment from hMRP-2 clone was determined (Fig. 2) and was found to be identical to the human MRP gene recently reported by Topper and Clayton [23]. Since the human genome appears to contain a single gene for MRP RNA (see below), the gene characterized in this study and by Topper and Clayton [23] may be the same. The MRP homologous sequence from the clone MRP-4 was also sequenced and the sequence corresponding to the MRP RNA and the flanking regions are shown in Fig. 2. This pseudogene was severely truncated and contained only a small portion (68 bp) corresponding to the 3' end of MRP RNA. The nucleotide sequence showed approx. 85~ homology to the MRP RNA. The long stretch of poly(A) sequence in the pseudogene immediately following the 3' end of MRP RNA sequence (Fig. 2) indicates that this pseudogene may have arisen by reverse transcription of an RNA and retroposition into the genome (reviewed in Refs. 24,25). Human genome contains one real gene and few pseudogenes for MRP / 7-2 RNA To understand the genomic organization of human MRP RNA gene, HeLa cell nuclear DNA was digested with several restriction enzymes, fractionated on an agarose gel, transferred onto a nitrocellulose sheet and hybridized with labeled MRP DNA probe. With every digestion, a strong hybridization signal and one or more band(s) with less intensity were observed (Fig. 3A). The sizes of genomic DNA fragment hybridizing with maximum intensity, such as 1.0 kb Pstl fragment, and 1.5 kb BamHi fragment, were consistent with the restriction maps of hMRP-1 and hMRP-2 clones (compare Fig. 1A and 3A). These data, and the correspondence of this DNA sequence (Fig. 2) with human MRP RNA [6], suggested that these strongly hybridizing bands may correspond to true human MRP RNA gene. The other DNA fragments hybridizing with the MRP DNA probe probably correspond to pseudogenes with partial homologies to MRP DNA sequence; one clone representing these homologies was characterized by sequencing (Fig. 2).
a-amanltln ( I*g/ml)
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1 2 3 4 5 6 7 8 9 10 11 12 Fig. 4. Effect of a-amanitinon the synthesisof MRP and 5S RNAs. Human MRP RNA gene and Syrian hamster 5S RNA gene (1.5 Fg each) were transcribedin the presenceof different concentrationsof a-amanitin.The RNAs synthesizedwere fractionatedon a 10~ polyacrylamidegel and subjectedto autoradiography.The finalconcentration of a-amanitin(/tg/ml) is indicatedon top of each lane.
The copy number of 3ene for MRP RNA was estimated by hybridization with cloned DNA digested with another set of restriction enzymes as an internal standard for copy number. Data shown in Fig. 3B indicated that the intensity of the 1 kb Pstl band corresponds to a single copy of MRP RNA gene in a human haploid genome. These data suggest that the human genome contains one real gene and a few pseudogenes for MRP RNA. M R P / 7-2 RNA gene is transcribed in vitro by RNA polymerase I11 The sensitivity of the MRP RNA synthesis to aamanitin was used to evaluate the type of RNA polymerase involved in its synthesis. The human MRP RNA gene and 5S RNA gene were transcribed in vitro in the absence or in the presence of different concentrations of a-amanitin. The synthesized RNAs were analyzed on a polyacrylamide gel and visualized by autoradiography (Fig. 4). The 1 kb Pstl fragment containing 737 bp upstream and 28 bp downstream of the MRP gene (Fig. 1A and Fig. 3) contained enough information to direct accurate transcription in vitro. The pattern of inhibition of the synthesis of both the 5S RNA and MRP RNA at different concentrations of a-amanitin was very similar (Fig. 4).
37
The RNAs corresponding to MRP RNA and 5S RNA were excised from the gel and the amount of radioactivity was quantitated. The extent of inhibition at different concentrations of a-amanitin was the similar for MRP RNA and 5S RNA (Table I). There was little inhibition of MRP RNA or 5S RNA synthesis at a-amanitin concentrations of up to 1 /~g/ml and the synthesis was completely inhibited at 2 0 0 / t g / m l concentration (Fig. 4 and Table I). These data show that the cloned MRP DNA contains a transcribable MRP RNA gene and MRP RNA, like 5S RNA, is synthesized by poi III.
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5" Flanking sequences are required and sufficient for transcription of MRP / 7-2 gene in vitro Several potential cis-acting elements, like TATA box corresponding to - 2 5 and - 3 4 , PSE corresponding to - 5 7 and - 6 7 , octamer corresponding to - 2 0 8 and - 2 1 5 and SP1 corresponding to - 2 2 5 and - 2 3 1 , were observed in the 5' flanking region of human MRP gene [23]. The organization of these cis-acting elements in the MRP gene is very similar to that of the mammalian U6 snRNA gene and the 7SK snRNA gene. Since mammalian U6 and 7SK snRNA genes require 5' flanking sequences for transcription and contain no internal promoters [26,27], we tested whether the human MRP RNA gene requires 5' flanking sequences for transcription. A series of 3' deletions were made and these mutant templates were tested for their transcriptional abilities in vitro (Fig. 5). The original MRP-2 clone, having 737 bp 5, upstream sequence and 28 bp 3' downstream sequence, and 5' deletion clone MRP ( - 841 + 28) were transcribed with nearly equal efficiency in vitro (Fig. 6B). The mutant DNAs with 3' deletions to nucleotide 226 (lane 2), to nucleotides 114 (lane 3), to nucleotides 14 (lane 4), and even to nucleotides 2 (lane 5) can direct transcription in vitro. The transcripts were of the expected length, reading through the vector sequence and terminating in the T cluster present on Bluescript vector. The size of the transcripts from MRP ( - 7 3 7 / 1 4 ) construct (Fig. 5, lane 4) and M R P ( - 8 4 / 2 ) construct (Fig. 5, lane 5) was consistent with correct initiation. The DNA construct, containing sequences corresponding to 1-270 bp of MRP RNA and 28 bp of the 3' flanking sequences, was not transcribed in vitro (lane 6).
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Fig. 5. Transcription of human MRP RNA gene and deletion mutants in vitro. 3/~g each of plasmid DNAs indicated above the lanes were transcribed in vitro as described in Materials and Methods. The synthesized RNAs were analyzed on 10~ polyacrylamide gels. mU6, mouse U6 snRNA gene [30]; ShSS, Syrian hamster 5S RNA gene.
These data show that DNA corresponding to MRP RNA is not sufficient to direct transcription, and that 5' flanking sequences are required and these flanking sequences corresponding to - 84/2 bp are sufficient for transcription in vitro.
Upstream sequences ( - 85 to -737) of the MRP gene enhance the transcription in frog oocytes in the case of both U6 and 7SK snRNA genes the DSE element in the - 200 region was found to enhance the transcription in vivo [26,27]. To test whether the
TABLE !
Quanutation of radioactivity in MRP/7-2 and 5S RNAs synthesized in the presence of different concentrations of a.amanitin DNAused Human MRP/7-2 gene Syrian hamster 5S gene
Concentration of a-amanitin ( p g / m l ) 0
0.l
1.0
l0
1~
2~
998 (100) 2083 (100)
925 (95.4) 1907 (91.6)
874 (87.6) 1620 (77.8)
338 (33.9) 992 (47.6)
22 (2.2) 150 (6.7)
8 (0.8) 19 (0.9)
38
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Discussion
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Fig. 6. Synthesisof MRP RNA in frog oocytes and in vitro. (A) The transcriptionof plasmid DNAs in frog oocyte nucleiwas carried out as described by Kressmannet al. [31]. The amount of 5S DNA injectedwas 1/20 of the MRP DNAs.The DNA constructsused are indicated above the lanes: the 5S gene used is a Syrian hamster 5S gene [26]. (B) 3/~g each of MRP DNA constructswas transcribedin vitro as describedin Materialsand Methods.[a-~2PIGTPwas used as precursor and the synthesizedRlqAswere analyzedon 10¢gpolyacrylamidegels.
sequences in the MRP gene have a similar enhancing effect, two constructs of the MRP gene were injected into frog oocytes and the efficiency of transcription was determined. The MRP gene containing 737 bp upstream was transcribed more efficiently (Fig. 6A, lane 1) when compared to MRP gene with only 84 bp of upstream sequences (Fig. 6A, lane 2). In both the cases a 5S RNA gene, co-injected as an internal control, was transcribed wRh nearly equal efficiency (Fig. 6A, lanes 1 and 2). In contrast, both - 7 3 7 / + 28 and - 8 4 / + 28 MRP gene constructs were transcribed with nearly equal efficiency in vitro (Fig. 6B, lanes I and 2). These data show that in vivo the sequences upstream of - 8 4 of the MRP gene have important role in the synthesis of MRP RNA.
Results obtained in this study showed the following: (1) The human genome contains one true gene and a few pseudogenes for MRP RNA; and (2) The MRP RNA gene is transcribed by pol 111 and 5' flanking sequences are required and sufficient for transcription in vitro. Topper and Clayton [23] isolated and characterized an MRP gene from the human genome; this gene has been localized to the chromosome 9 [28]. Since our data, obtained with the Southern blot (Fig. 3B), indicate that human genome contains only one gene for MRP RNA, it is likely that the gene reported by Topper and Clayton [23] is the same that has been analyzed in this study. Results obtained earlier showed that the mouse 7-2 RNA in isolated nucleus is synthesized by pol III [4]. This conclusion was based on the sensitivity of M R P / 7 2 RNA synthesis in vivo to a-amanitin and the association of MRP RNA with La antigen in who!,~ cells. Based on the similarity of promoters between U6, 7SK, and MRP RNA genes, Topper and Clayton [23] predicted that the MRP RNA gene is a pol III gene. Results obtained in this study confirm this prediction and are consistent with the data obtained with the isolated nuclei by Hashimoto and Steitz [4]. Eukaryotic pol III genes represent a spectrum of at least three different promoter types: (1) the Xenopus 5S RNA gene where an internal control region is sufficient to direct transcription: (2) human 7SL RNA, and EBER-RNA genes where transcription requires both internal and 5' flanking sequences, and (3) U6 and 7SK RNA genes where 5' flanking sequences are sufficient to direct accurate transcription. The ability of MRP gene construct with 5' flanking region and only two nucleotides corresponding to the MRP RNA ( - 84/2) to support transcription indicates that the 5' flanking sequences are essential and sufficient for MRP gene transcription (Fig. 5, lane 5). Data that a DNA construct lacking 5' flanking sequences was not able to support transcription in vitro, are consistent with this notion (Fig. 5 lane 6). Transcription of the MRP gene constructs in frog oocytes (Fig. 6) showed that the sequences upstream of - 8 4 have important role in the synthesis of MRP RNA. These results suggested that the regulatory mechanism of MRP RNA gene appears to be similar to that of U6 and 7SK RNAs genes [26,27]. The upstream sequences of human and mouse MRP genes were found to be highly conserved. In this study the MRP gene with only 84 bp of upstream sequences was capable of directing transcription both in vitro and in frog oocytes. These data suggest that most of these conserved upstream sequences are not required for transcription. Our in vitro studies do not rule out the role(s) of MRP intragenic sequences, for instance box A homology, in
39 r e g u l a t i n g e x p r e s s i o n o f t h e gene. A in vivo a n a l y s i s is r e q u i r e d to test w h e t h e r o r n o t t h e M R P i n t r a g e n i c s e q u e n c e s p l a y a n y role(s) in M R P R N A g e n e regulation. T h e a m o u n t o f M R P / 7 - 2 R N A is g r e a t e r in r a p i d l y g r o w i n g cells like N o v i k o f f h e p a t o m a cells c o m p a r e d to rat liver [8]. Since t h e m o u s e g e n o m e [29] a n d t h e h u m a n g e n o m e ( t h i s s t u d y ) c o n t a i n a single g e n e for M R P R N A , the levels o f M R P / 7 - 2 RNA may be r e g u l a t e d at t h e level o f t r a n s c r i p t i o n . Acknowledgements T h e s e s t u d i e s were c a r r i e d o u t b y a g r a n t f r o m National Cancer Institute CA-10893 awarded by NIH, D e p t . o f H e a l t h a n d H u m a n Services. W e t h a n k D r . W i l l i a m F o l k for t h e c l o n e d 5S g e n e , M s . M i n y o n e F i n l e y for H e L a cells, D r . S h a s h i G u p t a for H e L a cell e x t r a c t , Ms. L a u r a L e o for s u p e r b t e c h n i c a l a s s i s t a n c e , a n d D r . H a r r i s B u s c h for e n c o u r a g e m e n t .
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9 Yuan. Y., Singh, R. and Reddy, R. (1989) J. Biol. Chem. 264, 14835-14839. 10 Chang, D.D. and Clayton, D.A. (1987) EMBO J. 6, 409-417. il Chang. D.D. and Clayton, D.A. (1987) Science 235, 1178-1184. 12 Airman, S., Gold, H.A. and Bartkiewicz, M. (1988) in Structure and Function of Small Ribonucleoproteins Ed. (Birnstiel, M.. ed.), pp. 183-195, Springer Verlag, Berlin. 13 Gold, H.A., Craft, J., Hardin, J.A., Bartkiewicz. S. and Aftman, S. (1988) Proc. Natl. Acad. Sci. USA 85, 5483-5487. 14 Baer, M., Nilse, T.W., Costigan, C. and Airman. S. (1990) Nucleic Acids Res. 18, 97-103. 15 Bartkiewicz. M., Gold, H. and AItman, S. (1989) Genes Dev. 3, 488-499. 16 Topper, J.N. and Clayton, D.A. (1990) J. Biol. Chem. 265. 13254! 3262. 17 Forster, A.C. and AItman, S. (1990) Cell 62, 407-409. 18 Feinberg, A.P. and Vogelstein, B. (1983) Anal, Biochemistry 132. 6-13. 19 Benton, W.D. and Davis, R.W. (1977) Science 196, 180-183. 20 Southern. E.M. (1975) J. Mol. Biol. 98, 503-517, 21 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74. 5463-5467. 22 Weil. P.A., Segall, J., Harris, B., Ng, S. and Roeder, R. (1979) J. Biol. Chem. 234, 6163-6173. 23 Topper, J.N. and Clayton. D.A. (1990) Nucleic Acids Res. 18, 793-799. 24 Rogers, J. (1985) Int. Rev. Cytol. 93, 187-279. 25 Weiner. A.M., Deininger, P.L. and Efstratiadis. A. (1986) Annu. Rev. Biochem. 55, 631-661. 26 Das, G., Henning, D., Wright, D. and Reddy, R. (1988) EMBO J. 7, 503-512. 27 Murphy, S., DiLiegro, C. and Melli, M. (1987) Cell 51, 81-87. 28 Hsieh, C.L., Donlon, T,A., Darras, B.T., Chang. D.D., Topper, J.N., Clayton, D.A. and Francke, U. (1990) Genomics 6, 540-544. 29 Chang, D.D. and Clayton, D.A. (1989) Cell 56, 131-139. 30 Ohshima, Y., Okada, N., Tani, T.. ltoh, V. and Itoh, M. (1981) Nucleic Acids Rcs. 9, 5145-5158. 31 Kressmann, A., Clarkson, S.G., Pirrotta, V. and Birnstiel, M.L. (1982) Proc. Natl. Acad. Sci. USA 75, 1176-1180.
5' Flanking sequences of human MRP/7-2 RNA gene are required and sufficient for the transcription by RNA polymerase Ill Yan Yuan and Ram Reddy Department of Pharmacololo; Baylor College of Medicine. Houston. TX (U S.A.I (Received 17 September 19901 (Revised manuscript received 30 November ! 990)
Key words: Mitochondrial RNA processing RNA; Transcription: RNA polymerase i11: Cloning: (Human)
Human mitochondrial RNA processing (MRP) RNA is a 270 nucicotide-long small RNA found as ribonucleoprotein particles. In this study, we isolated four human genomic clones with homology to human MRP RNA. Two of these doues contained one copy each of the real gene coding for human MRP RNA; the other two clones represented a processed pseudogene. The Southern blot with the genumic DNA showed that the haploid human genome contains o n e copy of real gene and a few pseudogenes for M R P / 7 - 2 RNA. The human MRP RNA is synthesized by R N A polymerase !!i and the 5 ' flanking sequences - 8 4 to 1 of MRP RNA gene, containing "rATA and PSE-like elements, a r e required and sufficient for transeription in vitro.
Introduction In eukaryotic cells, nucleoli contain many small RNAs in the form of small ribonucleoprotein particles; these include U3, U8, U13, U14 and 7-2 RNAs. U snRNAs in animal nucleoli contain trimethylguanosine cap structures, and are bound to a common 34 kDa protein termed "Fibrillarin' [1-3]. In contrast, 7-2 RNA, as well as RNaseP RNA in mammalian cells, are bound to a 40 kDa To antigen and contain pppG or pppA on their 5' ends [1,4-6]. The U3 RNP particles are required components for a sequence-specific cleavage of preribosomal RNA in the nucleolus [7]. The functions of U8, UI3, and U14 RNPs are not known. 7-2 RNA was initially found as a nucleolar RNA [8]. Using immunoclectron microscopy, Reimer et al. [5] showed that the 7-2 RNA is associated with the To antigen and is localized to the granular compartment of the nucleolus. The nucleolar function of 7-2 RNPs is
Abbreviations: bp, base pair(s), MRP, mitochondrial RNA processing; nt, nucleotide(s); ~ IlL RNA polymerase i11: RNP(s), ribonucleoprotein(s); snRNA(s), small nuclear RNA(s). Correspondence: R. Reddy, Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, U.S.A.
not known, even though it is presumed to be involved in the processing of preribosomal RNA [9]. Pioneering studies carried out by Clayton and his associates with the MRP (mitochondrial RNA processing) RNase showed that the MRP RNase in mitochondria is responsible for a sequence-specific cleavage of the mitochondrial RNA; this cleavage generates an RNA primer that is used as a primer for mitochondriai DNA replication [10,11]. Results obtained in our laboratory showed that rat nucleolar 7-2 RNA is homologous to mouse MRP RNA [9], and it appears that nucleolar 7-2 RNA and MRP RNA are identical [6]. These data show that MRP RNase is functional in mitochondria as well as in nucleofi. MRP RNPs share several features with RNaseP [6]; RNaseP is an endoribonudease that processes precursor tRNAs to generate tRNAs with mature 5' termini [12]. Both RNPs are immunoprecipitabic with anti-To/Th antibodies [1,4,6,13] and are synthesized by RNA polymerase ill (this study; Ref. 14). Both RNAs show primary sequence homology to each other in four conserved blocks [6] and these conserved sequence elements appear to reside in similar stems in the proposed secondary structures of MRP and human RNaseP RNAs [15,16]. All the above properties show that MRP RNase is analogous to RNaseP in structure and in function 116,17]. In the present study, human MRP/7-2 RNA gene
34 was isolated and characterized. The synthesis of MRP/7-2 RNA in vitro was investigated. The results show that the human genome contains one copy of MRP gene and is transcribed by RNA polymerase ill in vitro. Upstream regulatory elements are necessary and sufficient for transcription of this gene in vitro.
Human MRP/7-2 RNA was isolated from HeLa cell nuclei and purified as described previously [9]. Doublestranded MRP cDNA was synthesized using the following deoxyoligonucleotides: 5' ccgaattcgtgctgaaaggcctg 3' corresponding to nueleotides 2 to 18 and 5' gaatgagccccgtgtggt 3' complementary to 237 to 254 nueleotides of MRP RNA. ccgaa sequence in one of the above oligos is an extra linker sequence. The cDNA was cloned into the SmaI site of the Bluescript plasmid vector. MRP cDNA insert was purified on polyacrylamide gels and 32p-labeled by either multiprimer procedure [18] or Polymerase Chain Reaction (PCR) with [a-S2pkICTP as the precursor. The PCR mixture (50 pl) contained 1 pM each of unlabeled dATE dGTP, dTTP, 100/tCi [a-32P]dCTP (3000 Ci/mmol), 300 rig each of deoxyoligonueleotide and 5 ng of DNA template. The labeled probe was purified on a Sephadex G-25 column and used for hybridization.
M R P ( - 84/226) were prepared by PCR using deoxyoligonueleotides corresponding to nucleotides - 8 4 to - 6 2 and deoxyoligonucleotides complementary to nueleotides 2 to - 1 9 , 114-101 or 226-209 of MRP RNA, respectively. The fragments were cloned into Bluescript vector at Smal site. 3' deletion construct M R P ( - 7 3 1 / 1 - 1 4 ) was made by using a convenient Stul restriction site. The 745 bp-long Pstl/Stul fragment was subcloned into Pstl/Smal digested Bluescript plasmid. Analysis of DNA by Southern blotting. 10 pg of HeLa nuclear DNA or 0.1-0.5 /tg of cloned DNA was digested with the appropriate restriction endonucleases, fractionated on agarose gels, blots were prepared and probed by the method of Southern [20]. The hybridization conditions were the same as those employed for screening the genomic library. In vitro transcriptions. Whole HeLa cell extract was prepared by the method of Weil et al. [22]. 3 /tg of supercoiled plasmid DNA was incubated at 30°C for 1-3 h in a 50/tl reaction mixture containing 50% (v/v) HeLa cell extracts, 0.6 mM each of unlabeled CTP, UTP, ATP, 0.025 mM GTP, 5-50/tCi of [a-32p]GTP, 6 mM creatine phosphate, 20 mM KCI, 1 mM DTT and 10 mM Tris-HCi (pH 8.1). The transcribed RNAs were subjected to SDS/phenol extraction and precipitated with ethanol and fractionated by electrophoresis on 10% polyacrylamide/7 M urea gels.
Isolation of genomic clones containing human MRP / 7-2 RNA sequences. A human genomic library (ACTT
Results
Materials and Methods
Preparation of cDNA probes for MRP/7-2 RNA.
57760), prepared from lymph node DNA by partial Alul/HaeIll digestion and cloning into charon 4A phage DNA, was screened by the method of Benton and Davis [19]. Hybridization was done in 5 x SSC, 0.05% sodium pyrophosphate, 2 × Denhardt's solution, 0.1% SDS and 50 p g / m l of Escherichia coil tRNA. The nitrocellulose filters were prehybridized for 6 h, hybridized for 16 h and washed in 2 x SSC, 1 × SSC and 0.5 × SSC containing 0.1% SDS each for 15 min at 42°C. Recombinant A phage DNAs, containing sequences complementary to MRP cDNA, were mapped by Southern hybridization [20] and appropriate DNA restriction fragments were subcloned into Bluescript plasmid vector. DNA sequences were determined by the chain-termination method of Sanger et al. [21] using MI3 universal, reverse or synthetic deoxyoligonueleotide primers.
Isolation of human MRP / 7-2 RNA gene MRP RNA cDNA insert was isolated, labeled by PCR in the presence of [a-32p]dCTP and 400000 phage plaques from a human genomic library were screened. After the first screening, three strong positives and four weak positives were observed. A total of four clones, representing two strong (hMRP-1, hMRP-2) and hMRP-2 -
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Construction of MRP / 7-2 RNA gene deletion mutants. 5' deletion DNAs M R P ( - 8 4 / + 28), M R P ( - 6 / + 28) and M R P ( 1 - 2 7 0 / + 28) were prepared by amplifying plasmid DNA by PCR using deoxyoligonucleotides corresponding to nueleotides - 8 4 to - 6 2 , - 6 to 15 or 2 to 18 of MRP RNA and the M13 universal primer, digesting the amplified DNA with Pstl and inserting into Bluescript vector digested with Sinai and Pstl. 3" deletion constructs M R P ( - 84/2), M R P ( - 84/114) and
hMRP-4
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Fig. 1. Restficti,~n maps of human MRP RNA gene and pseudogene.
The data obtained from the Southern blots of cloned phase MRP-2 and MRP.4 DNA as well as from the genomicblots were used to construct this restrictionpattern surroundingthe human MRP gene and a pseudogene,hMRP, humanMRP.
35 MRP-2
-160 -140 -120 -100 A G C T A T T C T G C T A G C C A C A A T G C C T C T G A A A G C C T A T A G T CTTAGAAAGTTATGCCCGAA AACGGTTTTTTTAATCTCAC
MRP-2
-80 -60 -40 -20 -I G C C A C C A A C T T T C T C A C C C T A A T C A T A A A A C A C A A T T T C T TTAGGGCTATAAAATACTAC T C T G T G A A G C T G A G G A C G ~
[qRP-2
1 20 40 60 80 G T T C G T G C T G A A G G C C T G T A T C C T A G G C T A C A C A C T G A G G ACTCTGTTCCTCCCCTTTCC GCCTAGGGGAAAGTCCCCGG
MRP-2 MRP-4
100 120 140 160 A C C T C G G G C A G A G A G T G C C A C G T G C A T A C G C A C G T A G A C A TTCCCCGCTTCCCACTCCAA AGTCCGCCAAGAAGCGTATC G G A C A G A G G C C T T A A G T T T A G T A A T A G T A G G G A G A G T A G G G G G G C C T G G C G G T G C A C A T G ATATGAGGAAATGGCATCCA
MRP-2 MRP-4
180 200 22O 240 C C G C T G A G C G G C G T G G C G C G G G G G C G T C A T C C G T C A G C T C C C T C T A G T T A C G C A G G C A G T GCGTGTCCGCGCACCAACCA CAGGATCTGGCAGCTAGTTA AATGATGGAATTCAGAGAAC
MRP-2 P,H P - 4
260 +1 C A C G G G G C T C A T T C T C A G C G CGG~r(.~'I-FI-t-FtGT'rCC~A *G****AACG********** * * * * * * e * * l * ~
+28
GACCACGGCTCACTGCAG AAG~GAAJU~GAAAA
÷5O AGAAGAAGAAGAAAAGAAAA
Fig. 2. Nucleotide sequences of human MRP gene and a pseudogene. The top line shows the sequence of the human MRP gene (clone hMRP-2); the sequence of MRP pseudogene (clone MRP-4) is shown below the real gene sequence. Stars indicate identical bases and dots indicate deletions. The 5" flanking sequences are numbered from - 1 to - 1 6 0 , the transcribed portion of MRP RNA is numbered from I to 270 and the 3' flanking region from + 1 to + 28.
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Fig. 3. Southern blot analysis of human genomic D N A and ~timation of the gene copy number. (A) l0 ~g each of HeLa cell nuclear D N A was digested with restriction endonucleases indicated above each lane, fractionated on a 0.8~ agarose gel, transferred to a nitrocellulose filter and probed with labeled MRP eDNA. (B) Each lane contained 10/tg of HeLa nuclear D N A digested with Pstl, and mixed with 13 pg. 40 pg, or 80 Pig of cloned hMRP-2 plasmid D N A digested with Pstl and BstN! corresponding to 1, 3, or 6 copy equivalents, respectively, per haploid human genome.
36 two weak hybridization signals (hMRP-3, hMRP-4), were plaque-purified and characterized. The restriction map of the phage DNAs indicated that both the clones with strong hybridization signal are identical to each other; similarly, the two clones with weak homology to MRP DNA probe appeared to be from the same genomic locus. The restriction maps surrounding the MRP related sequences are shown in Fig. 1. A 1 kb Pstl fragment from hMRP-2 DNA, and 2.8 kb Pstl fragment from hMRP-4 were suhcloned into the Pstl site of Biuescript vector. The nucleotide sequence of MRP / 7-2 gene and pseudogene The nucleotide sequence of a portion of the I kb Pstl fragment from hMRP-2 clone was determined (Fig. 2) and was found to be identical to the human MRP gene recently reported by Topper and Clayton [23]. Since the human genome appears to contain a single gene for MRP RNA (see below), the gene characterized in this study and by Topper and Clayton [23] may be the same. The MRP homologous sequence from the clone MRP-4 was also sequenced and the sequence corresponding to the MRP RNA and the flanking regions are shown in Fig. 2. This pseudogene was severely truncated and contained only a small portion (68 bp) corresponding to the 3' end of MRP RNA. The nucleotide sequence showed approx. 85~ homology to the MRP RNA. The long stretch of poly(A) sequence in the pseudogene immediately following the 3' end of MRP RNA sequence (Fig. 2) indicates that this pseudogene may have arisen by reverse transcription of an RNA and retroposition into the genome (reviewed in Refs. 24,25). Human genome contains one real gene and few pseudogenes for MRP / 7-2 RNA To understand the genomic organization of human MRP RNA gene, HeLa cell nuclear DNA was digested with several restriction enzymes, fractionated on an agarose gel, transferred onto a nitrocellulose sheet and hybridized with labeled MRP DNA probe. With every digestion, a strong hybridization signal and one or more band(s) with less intensity were observed (Fig. 3A). The sizes of genomic DNA fragment hybridizing with maximum intensity, such as 1.0 kb Pstl fragment, and 1.5 kb BamHi fragment, were consistent with the restriction maps of hMRP-1 and hMRP-2 clones (compare Fig. 1A and 3A). These data, and the correspondence of this DNA sequence (Fig. 2) with human MRP RNA [6], suggested that these strongly hybridizing bands may correspond to true human MRP RNA gene. The other DNA fragments hybridizing with the MRP DNA probe probably correspond to pseudogenes with partial homologies to MRP DNA sequence; one clone representing these homologies was characterized by sequencing (Fig. 2).
a-amanltln ( I*g/ml)
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1 2 3 4 5 6 7 8 9 10 11 12 Fig. 4. Effect of a-amanitinon the synthesisof MRP and 5S RNAs. Human MRP RNA gene and Syrian hamster 5S RNA gene (1.5 Fg each) were transcribedin the presenceof different concentrationsof a-amanitin.The RNAs synthesizedwere fractionatedon a 10~ polyacrylamidegel and subjectedto autoradiography.The finalconcentration of a-amanitin(/tg/ml) is indicatedon top of each lane.
The copy number of 3ene for MRP RNA was estimated by hybridization with cloned DNA digested with another set of restriction enzymes as an internal standard for copy number. Data shown in Fig. 3B indicated that the intensity of the 1 kb Pstl band corresponds to a single copy of MRP RNA gene in a human haploid genome. These data suggest that the human genome contains one real gene and a few pseudogenes for MRP RNA. M R P / 7-2 RNA gene is transcribed in vitro by RNA polymerase I11 The sensitivity of the MRP RNA synthesis to aamanitin was used to evaluate the type of RNA polymerase involved in its synthesis. The human MRP RNA gene and 5S RNA gene were transcribed in vitro in the absence or in the presence of different concentrations of a-amanitin. The synthesized RNAs were analyzed on a polyacrylamide gel and visualized by autoradiography (Fig. 4). The 1 kb Pstl fragment containing 737 bp upstream and 28 bp downstream of the MRP gene (Fig. 1A and Fig. 3) contained enough information to direct accurate transcription in vitro. The pattern of inhibition of the synthesis of both the 5S RNA and MRP RNA at different concentrations of a-amanitin was very similar (Fig. 4).
37
The RNAs corresponding to MRP RNA and 5S RNA were excised from the gel and the amount of radioactivity was quantitated. The extent of inhibition at different concentrations of a-amanitin was the similar for MRP RNA and 5S RNA (Table I). There was little inhibition of MRP RNA or 5S RNA synthesis at a-amanitin concentrations of up to 1 /~g/ml and the synthesis was completely inhibited at 2 0 0 / t g / m l concentration (Fig. 4 and Table I). These data show that the cloned MRP DNA contains a transcribable MRP RNA gene and MRP RNA, like 5S RNA, is synthesized by poi III.
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5" Flanking sequences are required and sufficient for transcription of MRP / 7-2 gene in vitro Several potential cis-acting elements, like TATA box corresponding to - 2 5 and - 3 4 , PSE corresponding to - 5 7 and - 6 7 , octamer corresponding to - 2 0 8 and - 2 1 5 and SP1 corresponding to - 2 2 5 and - 2 3 1 , were observed in the 5' flanking region of human MRP gene [23]. The organization of these cis-acting elements in the MRP gene is very similar to that of the mammalian U6 snRNA gene and the 7SK snRNA gene. Since mammalian U6 and 7SK snRNA genes require 5' flanking sequences for transcription and contain no internal promoters [26,27], we tested whether the human MRP RNA gene requires 5' flanking sequences for transcription. A series of 3' deletions were made and these mutant templates were tested for their transcriptional abilities in vitro (Fig. 5). The original MRP-2 clone, having 737 bp 5, upstream sequence and 28 bp 3' downstream sequence, and 5' deletion clone MRP ( - 841 + 28) were transcribed with nearly equal efficiency in vitro (Fig. 6B). The mutant DNAs with 3' deletions to nucleotide 226 (lane 2), to nucleotides 114 (lane 3), to nucleotides 14 (lane 4), and even to nucleotides 2 (lane 5) can direct transcription in vitro. The transcripts were of the expected length, reading through the vector sequence and terminating in the T cluster present on Bluescript vector. The size of the transcripts from MRP ( - 7 3 7 / 1 4 ) construct (Fig. 5, lane 4) and M R P ( - 8 4 / 2 ) construct (Fig. 5, lane 5) was consistent with correct initiation. The DNA construct, containing sequences corresponding to 1-270 bp of MRP RNA and 28 bp of the 3' flanking sequences, was not transcribed in vitro (lane 6).
1
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2
3
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5
6
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Fig. 5. Transcription of human MRP RNA gene and deletion mutants in vitro. 3/~g each of plasmid DNAs indicated above the lanes were transcribed in vitro as described in Materials and Methods. The synthesized RNAs were analyzed on 10~ polyacrylamide gels. mU6, mouse U6 snRNA gene [30]; ShSS, Syrian hamster 5S RNA gene.
These data show that DNA corresponding to MRP RNA is not sufficient to direct transcription, and that 5' flanking sequences are required and these flanking sequences corresponding to - 84/2 bp are sufficient for transcription in vitro.
Upstream sequences ( - 85 to -737) of the MRP gene enhance the transcription in frog oocytes in the case of both U6 and 7SK snRNA genes the DSE element in the - 200 region was found to enhance the transcription in vivo [26,27]. To test whether the
TABLE !
Quanutation of radioactivity in MRP/7-2 and 5S RNAs synthesized in the presence of different concentrations of a.amanitin DNAused Human MRP/7-2 gene Syrian hamster 5S gene
Concentration of a-amanitin ( p g / m l ) 0
0.l
1.0
l0
1~
2~
998 (100) 2083 (100)
925 (95.4) 1907 (91.6)
874 (87.6) 1620 (77.8)
338 (33.9) 992 (47.6)
22 (2.2) 150 (6.7)
8 (0.8) 19 (0.9)
38
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Discussion
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Fig. 6. Synthesisof MRP RNA in frog oocytes and in vitro. (A) The transcriptionof plasmid DNAs in frog oocyte nucleiwas carried out as described by Kressmannet al. [31]. The amount of 5S DNA injectedwas 1/20 of the MRP DNAs.The DNA constructsused are indicated above the lanes: the 5S gene used is a Syrian hamster 5S gene [26]. (B) 3/~g each of MRP DNA constructswas transcribedin vitro as describedin Materialsand Methods.[a-~2PIGTPwas used as precursor and the synthesizedRlqAswere analyzedon 10¢gpolyacrylamidegels.
sequences in the MRP gene have a similar enhancing effect, two constructs of the MRP gene were injected into frog oocytes and the efficiency of transcription was determined. The MRP gene containing 737 bp upstream was transcribed more efficiently (Fig. 6A, lane 1) when compared to MRP gene with only 84 bp of upstream sequences (Fig. 6A, lane 2). In both the cases a 5S RNA gene, co-injected as an internal control, was transcribed wRh nearly equal efficiency (Fig. 6A, lanes 1 and 2). In contrast, both - 7 3 7 / + 28 and - 8 4 / + 28 MRP gene constructs were transcribed with nearly equal efficiency in vitro (Fig. 6B, lanes I and 2). These data show that in vivo the sequences upstream of - 8 4 of the MRP gene have important role in the synthesis of MRP RNA.
Results obtained in this study showed the following: (1) The human genome contains one true gene and a few pseudogenes for MRP RNA; and (2) The MRP RNA gene is transcribed by pol 111 and 5' flanking sequences are required and sufficient for transcription in vitro. Topper and Clayton [23] isolated and characterized an MRP gene from the human genome; this gene has been localized to the chromosome 9 [28]. Since our data, obtained with the Southern blot (Fig. 3B), indicate that human genome contains only one gene for MRP RNA, it is likely that the gene reported by Topper and Clayton [23] is the same that has been analyzed in this study. Results obtained earlier showed that the mouse 7-2 RNA in isolated nucleus is synthesized by pol III [4]. This conclusion was based on the sensitivity of M R P / 7 2 RNA synthesis in vivo to a-amanitin and the association of MRP RNA with La antigen in who!,~ cells. Based on the similarity of promoters between U6, 7SK, and MRP RNA genes, Topper and Clayton [23] predicted that the MRP RNA gene is a pol III gene. Results obtained in this study confirm this prediction and are consistent with the data obtained with the isolated nuclei by Hashimoto and Steitz [4]. Eukaryotic pol III genes represent a spectrum of at least three different promoter types: (1) the Xenopus 5S RNA gene where an internal control region is sufficient to direct transcription: (2) human 7SL RNA, and EBER-RNA genes where transcription requires both internal and 5' flanking sequences, and (3) U6 and 7SK RNA genes where 5' flanking sequences are sufficient to direct accurate transcription. The ability of MRP gene construct with 5' flanking region and only two nucleotides corresponding to the MRP RNA ( - 84/2) to support transcription indicates that the 5' flanking sequences are essential and sufficient for MRP gene transcription (Fig. 5, lane 5). Data that a DNA construct lacking 5' flanking sequences was not able to support transcription in vitro, are consistent with this notion (Fig. 5 lane 6). Transcription of the MRP gene constructs in frog oocytes (Fig. 6) showed that the sequences upstream of - 8 4 have important role in the synthesis of MRP RNA. These results suggested that the regulatory mechanism of MRP RNA gene appears to be similar to that of U6 and 7SK RNAs genes [26,27]. The upstream sequences of human and mouse MRP genes were found to be highly conserved. In this study the MRP gene with only 84 bp of upstream sequences was capable of directing transcription both in vitro and in frog oocytes. These data suggest that most of these conserved upstream sequences are not required for transcription. Our in vitro studies do not rule out the role(s) of MRP intragenic sequences, for instance box A homology, in
39 r e g u l a t i n g e x p r e s s i o n o f t h e gene. A in vivo a n a l y s i s is r e q u i r e d to test w h e t h e r o r n o t t h e M R P i n t r a g e n i c s e q u e n c e s p l a y a n y role(s) in M R P R N A g e n e regulation. T h e a m o u n t o f M R P / 7 - 2 R N A is g r e a t e r in r a p i d l y g r o w i n g cells like N o v i k o f f h e p a t o m a cells c o m p a r e d to rat liver [8]. Since t h e m o u s e g e n o m e [29] a n d t h e h u m a n g e n o m e ( t h i s s t u d y ) c o n t a i n a single g e n e for M R P R N A , the levels o f M R P / 7 - 2 RNA may be r e g u l a t e d at t h e level o f t r a n s c r i p t i o n . Acknowledgements T h e s e s t u d i e s were c a r r i e d o u t b y a g r a n t f r o m National Cancer Institute CA-10893 awarded by NIH, D e p t . o f H e a l t h a n d H u m a n Services. W e t h a n k D r . W i l l i a m F o l k for t h e c l o n e d 5S g e n e , M s . M i n y o n e F i n l e y for H e L a cells, D r . S h a s h i G u p t a for H e L a cell e x t r a c t , Ms. L a u r a L e o for s u p e r b t e c h n i c a l a s s i s t a n c e , a n d D r . H a r r i s B u s c h for e n c o u r a g e m e n t .
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