Characterization of a ribonuclease III-like protein required for cleavage of the pre-rRNA in the 3'ETS in Arabidopsis

Abstract

Ribonuclease III (RNaseIII) is responsible for processing and maturation of RNA precursors into functional rRNA, mRNA and other small RNA. In contrast to bacterial and yeast cells, higher eukaryotes contain at least three classes of RNaseIII, including class IV or dicer-like proteins. Here, we describe the functional characterization of AtRTL2, an Arabidopsis thaliana RNaseIII-like protein that belongs to a small family of genes distinct from the dicer family. We demonstrate that AtRTL2 is required for 3'external transcribed spacer (ETS) cleavage of the pre-rRNA in vivo. AtRTL2 localizes in the nucleus and cytoplasm, a nuclear export signal (NES) in the N-terminal sequence probably controlling AtRTL2 cellular localization. The modeled 3D structure of the RNaseIII domain of AtRTL2 is similar to the bacterial RNaseIII domain, suggesting a comparable catalytic mechanism. However, unlike bacterial RNaseIII, the AtRTL2 protein forms a highly salt-resistant homodimer that is only disrupted on treatment with DTT. These data indicate that AtRTL2 may use a dimeric mechanism to cleave double-stranded RNA, but unlike bacterial or yeast RNase III proteins, AtRTL2 forms homodimers through formation of disulfide bonds, suggesting that redox conditions may operate to regulate the activity of RNaseIII.

Figure 1.

The A. thaliana genome encodes three RNase III-like proteins that belong to a distinct, non-dicer, gene family. (A) Schematic representation of RNaseIII and RNaseIII-like proteins from A. thaliana (AtRTL1, AtRTL2 and AtRTL3), S. cerevisiae (Rnt1), S. pombe (PacI), E. coli (Ec_RNaseIII) and A. aeolicus (Aa_RNaseIII). Gray boxes correspond to RNaseIII motifs, white boxes to double-strand RNA-binding domain (DS-RBD) and dotted white box in AtRTL1 to less conserved RBD. Black bars correspond to 100 amino acid length. (B) Phylogenetic relation of different RNaseIII and RNase-like proteins. Numbers represent the percentage value of Bootstrap. Accession numbers of sequences used in this analysis: A. thaliana DCL1 (At1g01040), DCL2 (At3g03300), DLC3 (At3g43920), DCL4 (At5g20320), AtRTL1 (this study), AtRTL2 (At3g20420) and AtRTL3 (At5g45150); O. sativa dicer-like proteins (available in the tree), OsRTL1 (Os06g0358800), OsRTL2 (Os05g0271300) and OsRTL3 (Os01g0551100), S. cerevisiae (AAB04172), S. pombe (NP_595292), E. coli (NP_417062), A. aeolicus (NP_213645); C. elegans (NP_501789.1), D. melanogaster (NP_477436.1) and H. sapiens (NP_037367.21).

Figure 2.

In silico analysis of the AtRTL2 sequence suggests a novel mechanism for RNaseIII activity in plants. (A) Comparison of the crystal structure of A. aeolicus RNaseIII protein domain (residues 1–147) and modeled AtRTL2 (residues 57–214). Blue boxes show residues E93, E119 and E165 (E37, E64 and E110 in A. aeolicus) and the red box shows residue D100 (D44 in A. aeolicus), located in the RNaseIII domain and proposed as RNA cutting sites. (B) Amino acid sequence alignment of AtRTL2, OsRTL2, bacterial RNaseIII (E. coli and A. aeolicus) and yeast Rnt1 proteins. Conserved amino acids are shaded black and gray. The 9 amino acid signature is indicated. The gray bar shows a potential nuclear export signal (NES), the double over lining shows predicted double-stranded RNA-binding domains (Ds-RBD) and the black bar the putative bi-partite nuclear localization signal (bi-NLS). The modeled AtRTL2 sequence (residues 57–214) is boxed. Black arrowheads show the two conserved cysteines in AtRTL2 and OsRTL2 and potentially required for protein dimerization. Black arrows show proposed RNA cutting site residues in the bacterial sequence. The gray arrow shows the glutamic acid residue shown in the predicted structure of RTL2.

Figure 3.

AtRTL1, AtRTL2 and AtRTL3 gene expression in A. thaliana plants. RT-PCR analysis of AtRTL1, -2 and -3 in: flowers buds (lane 1), roots (lane 2), seedlings (lane 3), leaves (lane 4), seeds (lane 5) and germinating seeds (lane 6). Arabidopsis thaliana elongation factor 1α (EF-1α) gene expression was analyzed as a PCR control to evaluate the amount of cDNA used in each reaction (lanes 1–6). Lane 7, PCR amplification control using genomic DNA.

Figure 4.

AtRTL2 protein is expressed during seed maturation and germination. Western blot analyses of AtRTL2 expression during seed development at 0, 4, 8, 10 13, 16 and 21 days after fertilization (lanes 1–7) and 6, 24 and 48 h after imbibition (lanes 8–10). Gel protein loading was verified by staining the gel with Coomassie blue. Seed development can be visualized by accumulation of 12S proteins (lanes 5–10).

Figure 5.

Nuclear and cytoplasmic localization of AtRTL2. (A) Cellular localization of AtRTL2::GFP fusion protein in transfected onion epidermis cells. Arrows point to the nucleus and cytoplasm visualized by GFP fluorescence (Upper panel); the nucleus can be easily observed by Nomarski (Middle panel). Onion epidermial cells transformed with GFP alone produce homogenous GFP fluorescence (Lower panel). (B) Immunolocalization of AtRTL2. Total soluble (lanes 1 and 3) and nuclear (lanes 2 and 4) protein extracts isolated from A. thaliana seedlings were separated by SDS–PAGE, transferred to nitrocellulose and incubated with α-AtRTL2 (left panel) or with α-AtNUC1 and α-NTR (right panel) antibodies. α-AtNUC1 antibody detects nuclear and nucleolar Arabidopsis nucleolin like-1 protein. α-NTR detects cytoplasmic Arabidopsis NADPH-thioredoxin reductase.

Figure 6.

AtRTL2 contains a nuclear export signal (NES). (A) Alignment of putative NES sequences from AtRTL2 with NES from Pap1; HIV-1Rev and mPKIα (top). Schematic representation of GUS, GUS::NLS and NES::GUS::NLS constructs (bottom). (B) Onion epidermal cell transformed with GUS alone (upper panel), GUS fused to the nuclear localization signal (NLS) of RPL13 (middle panel) and the GUS::NLS construct fused to the N-terminal sequence of AtRLT2 containing a putative NES (bottom panel). White arrows show three nuclear bodies containing unexported GUS activity.

Figure 7.

Redox regulation of AtRTL2 protein dimerization. (A) Gel filtration chromatographic analysis of AtRTL2 under 0.1 M KCl (top) or 0.5 M KCl (bottom) buffer conditions. Numbered lines correspond to the protein fractions (from 57 to 79). The peak position of alcohol dehydrogenase (ADH, 158 kDa) and bovine serum albumin (BSA, 67 kDa) markers are indicated by arrowheads. The arrow at the 110 kDa position indicates the estimated size of the RTL2 protein peak. (B) Recombinant His-AtRTL2 protein in sample buffer containing 120, 12, 1.2 and 0.12 mM DTT (lanes 1–4) was analyzed by SDS–PAGE and western blot with α-AtRTL2 antibodies. Arrows indicate positions of monomers, dimers and higher order structures according to standard molecular weight markers.

Figure 8.

Analysis of A. thaliana plants with an AtRTL2 gene disrupted by T-DNA insertion. (A) Diagram of AtRTL2 gene transcript including 5′UTR and 3′UTR. Gray boxes correspond to exons separated by three introns. The T-DNA insertion in AtRtl2 plants is indicated by a gray diamond. Positions of primers 5′rtl2 and 3′rtl2 used to detect AtRTL2 transcripts are indicated by black arrows. (B) Left panel: RT-PCR reaction using total RNA isolated from 15-day WT and AtRtl2 seedlings (lanes 1 and 2) to detect AtRTL2 transcripts. Detection of the U3snoRNA transcript was performed as an RT-PCR control to evaluate the amount of RNA used in each reaction (bottom). Lower panel: nuclear protein fractions from WT and Atrtl2 (lanes 1 and 2) were fractionated by SDS–PAGE and hybridized with α-AtRTL2 antibody. Right panel: RT-PCR reaction using total RNA isolated from 15-day seedlings (WT and AtRtl2 plants lanes 3–4) to detect monocistronic (snoRNA U3) and polycistronic snoRNA precursors (cluster 15), tsnoRNA R43.1 and the 3′ETS from pre-rRNA (At3′ETS). Schematic representation of genomic organization of snoRNA U3, cluster 15 snoRNA, tsnoRNA R43.1 and the 3′ETS to show positions of primers used for amplification in the RT-PCR reaction. Panel EF1α shows a PCR reaction using genomic DNA extracted from WT plants (lane 1) or total RNA extracted from WT (lane 2) or AtRtl2 (lane 3) plants.