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. 2008 Oct;36(18):5832-44.
doi: 10.1093/nar/gkn549. Epub 2008 Sep 12.

Dicistronic tRNA-5S rRNA genes in Yarrowia lipolytica: an alternative TFIIIA-independent way for expression of 5S rRNA genes

Joël Acker  1 , Christophe Ozanne, Rym Kachouri-Lafond, Claude Gaillardin, Cécile Neuvéglise, Christian Marck
Affiliations

Dicistronic tRNA-5S rRNA genes in Yarrowia lipolytica: an alternative TFIIIA-independent way for expression of 5S rRNA genes

Joël Acker et al. Nucleic Acids Res. 2008 Oct.

Abstract

In eukaryotes, genes transcribed by RNA polymerase III (Pol III) carry their own internal promoters and as such, are transcribed as individual units. Indeed, a very few cases of dicistronic Pol III genes are yet known. In contrast to other hemiascomycetes, 5S rRNA genes of Yarrowia lipolytica are not embedded into the tandemly repeated rDNA units, but appear scattered throughout the genome. We report here an unprecedented genomic organization: 48 over the 108 copies of the 5S rRNA genes are located 3' of tRNA genes. We show that these peculiar tRNA-5S rRNA dicistronic genes are expressed in vitro and in vivo as Pol III transcriptional fusions without the need of the 5S rRNA gene-specific factor TFIIIA, the deletion of which displays a viable phenotype. We also report the existence of a novel putative non-coding Pol III RNA of unknown function about 70 nucleotide-long (RUF70), the 13 genes of which are devoid of internal Pol III promoters and located 3' of the 13 copies of the tDNA-Trp (CCA). All genes embedded in the various dicistronic genes, fused 5S rRNA genes, RUF70 genes and their leader tRNA genes appear to be efficiently transcribed and their products correctly processed in vivo.

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Figures

Figure 1.
Figure 1.
Overview of the transcription mechanisms of tRNA, 5S rRNA and tRNA–5S rRNA dicistronic genes. (A) In S. cerevisiae, transcription of a tRNA gene by Pol III requires the assembly of transcription factor TFIIIC onto the tDNA (GC-rich) followed by that of TFIIIB (onto a AT-rich region) that finally recruits Pol III for multiple cycles of transcription. (B) A regular 5S rRNA gene is first recognized by its specific factor TFIIIA; then the 5S rRNA gene–TFIIIA complex is bound by TFIIIC and the next steps of transcription are identical to that of tRNA genes. In both cases, transcription stops and efficiently recycles when Pol III reaches the terminal T-track in the RNA-like strand. (C) Transcription of a dicistronic tRNA–tRNA gene by a unique TFIIIC molecule binding on the upstream gene. Assembly of TFIIIB onto the upstream gene (GC-rich) triggered by TFIIIC bound on the downstream gene to transcribe this only gene is penalized in vivo. (D) Hypothetical transcription of a dicistronic tRNA–5S rRNA gene may proceed similarly through the recognition of the promoter elements of the upstream gene by TFIIIC. In this case a single primary RNA (arrow) is produced and later matured into two functional products (tRNA and 5S rRNA) without the need of TFIIIA (see text for details).
Figure 2.
Figure 2.
Structural alignment of the 13 RUF70 genes located beyond the 13 copies of tDNA-Trp (CCA). (A) For sake of clarity, the DNA sequence, instead of RNA sequence, is used. Nucleotides 67–73 of the tDNA-Trp (CCA) are shown at left followed by the sequence of the RUF70 genes (sorted according to similarity). Coloured background in stems indicate correct base pairing (including GT pairs, GU in RNA). Stars above base pairing line (shown as opening and closing brackets) denote compensatory base pairing (e.g. TA or GC or CG pairs exchange) in stems. The length indicated at right is computed from the nucleotide following nt 73 of tDNA-Trp to the transcription termination signal (T-track, not included). The oligonucleotides used in northern experiments (see Figure 3D) hybridize with the underlined sequences. Detailed coordinates are given in Supplementary Data 3. (B) Alignment showing an alternative structure expanding the first stem at the expense of the tRNA acceptor stem. (C) 2D RNA structure corresponding to alignment shown in (A) for the first sequence. (D) Same for the alignment shown in (B).
Figure 3.
Figure 3.
Expression detected by RT–PCR, in vitro trancriptions and northern analyses. (A) Expression of the five different tRNA–5S rRNA, one tRNA–tRNA–5S rRNA and tRNA-Trp–RUF70 composite genes detected by RT–PCR. ‘RT’ refers to regular RT–PCR experiment, the reverse transcriptase was omitted in the lanes labelled ‘PCR’. The predicted length of the RT–PCR products is indicated at bottom. Only a faint band was obtained when testing the expression of the tRNA-Trp–RUF70 gene (arrow). (B) In vitro transcription of cloned copies of the same composite genes with WCE from either S. cerevisiae or Y. lipolytica as indicated. A longer exposure was used for the rightmost seven lanes. A tRNA-Ile (TAT) gene from S. cerevisiae (labelled ‘Sc’) is used as a control in the left lane. Expected lengths are indicated at bottom. Asterisks denote the faint bands obtained in the transcription experiments of tRNA-Trp–RUF70 gene. (C) In vitro transcription of the same genes using a fully recombinant TFIIIC reconstituted system from S. cerevisiae. (D) Northern blot analysis of the mature transcription products of the tRNA-Trp (CCA)–RUF70 composite genes. Total RNAs were extracted from the indicated strains growing in exponential phase. RUF70 genes were probed with a mixture of oligos targeted at the main stem-loop (sequences underlined in Figure 2A). The positive response of S. cerevisiae RNAs is due to the nearly perfect sequence conservation of the tRNA-Trp (CCA) genes between S. cerevisiae and Y. lipolytica.
Figure 4.
Figure 4.
Phenotypic and 5S rRNA analyses of Y. lipolytica ΔylTFIIIA mutants. (A) Total RNA content of wild-type S. cerevisiae strain (YPH500), wild-type Y. lipolytica strains (E150 and PO1d) and two ΔylTFIIIA Y. lipolytica deleted strains (ΔylTFIIIA C6 and ΔylTFIIIA C7, similar results were obtained for ΔylTFIIIA C8, not shown). Cells were grown in exponential phase, total RNA was extracted and analysed in 6% acrylamide–urea gel and stained with ethidium bromide. The migration of tRNA, 5.8S and the two distinct 5S rRNA species (arrows at right) are indicated. (B) Restriction pattern of the ylTFIIIA amplified locus of Y. lipolytica transformants. Only three out 10 transformants lost accurately the ylTFIIIA gene (C6, C7 and C8). Strain E150 was used as a control for the wild restriction pattern. M, molecular marker, 1 kb DNA ladder (New England Biolabs). (C) Effects of the disruption of the ylTFIIIA gene (YALI0F05104g) on growth ability. Serial dilutions (from 3 × 103 to 3 cells) of overnight cultures of the wild-type strains (E150 and PO1d) and of three independent (ΔylTFIIIA C6, C7 and C8) mutant strains were inoculated on YPD plates (or YNB supplemented, not shown) and incubated at 28°C (or 18°C, not shown) for 3 days.
Figure 5.
Figure 5.
Evolution of TFIIIA sequence throughout eukaryotes. The nine filled rectangles in the top sequence (X. laevis) symbolize the nine zinc fingers. The last zinc finger in S. cerevisiae TFIIIA is also traditionally numbered nine because this sequence was obtained prior to that of S. pombe that exhibits 10 zinc fingers. Multiple sequences alignment (not shown) confirm that finger 9 of S. pombe sequence corresponds to finger 9 of X. laevis sequence and that fingers 9 of S. cerevisiae and Y. lipolytica correspond to finger 10 of S. pombe. In TFIIIA from Y. lipolytica, an extra 10th zinc is present between finger 9 and the last block of homology common to all hemiascomytes (small dark vertical rectangle). See also alignment in Supplementary Data 5.
Figure 6.
Figure 6.
Structural alignment of the 10 C/D box snoRNA genes located beyond the tDNA-Gly (GCC) genes of P. trichocarpa. Ten dicistronic tRNA-Gly (GCC)–snoRNA genes were identified in the genome of P. trichocarpa. The 10 copies of tDNA are identical and are shown under the usual cloverleaf structure (top). The 10 C/D snoRNA gene sequences shown (bottom) immediately follows nt 73 of the tDNAs. Dashes indicate the antisense element and the asterisk indicates the nucleotide targeting the methylation. Most conserved residues (in bold) are displayed in the last line.
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