Degradation of a polyadenylated rRNA maturation by-product involves one of the three RRP6-like proteins in Arabidopsis thaliana

Abstract

Yeast Rrp6p and its human counterpart, PM/Scl100, are exosome-associated proteins involved in the degradation of aberrant transcripts and processing of precursors to stable RNAs, such as the 5.8S rRNA, snRNAs, and snoRNAs. The activity of yeast Rrp6p is stimulated by the polyadenylation of its RNA substrates. We identified three RRP6-like proteins in Arabidopsis thaliana: AtRRP6L3 is restricted to the cytoplasm, whereas AtRRP6L1 and -2 have different intranuclear localizations. Both nuclear RRP6L proteins are functional, since AtRRP6L1 complements the temperature-sensitive phenotype of a yeast rrp6Delta strain and mutation of AtRRP6L2 leads to accumulation of an rRNA maturation by-product. This by-product corresponds to the excised 5' part of the 18S-5.8S-25S rRNA precursor and accumulates as a polyadenylated transcript, suggesting that RRP6L2 is involved in poly(A)-mediated RNA degradation in plant nuclei. Interestingly, the rRNA maturation by-product is a substrate of AtRRP6L2 but not of AtRRP6L1. This result and the distinctive subcellular distribution of AtRRP6L1 to -3 indicate a specialization of RRP6-like proteins in Arabidopsis.

FIG. 1.

Analysis of RRP6-like proteins. (A) Conservation of functional domains in RRP6 and RRP6-like proteins. Comparison of S. cerevisiae ScRrp6p and Homo sapiens HsPM/Scl-100 and the three A. thaliana RRP6-like proteins (AtRRP6L1, AtRRP6L2, and AtRRP6L3). Percent identity and, in parentheses, similarity with ScRrp6p are given below each domain, drawn as boxes. (B) Phylogenetic analysis of RRP6-like proteins presented as an unrooted maximum-likelihood tree. Bootstrap values above 70 (using 100 replications) are indicated along the branches. The scale bar indicates the evolutionary distance (amino acid substitutions per site). Pt, P. trichocarpa; At, A. thaliana; Os, O. sativa; Sc, S. cerevisiae; Ce, Caenorhabditis elegans; Hs H. sapiens; Dm, Drosophila melanogaster.

FIG. 2.

Subcellular distribution of RRP6-like proteins. (A) GFP fluorescence (left) and Nomarski (right) images of tobacco BY2 cells transiently expressing EGFP and EGFP fusion proteins. β-Glucuronidase-GFP (GUS-GFP) is a large protein that cannot enter the nuclear compartment by passive diffusion. (B) Root tips of stably transformed Arabidopsis plants expressing RRP6L-EGFP fusion proteins. (C) Enlarged view of root tips of transformed Arabidopsis plants showing the intranuclear distribution of fusion proteins. Comparison of fluorescence and Nomarski panels shows that RRP6L1-EGFP (top) is mainly in the nucleoplasm and in the nucleolar vacuole. RRP6L2-EGFP is detected mainly inside the nucleolus (bottom). Cy, cytoplasm; Np, nucleoplasm; Nu, nucleolus. Size bar = 10 μm.

FIG. 3.

AtRRP6L1 complements yeast rrp6Δ. Growth at the nonpermissive temperature (37°C) of WT and rrp6Δ yeast strains harboring empty vector (vec.) or vectors encoding either S. cerevisiae Rrp6p (ScRRP6) or the indicated Arabidopsis RRP6-like proteins.

FIG. 4.

Characterization of rrpl1 and -2 TDNA insertion mutants. (A) Diagram of the intron-exon structure of RRP6L1. Exons are in black, introns in white. The 5′ and 3′ untranslated regions are drawn as gray blocks. T-DNA insertion sites for rrp6l1-1 and -2 are shown. In rrp6l1-1, the T-DNA insertion removed 29 nt from the genomic sequence. (B) Detection of RRP6L1 mRNA in WT and mutant plants by virtual Northern analysis, as described in Materials and Methods. A probe for RBP1A mRNA was used as a loading control. (C) Organi-zation and T-DNA insertion sites in RRP6L2. (D) Virtual Northern blot analysis of RRP6L2 mRNA in WT and mutant plants showed a size difference between transcripts in WT and rrp6b-2 mutants. (E) Western blots of proteins extracted from WT, rrp6l21-1, or rrp6l2-2 seedlings were probed with antibodies against AtRRP6L2 (left). The truncated protein encoded by the mutant rrp6b-2 allele is indicated by an arrow. As a loading control, the membrane was stained with Coomassie blue (right).

FIG. 5.

A polyadenylated rRNA maturation by-product accumulates in rrp6l2 mutants. (A) Diagram showing the 5′ region of the polycistronic rRNA transcript of A. thaliana. Distal (5′ dETS) and proximal (5′ pETS) RNA segments and processing sites P and P′, respectively, are indicated. The promoter is shown by a bent arrow. (B) Mapping of 5′ and 3′ ends of the 5′ pETS by cRT-PCR. Total RNA from rrp6l2-1 plants was self-ligated by T4 RNA ligase, and cDNA was synthesized using a gene-specific reverse primer. The same primer was combined with a gene-specific forward primer to amplify joined 5′ and 3′ ends by PCR. Primers are indicated on the diagram. 5′ ends are shown above the diagram, 3′ ends are shown below, and nonencoded nucleotides at the 3′ ends of 5′ pETS transcripts are indicated. (C) Characterization of the 5′ pETS by 3′ RACE. Oligo(dT)₁₂-primed cDNA was synthesized from total RNA from WT or mutant plants. 3′ ends were then amplified by PCR using a gene-specific forward primer (arrow above the diagram) and a reverse primer specific for the oligo(dT) primer adapter sequence. The PCR products were analyzed by electrophoresis (top). The PCR products obtained from rrp6l2 samples were cloned and sequenced to map polyadenylation sites (bottom). The locations and frequencies of polyadenylation sites are indicated on the sequence, and the sizes and nucleotide compositions of poly(A) tails are given below. Clones obtained in an A-rich region may correspond to artifacts and are indicated by a question mark. (D) Accumulation of the 5′ pETS in WT and rrp6l2 (left) or WT and rrp6l1 (right) plants as determined by virtual Northern blotting. Full-length oligo(dT)-primed cDNA was amplified by 16 PCR cycles, separated on an agarose gel, blotted, and hybridized to a DNA probe corresponding to the 5′ pETS.