Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305-8572, Japan1
Department of Molecular and Cellular Biology, The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, MA 01238, USA2
Author for correspondence: Kouji Nakamura. Tel: +81 298 53 6419. Fax: +81 298 53 7723. e-mail: nakamura.kouji{at}nifty.ne.jp
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ABSTRACT |
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Keywords: non-coding RNA, RNA processing, RNA secondary structure
Abbreviations: scRNA, small cytoplasmic RNA; sRNA, small RNA; tmRNA, transfer-message RNA
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INTRODUCTION |
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Signal recognition particle (SRP) RNA is involved in the translocation of proteins across the endoplasmic reticulum (Stroud & Walter, 1999 ). SRP RNAs are ubiquitous in nature and contain evolutionarily conserved structural features (Poritz et al., 1988
; Gorodkin et al., 2001
). The 4·5S RNA of E. coli and the small cytoplasmic RNA (scRNA) of Bacillus subtilis belong to a family of SRP RNAs that function in protein secretion as an integrated component of the SRP-like particle along with an Ffh protein that is a homologue of one of the mammalian SRP components, SRP54 (Bernstein et al., 1989
). However, the earliest defect seen in cells conditionally depleted of 4·5S RNA or scRNA is the inhibition of protein synthesis both RNA molecules are normally associated with ribosomes (Brown, 1987
; Bourgaize & Fournier, 1987
; Nakamura et al., 1999
). These results suggest a role for scRNA and 4·5S RNA in translation. Elongation factor G (EF-G) is a 4·5S RNA-binding protein, and a depletion of 4·5S RNA causes an increase in the amount of EF-G associated with ribosomes, suggesting that 4·5S RNA is involved in EF-G recycling (Brown & Fournier, 1984
; Jovine et al., 2000
; Shibata et al., 1996
). Therefore, 4·5S RNA affects both protein synthesis and secretion, but the exact mechanism of its action in each pathway is, as yet, unclear.
In contrast to E. coli, only three sRNAs besides 5S rRNA and tRNAs have been identified in B. subtilis. Genomic sequence data and new algorithms have allowed the development of systematic screens for non-coding sRNA genes (Le et al., 1989 ; Chen et al., 1990
; Dandekar & Hentze, 1995
; Lowe & Eddy, 1997
). The present study identifies and characterizes two novel sRNAs in B. subtilis named BS190 RNA and BS201 RNA and shows that the smaller, mature BS190 RNA is generated by processing its precursor, BS201 RNA, at the 5' end. This study also shows that a reduction in the level of BS190 RNA expression leads to defective vegetative growth.
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METHODS |
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Preparation and purification of RNAs from B. subtilis.
Total RNA was isolated from frozen cells essentially as described by Sambrook et al. (1989) and Nuyts et al. (2001)
. Frozen cells (0·1 g, wet weight) were vigorously shaken in 2 ml phenol/chloroform (1:1, v/v) containing 2·4 g glass beads. Total RNA was loaded into the wells of 6% denaturing polyacrylamide gels (48 cm height) in 6 M urea and then resolved by electrophoresis for 14 h at 500 V. After this time, the gels were stained with ethidium bromide and visualized under a UV light. Two bands of about 200 nt in size (estimated by comparison with molecular markers) were excised from the gels and dialysed against 0·8 ml of 0·5xTBE buffer [45 mM Tris/HCl (pH 8·0), 45 mM boric acid, 1 mM EDTA]. RNAs were electro-eluted from the gel and purified as described above.
Cloning and sequencing of the BS190 RNA and BS201 RNA genes.
Both the BS190 RNA and the BS201 RNA, which had been purified from denaturing polyacrylamide gels and polyadenylated at their 3' termini using poly(A) polymerase (Takara-Shuzo), were used as templates for the reverse transcription of single-stranded DNA copies using the oligomer (dT)25 as a primer. The first strand of DNA was synthesized using RNA-dependent DNA polymerase (reverse transcriptase; Takara-Shuzo). The product of the first-strand synthesis was treated with RNase H (Takara-Shuzo), and the second strand of DNA was constructed using E. coli DNA polymerase I (Takara-Shuzo). The DNA fragment generated was purified and then inserted into the HincII site of pUC118. The hybrid plasmid was used to transform E. coli JM109 as described above. Clones containing the BS190 RNA gene or the BS201 RNA gene were designated pTUE190 and pTUE201, respectively. Both strands of the DNA fragment were shotgun sequenced by chain termination using the Dye Terminator Cycle Sequencing Kit (Applied Biosystems). The two gene fragments that were cloned into pTUE190 and pTUE201 covered the 5' ends of BS190 RNA and BS201 RNA, respectively, according to a comparison with the 5' end of each RNA determined by primer extension.
Mapping the 5' ends of the BS190 RNA and BS201 RNA genes.
This was done by primer extension. Primer P1 (5'-CGCCATTTAAAAATGCGGGC-3'; positions 542523, Fig. 1B) was labelled by 5'-end phosphorylation using [
-32P]ATP in the presence of polynucleotide kinase (TOYOBO); the subsequent extension was done essentially as described previously (Nakamura et al., 1994
) in a reaction mixture containing 1 pmol of labelled P1, 1090 µg of total RNA or 0·2 µg of gel-purified RNA, 1 mM of each dNTP and 25 U of AMV reverse transcriptase (Takara-Shuzo) in 20 µl of the buffer supplied by the manufacturer. The oligonucleotide and the RNA were mixed and then ethanol precipitated. The precipitate was resuspended in 10 µl TE buffer [Tris/HCl (pH 7·5), 1 mM EDTA] and incubated at 70 °C for 5 min and slowly cooled to 42 °C. The other components of the reaction mixture were added to the suspension, and the mixture was incubated at 42 °C for 60 min. The reaction was stopped by a 5 min incubation at 95 °C. Formamide loading buffer was added to the samples; the reaction products were then separated through polyacrylamide sequencing gels containing 6 M urea. The gels were dried and the bands were visualized by autoradiography. Primer-extension products were identified on the gels by comparing the bands generated with those of sequencing reactions generated with the same labelled oligonucleotide and a Sequencing Kit (Takara-Shuzo). The templates for sequencing reactions were M13 phage clones containing the wild-type B. subtilis BS190 RNA gene.
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Northern blotting.
RNA was isolated from cell lysates prepared from B. subtilis cells at the vegetative phase of growth. After electrophoresis on a sequencing gel, the RNA was electrotransferred onto Gene Screen nylon membranes (NEN Research Products) and hybridized with the 5'-end 32P-labelled 300 bp DNA fragment containing the BS190 RNA coding sequence that was amplified by using the synthetic primers P3 (5'-ATTTGCAGTTCGATTC-3') and P4 (5'-ATAAAATGGCTGATCC-3').
Construction of the deletion mutant.
The BS190 RNA mutant strain was constructed as follows. A 378 bp fragment (UP; from +48 to +411, Fig. 1B
) of the BS190 RNA locus, including the 3' region of aspS, and a 576 bp fragment (DOWN; from +663 to +1218, Fig. 1B
) in front of the 5' region of yrvM were amplified from B. subtilis chromosomal DNA using Pup-1 (5'-GATCAAGCTTATGACCTCGTCTTAAAC-3') and Pup-2 (5'-GATCGAATTCCTGCAAATTCAGTTCTTAAC-3'), and Pdown-1 (5'-GATCGAATTCGTGCGCAGAAAAAACGGCTG-3') and Pdown-2 (5'-GATCGGATCCTATCAGCGATTTGGAAGC-3'), respectively (Fig. 1
). After amplification, the UP fragment was digested with HindIII and EcoRI and the DOWN fragment was digested with EcoRI and BamHI restriction sites for these enzymes were located within each primer used for PCR (shown in bold). The two fragments were ligated and inserted into the HindIII and BamHI sites of pBluescript II SK-. The resulting recombinant plasmid DNA was digested with EcoRI and then ligated with a kanamycin-resistance cassette that was prepared from pDG783 (Guerout-Fleurgy et al., 1995
) by digestion with EcoRI. Plasmid DNA from positive clones selected in E. coli for kanamycin-resistance were isolated. The DNA fragment containing the BS190 RNA gene locus in which the BS190 RNA gene was replaced with the kanamycin-resistance gene was isolated by PCR using primers Pup-1 and Pdown-2 and used to transform competent B. subtilis wild-type cells. Isolates exhibiting kanamycin-resistance resulted from double-crossover events and were confirmed by Southern hybridization (data not shown). Northern blots demonstrated the absence of BS190 RNA in these isolates (Fig. 2A
), which were referred to as the
BS190 RNA mutant.
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RESULTS AND DISCUSSION |
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Subcellular localization of BS190 RNA
Among the sRNAs that have been characterized previously, several have been implicated in translation (Altuvia et al., 1998 ; Majdalani et al., 1998
; Muto et al., 1998
; Tetart & Bouche, 1992
). To examine the possibility that either BS190 RNA or BS201 RNA is associated with ribosomes, cell lysates prepared from exponentially growing cells were separated by sucrose density-gradient centrifugation. RNA was prepared from each fraction and Northern blotted to detect BS190 RNA and BS201 RNA. Quantitative densitometric data indicated that approximately 60% of the total BS190 RNA sedimented with polysomes (Fig. 6B
, inset) and about 30% was detected in the 70S monomeric ribosomes (Fig. 6
). No band corresponded to BS201 RNA in neither the 70S monosomal (Fig. 6
) nor the polysomal fraction (Fig. 6
, lane 2, inset). These results suggest that BS190 RNA specifically associates or interacts with ribosomes during translation. Consequently, BS190 RNA, rather than BS201 RNA, is active or functional.
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To date, at least 10 sRNAs have been identified in E. coli. Recently, 24 candidate intergenic regions in E. coli have been proposed as possibly encoding novel sRNAs (Wassarman et al., 2001 ). Of these 24 intergenic regions, Wassarman et al. (2001)
confirmed that 14 encode sRNAs, by using micro-array technology and Northern hybridization. Based on the DNA sequence data for the whole genome of B. subtilis, we selected 123 regions of >500 nt in length as candidates for non-coding RNA-encoding regions in B. subtilis. These regions were located between two ORFs, but they lacked a protein-coding capacity and a ShineDalgarno sequence. Among these regions, we have found (by using Northern blotting) that the yocIyocJ intergenic region can produce an sRNA of 203 nt (Ando et al., 2002
). However, we have not found a sequence corresponding to this RNA adjacent to the E. coli homologues of yocI and yocJ.
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ACKNOWLEDGEMENTS |
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Received 6 February 2002;
revised 25 April 2002;
accepted 1 May 2002.