Plant dicistronic tRNA-snoRNA genes: a new mode of expression of the small nucleolar RNAs processed by RNase Z

Abstract

Small nucleolar RNAs (snoRNAs) guiding modifications of ribosomal RNAs and other RNAs display diverse modes of gene organization and expression depending on the eukaryotic system: in animals most are intron encoded, in yeast many are monocistronic genes and in plants most are polycistronic (independent or intronic) genes. Here we report an unprecedented organization: plant dicistronic tRNA-snoRNA genes. In Arabidopsis thaliana we identified a gene family encoding 12 novel box C/D snoRNAs (snoR43) located just downstream from tRNA(Gly) genes. We confirmed that they are transcribed, probably from the tRNA gene promoter, producing dicistronic tRNA(Gly)-snoR43 precursors. Using transgenic lines expressing a tagged tRNA-snoR43.1 gene we show that the dicistronic precursor is accurately processed to both snoR43.1 and tRNA(Gly). In addition, we show that a recombinant RNase Z, the plant tRNA 3' processing enzyme, efficiently cleaves the dicistronic precursor in vitro releasing the snoR43.1 from the tRNA(Gly). Finally, we describe a similar case in rice implicating a tRNA(Met-e) expressed in fusion with a novel C/D snoRNA, showing that this mode of snoRNA expression is found in distant plant species.

Fig. 1. Genomic organization of the tsnoR43 dicistronic genes. (A) Sequence alignment of the tsnoR43 genes. The tRNA^Gly sequences are in the Arabidopsis tRNA databanks (see Materials and methods for addresses). SnoRNAs were predicted by SnoScan (Lowe and Eddy, 1999). Divergent nucleotides are in red. Grey boxes indicate functional elements on the tRNA and snoRNA. Consensus sequences for tRNA A and B boxes (Geiduschek and Tocchini-Valentini, 1988) and snoRNA C and D boxes (Bachellerie and Cavaillé, 1998) are indicated below. The blue overline indicates the rRNA antisense element of snoR43.1 targeting 18S:A543 and blue nucleotides display rRNA/snoRNA pairing. IR indicates inverted repeats forming a terminal stem on C/D snoRNAs. The RT–PCR sense primer p1 annealing to the tRNA and the 5′ ends of the reverse primers p1r, p11 and p12 are underlined with arrows. (B) Chromosomal location of tsnoR43 genes. The 12 dicistronic tsnoR43 genes are shown in red. The 13 single tRNA^Gly genes are shown in grey. The name of the BAC containing these genes is given in parentheses in each case. Lines link isoforms located in large chromosomal duplications shown by the shaded regions (Blanc et al., 2000). (C) The 18S:A543 is 2′-O-methylated in the 18S rRNA. Methylation of 18S:A543 was detected by primer extension on total RNA at low dNTP concentration (Maden, 2001). Prematurely reverse transcriptase arrest one nucleotide before the methylated residue is shown by an asterisk. The 18S rRNA/snoR43.1 duplex is shown, and the target A543 is indicated by a black circle. (D) Expression of tsnoR43 genes detected by RT–PCR. The RT–PCR on total RNA from Arabidopsis seedlings was performed with p1 and each of the reverse primers as indicated: +RT and –RT refer to the presence and absence, respectively, of reverse transcriptase in the RT–PCR assay. The gDNA is a PCR reaction performed with genomic DNA as template for a size control to compare with the RT–PCR product. M is a DNA 100 bp ladder.

Fig. 1. Genomic organization of the tsnoR43 dicistronic genes. (A) Sequence alignment of the tsnoR43 genes. The tRNA^Gly sequences are in the Arabidopsis tRNA databanks (see Materials and methods for addresses). SnoRNAs were predicted by SnoScan (Lowe and Eddy, 1999). Divergent nucleotides are in red. Grey boxes indicate functional elements on the tRNA and snoRNA. Consensus sequences for tRNA A and B boxes (Geiduschek and Tocchini-Valentini, 1988) and snoRNA C and D boxes (Bachellerie and Cavaillé, 1998) are indicated below. The blue overline indicates the rRNA antisense element of snoR43.1 targeting 18S:A543 and blue nucleotides display rRNA/snoRNA pairing. IR indicates inverted repeats forming a terminal stem on C/D snoRNAs. The RT–PCR sense primer p1 annealing to the tRNA and the 5′ ends of the reverse primers p1r, p11 and p12 are underlined with arrows. (B) Chromosomal location of tsnoR43 genes. The 12 dicistronic tsnoR43 genes are shown in red. The 13 single tRNA^Gly genes are shown in grey. The name of the BAC containing these genes is given in parentheses in each case. Lines link isoforms located in large chromosomal duplications shown by the shaded regions (Blanc et al., 2000). (C) The 18S:A543 is 2′-O-methylated in the 18S rRNA. Methylation of 18S:A543 was detected by primer extension on total RNA at low dNTP concentration (Maden, 2001). Prematurely reverse transcriptase arrest one nucleotide before the methylated residue is shown by an asterisk. The 18S rRNA/snoR43.1 duplex is shown, and the target A543 is indicated by a black circle. (D) Expression of tsnoR43 genes detected by RT–PCR. The RT–PCR on total RNA from Arabidopsis seedlings was performed with p1 and each of the reverse primers as indicated: +RT and –RT refer to the presence and absence, respectively, of reverse transcriptase in the RT–PCR assay. The gDNA is a PCR reaction performed with genomic DNA as template for a size control to compare with the RT–PCR product. M is a DNA 100 bp ladder.

Fig. 2. Rice tRNA^Met-e–snoRNA genes. (A) Chromosomal organization of tRNA^Met-e–snoR1/snoR2 genes in chromosome 1. The putative TIS and terminator elements of Pol III are indicated by CAA and TT, respectively. Arrows indicate primers used for RT–PCR and the size of the expected product is given in nucleotides. The figure is not to scale. (B) Alignment of tsnoR1 and tsnoR2 gene sequences. Large white and grey boxes indicate the mature tRNA^Met-e and snoRNAs, respectively. Conserved functional elements of the tRNA and snoRNA are depicted as in Figure 1A. The guide element of snoR1 with nucleotides pairing to rRNA is shown by thick overline. No rRNA antisense element can be found in snoR2. Primers used for RT–PCR are underlined by arrows. (C) Detection of the tRNA^Met-e–snoR1 dicistronic precursor. RT–PCR with total RNA from rice seedlings was performed with the pair of primers indicated in (A); +RT and –RT refer to the presence and absence, respectively, of reverse transcriptase in the RT–PCR assay. gDNA indicates a PCR using genomic DNA from rice as template. M is the DNA 100 bp ladder.

Fig. 3. Characterization of tsnoR43.1 gene transcripts produced in vivo. (A) Schematic structure of the cloned tsnoR43.1 gene. Genomic sequences from –85 to +212 relative to TIS were inserted in pGEM-T Easy vector. Black boxes indicate vector sequences. White and grey boxes indicate the tRNA^Gly and predicted snoR43.1 with their terminal sequences. CAA and T–T indicate the predicted TIS and T-run terminator. For RNase protection the RNA probe complementary to the genomic sequence was transcribed from the SP6 promoter to the SphI site, as indicated. The fragments protected from RNase digestion are shown by horizontal lines with their sizes given in nucleotides. The primer used for reverse transcription is shown by an underlining arrow annealing to AtsnoR43.1. The positions of the primer extension stops are indicated by asterisks. Letters p and m indicate the 5′ end of precursor and mature products, respectively. The figure is not to scale. (B) RNase A/T1 mapping. For RNase protection assay the antisense RNA probe was hybridized to total RNA from Arabidopsis seedlings (At) or yeast tRNA (Sc). Upon RNase treatment, products were separated on a sequencing gel. C is a control with an untreated full-length probe. M shows DNA size markers in nucleotides. A shorter time exposure at the bottom of the gel is shown. (C) Primer extension analysis. Reverse transcription was performed from a radiolabelled primer annealing to snoR43.1 in total RNA from Arabidopsis seedlings (At) or yeast tRNA (Sc). A shorter time exposure at the bottom of the gel is shown to improve visualization of the mature snoRNA ends. A gel with a longer electrophoresis run was done to map the precursor 5′ end better. Letters p and m indicate precursor and mature products, respectively. The position of the primer extension signal is shown by an asterisk. X is an unidentified signal. CTAG is the sequence of the tsnoR43.1 gene. The tRNA and snoRNA terminal sequences are indicated by white and grey boxes, respectively, on the right of the gels.

Fig. 4. Expression of tagged tRNA^Gly.t–snoR43.s in transgenic Arabidopsis lines. (A) Structure of the tagged tRNA^Gly.t–snoR43.s transgene. The TIS, nucleotide +1, is indicated by an arrow. The conserved elements controlling tRNA gene expression and snoRNA accumulation are indicated. White and grey boxes indicate the mature tRNA and snoRNA with their terminal sequences, respectively. The terminal inverted repeats of snoR43.1 are indicated by thick overlines. The tag t inserted in the GCC anticodon and the tag s replacing the antisense rRNA element adjacent to the D′ box are in lower-case letters. Black boxes indicate T-DNA flanking sequences. Four transgenic lines were produced: s-tagged L1.s and L2.s and ts-tagged L3.ts and L4.ts. Primers used for RT–PCR and primer extension analysis are indicated by p1, pt and ps. The asterisks indicate the positions of the primer extension signals detected in (C). (B) Detection of the dicistronic tagged precursors by RT–PCR. The assay was performed using primers p1/ps on total RNA extracted from transgenic lines L1.s, L2.s, L3.ts and L4.ts or wild-type (WT) non-transgenic plants as indicated. C is a control RT–PCR using total RNA from L1.s as substrate but omitting reverse transcriptase in the reaction. M indicates 100 bp DNA size markers. (C) Detection of tagged AtsnoR43.s and tRNA^Gly.ts by primer extension. Reverse transcription was performed with primers ps or pt and total RNA from transgenic lines L1.s and L3.ts and non-transgenic lines WT, as indicated. Sc is a control with yeast tRNA. CTAG is the sequence ladder of tRNA^Glyt–snoR43.s gene. The tRNA and snoRNA sequences are indicated by white and grey boxes, respectively, on the left of the gels. The asterisks indicate the position of the extension signals.

Fig. 5. Cleavage of the tRNA–snoRNA precursor by recombinant RNase Z in vitro. (A) Structures of RNA substrates used for processing. All substrates contain tRNA (grey cloverleaf) and snoRNA (black stem loop). Substrates differ in the presence of the 5′ leader and 3′ trailer sequences shown by thin lines. (B) Processing of RNA substrates Rib3 and Rib4. Precursor RNAs Rib4 (5′ and 3′ end matured) and Rib3 (5′ matured) were incubated with rRNase Z for 10 min and 15 min as indicated. C are control reactions performed without addition of proteins. M is the DNA size marker given in nucleotides on the left. Precursor and products are shown schematically at the sides. (C) Primer extension analysis of the rRNase Z cleavage site on Rib4 substrate. Unlabelled Rib4 substrate was incubated in vitro with or without rRNase Z as indicated. After incubation, products of the reaction were extracted and analysed by primer extension with a radiolabelled primer snoR4 annealing to the snoRNA. ACGT refers to sequencing reactions started from primer sno4. The position of the cleavage site on the coding strand is indicated with an arrow. The small arrow indicates a minor cleavage site.

Fig. 6. Model for the expression of tRNA–snoRNA dicistronic genes in plants. The different activities predicted as participating in gene transcription and processing of the tRNA^Gly–snoR43.1 precursor are indicated. The assembly with the core conserved C/D snoRNP proteins may occur prior to 3′ end trimming to protect snoRNA from exonucleolytic degradation (Terns and Terns, 2002).