Bennett JL et al. (1992), Characterization of a Xenopus laevis ribonucleo...

XB-ART-23268

J Biol Chem 1992 Oct 25;26730:21765-72.

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Characterization of a Xenopus laevis ribonucleoprotein endoribonuclease. Isolation of the RNA component and its expression during development.

Bennett JL , Jeong-Yu S , Clayton DA .

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In order to facilitate studies of the assembly and transport of the site-specific RNase mitochondrial RNA processing (MRP) ribonucleoprotein, we have characterized it from Xenopus laevis cells. X. laevis RNase MRP displayed a similar spectrum of cleavage activity to that produced by previously isolated mammalian nuclear enzymes. A 277-nucleotide RNA component of the ribonucleoprotein was identified; the gene for the RNA was isolated, sequenced, and found to be 66 and 63% similar to mouse and human RNase MRP RNAs, respectively. Despite the evolutionary distance from its mammalian counterparts, X. laevis RNase MRP RNA contains five regions of homology to the mammalian RNase MRP RNA. Four of these regions correspond to those previously identified as conserved between RNase MRP and RNase P RNAs; the fifth encompasses nucleotides recently discovered to be sufficient for autoantigen binding. The expression and assembly of Xenopus RNase MRP RNA were examined in frog oocytes and developing embryos. RNase MRP RNA was expressed throughout oogenesis; it started to accumulate at stage I and reached a maximum in stage IV. During embryogenesis RNase MRP RNA expression began to elevate at approximately stage 22 and continued to rise through the swimming tadpole stage. When injected into the nucleus of mature oocytes, the X. laevis RNase MRP RNA gene was expressed accurately, and transcripts were packaged into immunoprecipitable particles.

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Species referenced: Xenopus laevis
Genes referenced: abcc1 dse pdgfb sp1 wls

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	FIG. 1. Xenopus nuclear extracts contain an RNase MRP cleavage activity. XenopuT nuclear extracts were subjected to glycerol gradient sedimentationa s described under “Materials and Methods.” Aliquots from individual fractions were assayed for RNase MRP activity on the standard mouse mtRNA substrate. The full length mouse mtRNA substrate, as well as the major cleavage products, are depicted schematically on the left as previously described (Karwan et al., 1991). The radioactive end label is schematically depicted at the 3’-end of the RNA. The two arrowheads denote cleavage products produced by the Xenopus RNase MRP activity on m tohues e substrate that are not seewn ith mouse RNase MRP; thbea sis fort his difference is currently unknown. Lune M, HpaII-digested pBR322 DNA markers. The directiono f sedimentation in the gradient isd epicted below.
	FIG. 2. Xenopus cells contain a Th-immunoprecipitable RNA which cosediments with RNase MRP activity. A, Xenopus nuclear extracts were prepared as described under “Materials and Methods.” Extracts (100 pl) were mixed with either normal human serum or Th autoantiserum, washed extensively in either 150 or 500 mM NaCI, and the coprecipitating RNAs were 3”end-labeled with pCp and separated on a 6% acrylamide-7 M urea denaturing gel. Lane 1, antibody: normal human serum, wash: 150 mM NaCI; lane 2, Th- 150 mM NaCI; lane 3, normal human serum-500 mM NaCI; lane 4, Th-500 mM NaCI; lane C, Th immunoprecipitation of mouse nuclear extract. Arrowhead denotes the -280-nucleotide RNA precipitated and RNase MRP RNAs are denoted at left. R, 150-pl aliquots of from Xenopus nuclear extracts with Th antiserum. Mouse RNase P individual fractions from the glycerol gradient in Fig. 1 were subjected to anti-Th immunoprecipitation, and the resulting RNAs were PCPend labeled and separated on a denaturing gel; lane L, Th immunoprecipitation of the total nuclear extract; lane M, HpalI-digested pBR322 DNA markers. The directioonf sedimentation in the gradient is depicted below. Arrowhead denotes the 280-nucleotide RNA.
	FIG. 3. Nucleotide sequence of the sequence of a 983-bp region corresponding to nucleotides -490 to +493 of the Xenopus RNase MRP RNA gene is shown. The 277-nucleotide MRP RNA is depicted in uppercase letters; the transcriptional start site is marked by a bent arrow. Putative transcriptional control elements are labeled with brackets as follows: SPI, SP1 transcription factor binding site; DSE, distal sequence element; PSE, proximal sequence element; TATA, TATA box; BOXA, RNA polymerase I11 box A sequence. Boxed sequences marked I, 11, 111, IV, and V are regions highly conserved among RNase MRP RNAs sequenced to date (see text for discussion). Stars highlight nucleotides proposed to be involved in a pseudoknot (Topper and Clayton, 1990b).
	FIG. 4. Expression of MRP RNA. A, Northern blot analysis of MRP RNA expression during Xenopus development. All lanes contain total RNA from 2.5 oocytes or 10 pg of total RNA from embryos. Total RNA was fractionated in a 6% polyacrylamide-7 M urea gel and blot-hybridized with MRP RNA antisense probe as described under “Materials and Methods” (upper panel)T. he full length MRP RNA is shown in most lanes; some degradation products are seen in oocyte lanes. The same membranes were rehybridized with the 5.8s rRNA antisense probe (lower panel). Specific activities of the two probes were similar; however, exposure times were 1-2 days for the MRP probe hybridization and 10-20 min for the 5.8s rRNA probe. Oocyte stages are according to Dumont (stages I thru VI) (Dumont, 1972); total ovary (0) was also loaded. Embryo stages are as follows: E, unfertilized eggs; 9, blastula; 11, gastrula; 13-16, neurula; 22, late neurula; 38 and 45, tadpoles. €3, graphic representation of MRP RNA expression. Northern blots as shown in A were scanned with a densitometer. Relative amounts of MRP transcripts were compared to that of stage VI oocytes and plotted at the various stages of development (MRP, A). The amount of MRP transcript was normalized to the amount of 5.8s rRNA in each stage from the same membrane, then compared with the value from stage VI oocytes (MRP/5.8S, W). In the oocyte graph, three independent experiments were pooled, and the mean values are plotted along with the standard deviation. A representative set of data from three different frog developments is shown in the embryo graph.
	FIG. 5. Immunoprecipitation of the RNase MRP particle from stages I and VI oocytes and tissue culture cells. Whole cell extracts were prepared from 75 stage I and 10 stage VI oocytes and immunoprecipitated with normal human serum (NHS lanes) or anti-Th human autoimmune serum (7% lanes). Ten pl each of the nuclear extracts of Xenopus (XTC) and mouse (LA9) cells were also immunoprecipitated with Th antiserum as controls. Pansorbin was used to precipitate immune complexes in this experiment. Bound (P, pellet) and unbound (S, supernatant) fractions were extracted, and coprecipitating RNAs were electrophoresed in a 6% polyacrylamide- 7 M urea gel and subsequently blot-hybridized with a MRP RNA probe. Arrowheads indicate the full length MRP RNA.
	FIG. 6. Analysis of transcripts after injection oft he MRP RNA gene intoo ocytes. A , Northern blot of time-course injection. DNA was injected into the nuclei of oocytes, and total RNA preparations were isolated after the indicated times of incubation. In each case RNA from four oocytes was isolated, fractionated in a 6% polyacrylamide-7 M urea gel, and blot-hybridized with the digoxigenin-labeled MRP antisense probe as described under “Materials and Methods.” Since a short exposure is shown here, a very low level of endogeneous MRP RNA is visible in this figure (see 0-h time point). The full length MRP RNA is indicated by the arrowheads. B, primer extension of MRP RNA transcribed from the injected DNA. Total RNA was prepared from the oocytes 5 h after injection and subsequently extended with reverse transcriptase after hybridization to the XLPE oligonucleotide. Lune I , 12 pg of XTC total RNA; lane 2, 2 pg of injected oocyte total RNA. Sequencing ladders were generated by primer extension using the oligonucleotide XLPE. The 5’-end guanosine nucleotide is indicated by the asterisk. C, immunoprecipitation of MRP RNA from injected oocytes. Either nucleus (NIX)cy,to plasmic (Cyt), or whole cell ( WC) extracts from six oocytes were prepared 16 h after injection of the MRP RNA gene. Immunoprecipitation with Th-antiserum and RNA detection were as described for Fie. 5. MRP RNA is visualized bv Northern hybridization. Both pellet (P) and supernatant (S) fractions are shown. Arrowheads indicate the fill length MRP RNA.