INTRODUCTION

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RNase III is a double-stranded-RNA (dsRNA)-specific endoribonuclease that introduces staggered cuts on each side of the RNA helix (28). In bacteria, RNase III is involved in processing pre-rRNA, tRNA, and phage polycistronic mRNA (7). Depletion of RNase III perturbs the expression level of about 10% of the bacterial proteins, suggesting a global role in gene regulation (10). Two eukaryotic homologues of RNase III were experimentally identified in Saccharomyces cerevisiae (Rnt1) (2) and Schizosaccharomyces pombe (Pac1) (14, 35, 41). In addition, database searches revealed homologues in the worm, mouse, and human (5, 35). Rnt1 was shown both in vivo and in vitro to process pre-rRNA (2, 18), three small nuclear RNAs (snRNAs) (1, 4, 40), and several small nucleolar RNAs (snoRNAs) (5, 6, 31). Similarly, Pac1 cleaves the 3' end of U2 snRNA and the 3' end of 25S rRNA (36, 37, 43). Also, it has been suggested that Pac1 plays a role in cell division, mating, and sporulation (14, 41). RNase III, Rnt1, and Pac1 cleave duplex RNAs longer than 20 nucleotides in vitro while their primary targets in vivo are intramolecular stem-loop structures (2, 33, 37). The basic features of the RNA cleavage mechanism appear to be similar for all three ribonucleases, but differences also exist that prevent free substrate exchange and genetic complementation (37).

Bacterial RNase III has two functionally and structurally separable subdomains: a C-terminal dsRNA-binding domain (dsRBD) and an N-terminal nuclease domain (8, 17). The dsRBD motif is located in the last 74 amino acids (aa) and adopts a tertiary fold consisting of two helices separated by three -strands (17). This tertiary structure is conserved throughout the family of dsRNA binding proteins including the RNA-dependent kinase (PKR) (27) and the Drosophila staufen protein (3). The isolated dsRBD from Escherichia coli RNase III binds RNA to form a RNA-protein complex (17; A. Nicholson, personal communication). The solution structure of the bacterial RNase III dsRBD (17) and the protein-RNA cocrystal structure of frog dsRNA binding protein A (38) suggest multiple RNA-protein contacts involving the two -helices and the loop between the first two -strands of the dsRBD. The structure of the N-terminal nuclease domain of RNase III is not known, but many mutations have helped identify the main features required for RNA cleavage (8, 28). The nuclease domain contains two stretches of conserved acidic amino acid residues at positions 37 to 47 and positions 60 to 74 (7, 28). These amino acids play either a key role in catalysis or an essential structural role. Mutations in these two regions abolish RNA cleavage without affecting RNA binding (21, 28).

Yeast Rnt1 shares with bacterial RNase III the main structural features of the nuclease domain and dsRBD, suggesting that they have similar functions (2). However, unlike the bacterial enzyme, eukaryotic Rnt1 possesses an N-terminal domain. The N-terminal domain constitutes 36% of the total Rnt1 protein with no significant homology to other eukaryotic homologues of RNase III, and it has no known function. To determine the function of the N-terminal domain and verify the activities of dsRBD and the nuclease domain, we have constructed a series of deletions separating the different domains of Rnt1 and tested them for RNA binding and cleavage. Here we show that dsRBD is sufficient for RNA binding and that the nuclease domain is required for RNA cleavage. Direct analysis of the N-terminal deletion effects on RNA binding and cleavage reveals an auxiliary role ensuring efficient RNA cleavage. Deletion of the N-terminal domain reduces the processing of the 25S rRNA 3' end by about 30% in vivo and slows growth by 35 to 40%. Biochemical and genetic assays suggest that the N-terminal domain influences Rnt1 function by mediating both inter- and intramolecular interactions.

	MATERIALS AND METHODS

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Strains and plasmids. Yeast was grown and manipulated by standard procedures (11, 34). The RNT1 cell is the haploid BMA64 strain carrying chromosomal disruption of RNT1 (6). Yeast PJ69-4A (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4 gal80 (LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ) was used for the yeast two-hybrid assays (15). KH54, the E. coli strain AG1688 (MC1061 F'128 lacI^q lacZ::Tn5), and plasmids pJH391, pFG157 and pKH101 (42) were used in the  repressor system.

Protein purification. All recombinant proteins in this work were produced either in E. coli BL21(DE3)pLysS (Promega Corp., Madison, Wis.), E. coli M15(pREP4) (Qiagen Inc.), or E. coli DH5F' (Life Technologies, Burlington, Ontario, Canada). Recombinant proteins were purified on a Ni-nitrilotriacetic acid agarose column (Pharmacia Biotech Inc., Baie d'Urfé, Québec, Canada) as described previously (16) with the following modifications. The first purification step was performed with Nickel buffer (25% glycerol, 1 M NaCl, 30 mM Tris [pH 8.0]). Protein fractions were pooled and passed through a second column with Nickel buffer without glycerol. Further purification of the N-terminal protein was performed on an HIC ISO column (Pharmacia Biotech Inc.) with a 0.05 to 1.5 M gradient of (NH₄)₂SO₄ and 50 mM sodium phosphate buffer at pH 7.5. The pure protein was collected in the unbound fraction. All purifications were conducted using the AKTA explorer fast protein liquid chromatography system (Pharmacia Biotech Inc.). The protein fractions were dialyzed against dialysis buffer (50% glycerol, 0.5 M KCl, 30 mM Tris [pH 8.0], 0.1 mM dithiothreitol [DTT], 0.1 mM EDTA [pH 8.0]) and stored at 20 or 80°C for long-term storage. The identity of the two proteins produced by the plasmid pQE30/dsRBD (Fig. 1C) was confirmed by monitoring the expression patterns of the two proteins and using Western blot analysis. Tests for RNA binding and dimerization confirm that the two proteins have similar activities.

Enzymatic assays. The radiolabeled RNA used as a substrate in the enzymatic assays was generated by T7 RNA polymerase in the presence of [-³²P]UTP. The RNA substrate was produced from a T7 promoter of plasmid pRS315/U5. To make this plasmid, a blunt-ended NheI fragment generated by PCR with primers 5'-CTTTTCTATTGCTAGCTTTCTAC-3' and 5'-GCTAGCAAATGCTTCAATGAG-3' was cloned in the blunt-ended XbaI site of pRS315. For the in vitro cleavage, 200 fmol of substrate was incubated for 10 min at 30°C in 10 µl of reaction buffer (30 mM Tris [pH 7.5], 5 mM spermidine, 10 mM MgCl₂, 0.1 mM DTT, 0.1 mM EDTA [pH 7.5]). The general effects of salt, N-terminal deletion, or N-terminal addition were confirmed using a wide range of substrate and protein concentrations. The amount of KCl used is indicated in the description of each experiment. The reaction was stopped by addition of a stop buffer (20 mM EDTA [pH 7.5] and 0.1% bromophenol blue in formamide) and directly loaded on denaturing 8% polyacrylamide gel. The cleavage rate was calculated using the Molecular Analyst programs (Bio-Rad Industries, Hercules, Calif.).

Gel mobility shift assay and in-gel cleavage assay. RNA binding reactions were performed using 2 fmol of radiolabeled RNA in 20 µl of binding buffer (20% glycerol, 30 mM Tris [pH 7.5], 5 mM spermidine, 0.1 mM DTT, 0.1 mM EDTA [pH 7.5]) for 10 min on ice. The amount of KCl and protein are indicated for each experiment. The reactions were fractionated on a 4% nondenaturing polyacrylamide gel at 0.5 V/cm² and 4°C. The in-gel cleavage assay was performed by cutting the bands corresponding to different complexes formed in the gel mobility shift assay and incubating them in a cleavage buffer (30 mM Tris [pH 7.5], 5 mM spermidine, 0.1 mM DTT, 0.1 mM EDTA [pH 7.5], 20 mM MgCl₂) at 30°C for 40 min. After the incubation period was complete, the gel pieces were removed and the RNA was extracted and loaded on 8% denaturing polyacrylamide gels.

RNase protection assay. A probe complementary to the 3' end of 25S rRNA and the 3' external transcribed spacer (ETS) was produced by T7 transcription (2). Total RNA (10 µg) was incubated at 42°C for 12 h with 10⁵ cpm of probe in 80% formamide hybridization buffer (25). The hybridization mix was digested with 2 µg of RNase T₁ per ml for 1 h at 30°C, extracted with phenol-chloroform, ethanol precipitated, and loaded on a 6% polyacrylamide gel.

Gel filtration assay. A Superdex 200 HR 10/30 column (Pharmacia Biotech Inc.) (10 by 300 to 310 mm) was equilibrated in gel filtration buffer (50 mM sodium phosphate [pH 7.5], 2 mM EDTA [pH 7.5], 0.5 M KCl) at 23°C and calibrated with low- and high-molecular-weight markers (Pharmacia Biotech Inc.). For sample application, 245 µg of each protein was applied to the column and 250-µl fractions were collected and analyzed on sodium dodecyl sulfate (SDS) gels (20).

Protein cross-linking. Cross-linking experiments were performed as described previously (24). Purified proteins (0.3 µg) were incubated for 10 min at 30°C in 10 µl of gel filtration buffer with increasing concentration of freshly diluted glutaraldehyde (Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada). The cross-linked proteins were analyzed by standard SDS-polyacrylamide gel electrophoresis (PAGE) and detected by silver staining.

Yeast two-hybrid assays. Plasmids encoding the appropriate AD- and BD-RNT1 fusion were cotransformed in yeast PJ69-4A using a modified lithium acetate method (39). The cells harboring both plasmids were selected on SCD medium (11) lacking lysine, uracil, and leucine. The two-hybrid interactions were indicated by the ability of a pair of plasmids to activate the three test promoters in PJ69-4A (15). Three or four independent transformants for each plasmid pair were tested on medium lacking either adenine or histidine. The histidine-containing media were supplemented with 10 mM 3-aminotriazole to avoid basal expression of histidine (15). The activation of the third reporter gene was tested by -galactosidase liquid assay as described earlier (32). Cells were harvested in mid-logarithmic phase, and their ability to hydrolyze o-nitrophenyl--D-galactopyranoside was measured as previously described (26).

repressor system. The  repressor assay was performed essentially as described previously (42). For the dot plaque assay, E. coli AG1688 transformed with either pJH/RNT1, pFG157, or pKH101 was grown to saturation in  broth (1% tryptone, 0.25% NaCl, 0.2% maltose, 10 mM MgSO₄, 50 µg of ampicillin per ml). A 300-µl volume of this bacterial culture was mixed in 3 ml of  top agar (0.5% yeast extract and 0.7% agar in  broth) and poured on a fresh plate of  agar ( broth, 1% agar), forming a bacterial lawn. Each lawn of bacteria was infected with a serial dilution of KH54 phage lysate containing between 5 × 10⁴ and 5 × 10⁸ PFU. Infected lawns were incubated for 18 h at 30°C, and the sizes of the resulting plaques were measured.

Western blot analysis. Yeast cells were grown to stationary phase in the appropriate SCD medium (11), and cellular proteins were extracted as previously described (39). Total proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (MSI, Westborough, Mass.). Western blot analysis was performed as described previously (12). Proteins were visualized using either monoclonal antibody against the Gal4 DNA binding domain or polyclonal antibody against the Gal4 activation domain (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.). The protein bands were visualized by enhanced chemiluminescence (ECL kit; Amersham, Arlington Heights, Ill.). The expression value of each fusion protein was estimated using the Molecular Analyst programs (Bio-Rad Industries).

	RESULTS

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The N-terminal domain of yeast RNase III is not essential for RNA binding and cleavage. Analysis of the Rnt1 sequence reveals three distinct domains; a 127-aa C-terminal domain containing a 74-aa dsRBD motif, a 154-aa central domain containing the RNase III nuclease motif, and a 191-aa N-terminal domain lacking significant homology to known proteins (Fig. 1A). To determine the contribution of the various domains to Rnt1 function, we expressed them individually in bacteria and assayed their activity in vitro. Five different segments of Rnt1 were expressed as N-terminal His₆-tagged proteins. The five proteins are full-length Rnt1 (Rnt1), Rnt1 lacking the C-terminal 150 aa including the 74-aa dsRBD motif (dsRBD), the 191-aa protein representing the N-terminal domain (N-term), a protein lacking the first 171 aa of the N-terminal domain (N-term), and a 127-aa protein containing the full dsRBD motif (dsRBD). Following expression in bacteria, these proteins were purified on two successive nickel affinity columns, with the exception of the N-term protein, which was repurified by hydrophobic interaction chromatography. All proteins were expressed in soluble form and were purified under native conditions to a purity of 85 to 95%, as judged by Coomassie blue-stained gels (Fig. 1C).

View larger version (49K): [in this window] [in a new window]   FIG. 1.   Purification and in vitro analysis of Rnt1 domains. (A) On top is shown a comparison of the functional domains of RNase III between bacteria, worms, and yeast. All members of the RNase III family possess a nuclease domain (NUCD) and a dsRNA binding domain (dsRBD). The N-terminal domain (NTD) is unique to the eukaryotic members of the RNase III family. Sequence alignment was performed using CLUSTALW (13). A schematic representation of Rnt1 fragments and the associated functional domains is shown at the bottom. The expressed segments of Rnt1 are indicated in amino acids on the left. (B) Illustration of the U5 3'-end model substrate. The cleavage sites are indicated by arrows. The numbers are relative to the U5 snRNA mature 3' end. The dotted line represents the vector sequence (pRS315). (C) Purification of the N-terminal His6-tagged Rnt1 fragments. All proteins were purified on metal chelating affinity columns. The N-term protein was repurified using hydrophobic interaction chromatography. Aliquots from the last purification step of each protein were fractionated on SDS-PAGE and stained with Coomassie brilliant blue R. The upper band in the dsRBD lane corresponds to a readthrough of the natural stop codon of RNT1 to a bacterial stop codon downstream. The protein molecular weight markers are indicated on the left in thousands. (D) In vitro cleavage assay of Rnt1 derivatives, using the 3' end of U5 as a model substrate. The 138-nucleotide substrate was incubated with no protein (lane 1), GST (lane 2), Rnt1 (lane 3), one of the four different Rnt1 deletions (lanes 4, 5, 7, and 8), or combinations of two different deletions (lanes 6 and 9). On the right, the position of the substrate and the different cleavage products are indicated as follows: S, full-length 138-nucleotide substrate; P1, 62-nucleotide 3'-end cleavage product; P2, 45-nucleotide 5'-end cleavage product; P3, middle 31-nucleotide cleavage product. The DNA molecular weight markers are indicated on the left. (E) Gel retardation assay of Rnt1 derivatives. The RNA was incubated with no protein (lane 1), Rnt1 (lane 2), one of the four different deletions (lanes 3, 4, 5, and 6), or GST (lane 7). The reaction was carried out in 25 mM KCl at 4°C, and the products were loaded on a 4% native gel. The positions of the shifted RNAs are indicated by solid arrowheads (Rnt, NT, and DS) on the right. The positions of supershifted RNAs are indicated by open arrowheads (Rnt1S, NTS, and DSS) on the left. The band indicated by the asterisk is a differently folded form of single-stranded RNA. The positions of the origin (ori) and unbound (Un) RNA are indicated on the right.

View larger version (76K): [in this window] [in a new window]   FIG. 2.   The N-terminal domain of Rnt1 is not essential for growth at 37°C. (A) Cells lacking the RNT1 gene were transformed with a series of RNT1 deletions fused with a nuclear localization signal from the GAL4 BD. The transformed yeast cells were streaked on minimal medium without uracil and incubated at 25 or 37°C. The position of each deletion is indicated on the right. The boxes illustrate deletions of the N-terminal domain (white), nuclease domain (light gray), and dsRBD (black). (B) Expression of the N-terminal deletion of Rnt1 from an inducible promoter without a nuclear localization signal. Segment 172 to 471 of Rnt1 was cloned under a copper-inducible promoter and transformed in cells lacking Rnt1. The cells were grown on minimal medium containing 100 µM Cu2+ at either 25 or 37°C. Boxes represent Rnt1 segments as described in panel A. (C) Mapping the 3' end of 25S rRNA using an RNase protection assay. The RNA was extracted from cells lacking Rnt1 (lanes 4, 5, and 6), expressing Rnt1 (lane 3), or expressing N-terminal deletions (lanes 7, 8, and 9) with or without a nuclear localization signal. The RNA was hybridized to a probe complementary to the 3' end of the 25S pre-rRNA and digested with RNase T1. The probe was also hybridized to E. coli tRNA as a control (lane 2). The positions of mature and extended 3' ends are indicated on the right. The DNA molecular weight markers are indicated on the left. (D) Western blot analysis of Rnt1 and N-term proteins. Proteins were extracted from RNT1 or RNT1 cells expressing different deletions of Rnt1p fused to the Gal4 BD. Equal amounts of proteins were separated on an SDS-polyacrylamide gel and examined using monoclonal antibodies against the Gal4 BD. Protein extracts from untransformed cells (Un) were included as control. The upper protein band in each lane corresponds to the expected size of the fusion protein. The lower band corresponds to a smaller protein that may result from either a pre-mature stop or degradation at the protein C terminus.

Deletion of the N-terminal domain impairs dsRNA cleavage at physiological salt concentrations in vitro. The apparent difference between the in vitro (Fig. 1) and in vivo (Fig. 2) activities of the N-term protein may reflect differences in the reaction conditions. Physiological salt concentrations in yeast range between 150 and 200 mM (30), while in vitro cleavage tests were normally conducted at concentrations lower than 50 mM (Fig. 1) to allow maximal activity (1, 2). To examine this possibility, we assayed Rnt1 or N-term cleavage of U5 over a range of KCl concentration from 10 to 400 mM. As shown in Fig. 3A, the Rnt1 cleavage rate diminished at KCl concentrations above 100 mM. From 150 to 400 mM KCl, the cleavage rate of Rnt1 remained more or less constant, with 60% of the substrate being cleaved. In contrast, N-term cleaved the RNA substrate at a rate similar to that of Rnt1 at KCl concentrations below 50 mM and cleavage was suppressed completely at concentrations higher than 200 mM (Fig. 3B). At physiological salt concentrations (150 to 200 mM KCl), N-term was about 30% to 40% less active than Rnt1 (Fig. 3C). This result is consistent with the reduced activity of N-term observed in vivo (Fig. 2C). Thus, although we observed previously that the N-terminal domain did not affect Rnt1 activity at low salt concentration (Fig. 1), we conclude that the N-terminal domain is required for Rnt1 function in vitro at high concentrations of monovalent salt.

View larger version (50K): [in this window] [in a new window]   FIG. 3.   Deletions of the N-terminal domain of Rnt1 impairs RNA cleavage at physiological KCl concentrations. Cleavage of the U5 snRNA 3' end by Rnt1 (A) or N-term (B) in increasing salt concentrations is shown. In each panel, RNA incubated with GST under the same reaction conditions is included as a control (lane 1). The positions of the RNA substrate (S) and the cleavage products (P1, P2, and P3) are indicated on the right, and the DNA markers are indicated on the left. (C) Percent cleavage rate of Rnt1 () and N-term () versus concentration of KCl. Autoradiographs of gels similar to these in panel A and B were scanned and quantified using the Bio-Rad gel analysis system. The data points shown are the average of two different experiments.

View larger version (48K): [in this window] [in a new window]   FIG. 4.   Deletion of the N-terminal domain influences Rnt1 binding affinity. (A and C) Increasing concentrations of Rnt1, N-term, or dsRBD were incubated with 2 fmol of U5 3'-end model substrate in 5 mM KCl (A) or 100 mM KCl (C). RNA incubated with GST under the same conditions is included as a control. The position of the gel origin (Ori) and unbound RNA (Un) are indicated on the left. (B and D) Quantitative analysis of RNA binding to Rnt1 (), N-term (), or dsRBD () were carried in either 5 mM KCl (B) or 100 mM KCl (D). The binding percentage was plotted versus the protein concentration. Each data point is the average of three experiments.

View larger version (44K): [in this window] [in a new window]   FIG. 5.   In-gel cleavage assay of Rnt1 and N-term RNA-protein complexes. (A) A gel shift assay was conducted at 125 mM KCl as described in Materials and Methods. (B and C) The gel bands corresponding to each complex were cut and incubated in either 50 mM KCl (B) or 250 mM KCl (C) in presence of MgCl2 to allow RNA cleavage. At the end of the incubation period, the gel pieces were removed and the RNA was extracted and loaded on an 8% denaturing polyacrylamide gel (B and C). In panel A, the position of each Rnt1 complex is indicated on the left (bands 2 and 3) and the position of each N-term complex is indicated on the right (bands 4 and 5). The band corresponding to the input RNA (band 1) was used as control. In panels B and C, the substrate and cleavage products are indicated on the right. The DNA molecular weight markers are indicated on the left.

Biochemical evidence for N-terminal domain- and dsRBD-mediated dimerization of Rnt1. To examine the role of the N-terminal domain in the formation of active Rnt1 protein, we analyzed the conformation of Rnt1 and its derivatives in solution by size exclusion chromatography. Each protein was expressed in bacteria and purified as described in Fig. 1 before being loaded on a gel filtration column. Each column was calibrated with high- and low-molecular-weight markers prior to the sizing of each protein. Rnt1 eluted in three major peaks, the smallest corresponding to a dimer form and the other two corresponding to a tetrameric and a multimeric form (Fig. 6A). These protein complexes are not aggregates of denatured proteins, since all three forms were equally capable of cleaving the substrate RNA (data not shown). This result suggests that, similar to the bacterial RNase III (9, 22, 23), Rnt1 self-interacts to form a dimer in solution. Gel filtration of the N-term resulted in only two peaks, one of which corresponded to the monomer form while the other corresponded to the dimer form (Fig. 6B). This result suggests that N-term cannot self-interact as efficiently as Rnt1. For the dsRBD, only one peak corresponding to the dimer size was observed (Fig. 6C). This result suggests that a dimerization domain exists within the dsRBD, as observed with other dsRNA binding proteins (29). Notably, dsRBD that lacks the dsRBD motif migrated as one large peak beyond the range of the column (data not shown). The N-term protein migrated on the column in the same fashion as the dsRBD, forming only one peak of high molecular weight corresponding to a multiple protein complex (Fig. 6D). This result suggests that the N-terminal domain acts as a second dimerization signal for Rnt1.

View larger version (32K): [in this window] [in a new window]   FIG. 6.   Rnt1 dimerization is mediated by N-terminal and C-terminal signals. Size exclusion chromatography was carried out with Rnt1 (A), N-term (B), dsRBD (C), and N-term (D) using a Superdex 200 HR 10/30 column. Each protein was loaded on columns precalibrated with 500 mM KCl. The protein content of each peak was verified by loading the corresponding fractions on SDS-polyacrylamide gels (shown below each chart). The size of each peak reflects the light absorbancy and not the amount of each protein. Peaks corresponding to the column end (end) are identified on top of each chart. The molecular weight marker is shown as a dotted line, and the size corresponding to each peak is indicated on top.

View larger version (70K): [in this window] [in a new window]   FIG. 7.   Glutaraldehyde cross-linking analysis of Rnt1 derivatives. Pure Rnt1 (A), N-term (B), N-term (C), and dsRBD (D) were incubated in increasing concentrations of glutaraldehyde-500 mM KCl. The reaction mixtures were incubated for 10 min and then loaded on SDS-polyacrylamide gels and silver stained. The positions of monomer, dimer, and multimer are shown on the right. The molecular weight markers are indicated on the left in thousands.

In vivo evidence for N-terminal-mediated self-interactions of Rnt1. To test Rnt1 dimerization in vivo and map its dimerization signals, we used the yeast two-hybrid assay. Two sets of plasmids carrying various segments of Rnt1 either fused to the Gal4 activation domain (AD) expressed from ADH1 promoter or fused to the Gal4 BD expressed from a truncated ADH1 promoter were used (Fig. 8A). The different plasmids were transformed in all pairwise combinations into yeast strain PJ69-4A (15) containing three different marker genes (HIS3, ADE2, and lacZ) under the control of three different test promoters (GAL1, GAL2, and GAL7, respectively). Real interactions can be scored using all three markers and may be quantified using a -galactosidase liquid assay. The results shown in Fig. 8A indicate that the N-terminal domain fused to Gal4 AD (AD-NT2/1-191) can interact with itself (BD-NT2/1-191), the dsRBD (BD-DS1/344-471), and Rnt1 (BD-RNT/1-471). The N-term protein appears to interact directly with the dsRBD because they can be cross-linked in vitro in the absence of any other factors (data not shown). These results suggest that the enzyme may self-interact through interactions between the two N-terminal domains, the two dsRBDs, or the dsRBD and the N-terminal domain.

View larger version (45K): [in this window] [in a new window]   FIG. 8.   Yeast two-hybrid analysis of interactions between Rnt1 domains. (A) Summary of Rnt1 inter- and intramolecular interactions determined by two-hybrid analysis. Two sets of plasmids carrying the indicated segments of Rnt1 fused to either GAL4 BD (BD-fusion) or GAL4 AD (AD-fusion) were constructed. Different combinations of the two sets were introduced into yeast strain PJ69-4A carrying three reporter genes (ADE2, HIS, and lacZ) under the control of three different promoters. Interaction between any pair of BD and AD fusion proteins will lead to the activation of all three markers with different efficiencies depending on the promoter stringency. The expression level (PE) of each fusion protein was assayed by Western blot analysis of whole-cell extract with anti-BD or anti-AD antibodies. The expression level of each fusion is indicated as a percentage of the wild-type BD or AD expression level. The interaction level of each two plasmids is indicated as weak (+), moderate (++), and strong (+++). The strength of the interaction was also measured using the -galactosidase liquid assay (LZ), and the average Miller units of three experiments are indicated. A schematic representation of the different constructs is shown on top and on the right of the table. White boxes indicate the N-terminal domain, light gray boxes indicate the nuclease domain, and black boxes indicate the dsRBD. (B) Comparison of the interaction strength between the different functional domains of Rnt1 using liquid -galactosidase assays. An average of three experiments of each pair of plasmids was plotted using Miller points. (C)  repressor assay of Rnt1 dimerization. Dot plaque assay of Rnt1 dimerization was conducted using E. coli AG1688 transformed with either N or N-Rnt1 fusion. The cells were poured as a lawn and infected with a 5-µl dilution of KH54 lysates. The ability of Rnt1 to dimerize is measured by its ability to suppress  infection and reduce bacterial lysis. The titer is indicated beside each spot.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Yeast Rnt1 and the bacterial RNase III share the basic features required for dsRNA binding and cleavage (2). In addition to the nuclease domain and dsRBD, the yeast enzyme contains a 191-aa extension at the N terminus unique to the eukaryotic homologues of RNase III. We have found that the N-terminal domain favors the formation of stable or functional Rnt1 protein complexes. Deletion of the N-terminal domain reduces the processing activity of Rnt1 by 35 to 40% and makes it hypersensitive to monovalent salt (Fig. 2 and 3). Biochemical (Fig. 6 and 7) and genetic (Fig. 8) evidence indicates that the N-terminal domain can interact with itself and with the dsRBD. Together, our results suggest that the eukaryotic N-terminal domain enhances Rnt1 function by mediating the formation of optimum protein conformations.

Yeast RNase III has a novel functional domain. The dsRBD and the nuclease domain of E. coli RNase III can be structurally and functionally separated (17; Nicholson, personal communication). The conserved C-terminal dsRBD is sufficient for dsRNA binding, and the N-terminal nuclease domain is sufficient for RNA cleavage. Yeast Rnt1 contains sequences homologous to both dsRBD and nuclease domain in addition to a unique 191-aa N-terminal domain. Here we provide evidence that yeast RNase III dsRBD is sufficient for dsRNA binding and that the nuclease domain is required for RNA cleavage. However, unlike the bacterial enzyme, deletion of the C-terminal dsRBD abolishes all RNA binding activity (Fig. 1) and the nuclease domain cannot cleave the substrate without the dsRBD, even with different divalent metal ions (Fig. 1 and data not shown). Deletion of the N-terminal domain did not affect the basic functions of Rnt1, suggesting that the protein structural and sequence elements required for RNA binding and cleavage are conserved among prokaryotes and eukaryotes. However, the efficiency of the RNA cleavage is diminished by the deletion of the N-terminal domain without significantly affecting the RNA binding efficiency (Fig. 3 and 4). Thus, deleting the N-terminal domain results in the formation of RNA-protein complexes in vitro that are either less productive or unstable under RNA cleavage conditions. These results suggest that the N-terminal domain is a functionally and structurally separate domain required for normal cell growth and efficient RNA cleavage. Database searches reveal the presence of two types of N-terminal domains among the eukaryotic homologues of RNase III (35). The first has homologies to the DEAD box ATPase-dependent helicase family and may be found in S. pombe (Pac 8), Caenorhabditis elegans, and Homo sapiens but not in the S. cerevisiae genome. The second type has no significant homology to known proteins and can be found in S. cerevisiae (Rnt1), S. pombe (Pac1), C. elegans, and H. sapiens genomes. Deletion of the Pac1 N-terminal domain appears to inhibit RNA cleavage, but the mechanism and extent of inhibition are not clear (14). The activity of the Pac1 N-terminal deletion was tested in crude bacterial extracts, preventing accurate measurements, and its ability to bind RNA was not examined. More tests with Pac1 and other eukaryotic homologues of Rnt1 are required to identify possible conserved functions of the N-terminal domain. Meanwhile, the results presented here suggest that in yeast the N-terminal domain of the eukaryotic RNase III is a distinct functional domain required for efficient RNA cleavage under physiological conditions.

Yeast RNase III self-interaction is mediated by N-terminal and C-terminal signals. RNase III forms a dimer in solution and appears to function as a dimer (9, 20). Here we show that Rnt1 also forms an intermolecular complex mediated by signals located at the N-terminal and C-terminal domains. However, the mechanism of Rnt1 self-interaction and binding to dsRNA appears different from that of RNase III. Unlike RNase III, Rnt1 forms multiple protein complexes at high salt concentrations (Fig. 6 and 7). Multiple interactions may also occur after binding to RNA at high protein concentrations, suggesting that these protein interactions do not interfere with RNA association. In addition, the nature of Rnt1-RNA complexes appears to be different from those of RNase III, since their stability is not dependent upon divalent metal ions (19). These differences between RNase III and Rnt1 may be caused at least in part by the N-terminal domain of Rnt1. Analysis of Rnt1 derivatives suggests that the multiple protein complexes formed in solution are mediated in part by the N-terminal domain (Fig. 6 and 7). N-terminally deleted Rnt1 or a protein containing only the dsRBD multimerize less readily and form mainly dimers or remain as monomer in solution, similar to the bacterial RNase III (21). In contrast, proteins containing the N-terminal domain or lacking the dsRBD tend to form higher-molecular-weight complexes. These observations suggest that Rnt1 possesses two dimerization signals that may provide a dynamic switch between different complexes (see below).

View larger version (22K): [in this window] [in a new window]   FIG. 9.   The N-terminal domain is a modulator of yeast RNase III activity. Hypothetical models of Rnt1 and N-term function. (A) Yeast RNase III. The intramolecular interaction of the dsRBD and N-term stabilizes the inactive protein, while the intermolecular interaction mediated by N-term and dsRBD stabilizes the protein complex on the RNA. (B) N-term. The N-terminal domain deletion from Rnt1 destabilizes the protein and weakens the protein-RNA complex, reducing the cleavage efficiency.

Is the N-terminal domain a regulator of Rnt1 function? In addition to its role in Rnt1 dimerization, the N-terminal domain appears to influence RNA binding and cleavage. Accordingly, the physical interaction that we detected between the N-terminal domain and dsRBD could be related to a regulatory function. One interesting possibility illustrated in Fig. 9 is that the N-terminus-mediated protein interactions modulate Rnt1 function. The N-terminal domain may interact intramolecularly with the dsRBD. This interaction would be disrupted upon binding of the RNA substrate to trigger conformational changes leading to intermolecular interaction between the two N-terminal domains and RNA cleavage. This model would explain why the deletion of the N-terminal domain promotes dsRNA binding without increasing the cleavage rate (Fig. 3 and 4). We therefore suggest that the N-terminal domain has dual functions, as depicted in Fig. 9. The first function is to interact with the dsRBD to form a compact protein structure that would be stable in the absence of the RNA, and the second function is to self-interact upon RNA binding to stabilize the ribonucleoprotein complex leading to efficient RNA cleavage. However, other in vivo functions of the N-terminal domain such as the regulation of Rnt1 interaction with other cellular proteins remains a possibility.