Mouse ribonuclease III. cDNA structure, expression analysis, and chromosomal location
© Fortin et al; licensee BioMed Central Ltd. 2002
Received: 23 May 2002
Accepted: 21 August 2002
Published: 21 August 2002
Members of the ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoribonucleases participate in diverse RNA maturation and decay pathways in eukaryotic and prokaryotic cells. A human RNase III orthologue has been implicated in ribosomal RNA maturation. To better understand the structure and mechanism of mammalian RNase III and its involvement in RNA metabolism we determined the cDNA structure, chromosomal location, and expression patterns of mouse RNase III.
The predicted mouse RNase III polypeptide contains 1373 amino acids (~160 kDa). The polypeptide exhibits a single C-terminal dsRNA-binding motif (dsRBM), tandem catalytic domains, a proline-rich region (PRR) and an RS domain. Northern analysis and RT-PCR reveal that the transcript (4487 nt) is expressed in all tissues examined, including extraembryonic tissues and the midgestation embryo. Northern analysis indicates the presence of an additional, shorter form of the transcript in testicular tissue. Fluorescent in situ hybridization demonstrates that the mouse RNase III gene maps to chromosome 15, region B, and that the human RNase III gene maps to a syntenic location on chromosome 5p13-p14.
The broad transcript expression pattern indicates a conserved cellular role(s) for mouse RNase III. The putative polypeptide is highly similar to human RNase III (99% amino acid sequence identity for the two catalytic domains and dsRBM), but is distinct from other eukaryotic orthologues, including Dicer, which is involved in RNA interference. The mouse RNase III gene has a chromosomal location distinct from the Dicer gene.
The enzymatic cleavage of double-stranded(ds) RNA structures is an essential step in the maturation and decay of many eukaryotic and prokaryotic RNAs. Members of the ribonuclease III superfamily of endoribonucleases  are the primary agents of dsRNA cleavage . RNase III orthologues are conserved in eukaryotes and in bacteria, with Escherichia coli RNase III  as the best characterized member. E. coli RNase III is active as a homodimer, and requires a divalent metal ion (preferably Mg2+) to hydrolyze phosphodiesters, creating 5'-phosphate, 3'-hydroxyl product termini . E. coli RNase III cleaves rRNA and mRNA precursors as part of the respective maturation pathways. E. coli RNase III also initiates mRNA degradation, and participates in antisense RNA action [2, 5–8].
Two mammalian RNase III orthologues have been identified, and exhibit common domains as well as apparently unique features. The RNase III orthologue "Dicer" (Figure 1) plays a central role in RNA interference (RNAi) by cleaving dsRNAs to ~21 bp fragments, termed small interfering(si) RNAs. The siRNAs are incorporated into a macromolecular complex which carries out degradation of homologous RNA sequences . Dicer action serves to inhibit viral infection and retroposon movement, and also plays a role in developmental pathways by cleaving precursors to small regulatory RNAs (reviewed in [14, 15]). The predicted sequence of the mouse Dicer polypeptide exhibits a single dsRBM and tandem catalytic domains. In addition, the N-terminal region contains a DExH/DEAH RNA helicase motif and a PAZ (Pinwheel-Argonaut-Zwille) domain, which is also present in other proteins involved in RNAi [16, 17].
The second mammalian RNase III orthologue also exhibits a single C-terminal dsRBM and tandem catalytic domains, but is otherwise structurally distinct from Dicer as it lacks the helicase and PAZ domains (Figure 1) [18, 19]. Preliminary evidence indicates that this RNase III orthologue participates in rRNA maturation. Thus, a reduction in human RNase III levels in vivo causes the accumulation of specific rRNA processing intermediates . Consistent with this functional role, human RNase III localizes to the nucleolus in a cell-cycle-dependent manner . A truncated form of human RNase III has been purified and shown to cleave dsRNA in vitro . However, little else is known of the functional roles or the mechanistic features of this enzyme. We report here (i) the characteristics of the cDNA sequence and predicted polypeptide of mouse RNase III, (ii) demonstrate transcript expression patterns, and (iii) report the chromosomal locations of the mouse and human RNase III genes.
Results and Discussion
Features of the mouse RNase III cDNA sequence
Features of the predicted RNase III polypeptide
The dsRBM and catalytic domains
The Proline-rich region
A proline-rich region (PRR) is present in the N-terminal portion of the polypeptide (see Additional File 1). Of the 63 prolines occurring in the PRR, 62 (98%) are conserved in the corresponding region of human RNase III. Repetitive proline sequences tend to adopt a polyproline II (PPII) helix, consisting of an extended structure with three residues per turn. A proline at every third position serves to stabilize the structure , and also participates in hydrogen bonds as well as in hydrophobic interactions . The presence of a PRR in mouse RNase III suggests protein-protein interactions important for function. PRR-mediated interactions are relatively weak and reversible, and occur with the PPII helix in the C-terminal domain of RNA polymerase II during transcription initiation and elongation .
The Arginine/Serine (RS) domain
The mouse RNase III polypeptide contains an RS domain adjacent to the PRR (see Additional File 1). The 13 positions containing the RS/SR dipeptide motif are shared between the mouse and human polypeptides, with an overall 87% sequence identity, with the human sequence containing an additional SR dipeptide. RS domains participate in protein-protein interactions, and RS domain-containing proteins play essential roles in constitutive or alternative mRNA splicing [23–25]. RS proteins bind RNA via an RNA recognition motif (RRM), allowing subsequent recruitment of splicing components via RS domain interactions. The presence of this domain in mouse RNase III suggests similar protein-protein interactions involved in RNA maturation, which may be functionally associated with components of the RNA splicing and transport machinery.
Potential sites of post-translational modification
Psort analysis of the predicted mouse RNase III amino acid sequence indicates three potential nuclear localization signals positioned at residues 254, 355 and 508 (see Additional File 1). A Prosite scan identified multiple potential phosphorylation sites for protein kinase C, casein kinase II and cAMP-dependent protein kinase (data not shown). Some of these signals may be involved in protein localization, as it has been shown that human RNase III localizes to the nucleolus during the cell cycle S phase .
Mouse RNase III transcript expression patterns
Chromosomal locations of the mouse and human ribonuclease III genes
Sequence database searches and other studies  have revealed only two RNase III orthologues (Dicer and RNase III) in the mouse and human genomes. However, the presence of an additional RNase III-specific transcript in at least one tissue indicates the possibility of alternative forms of RNase III. The broad expression pattern suggests a conserved cellular function for mouse RNase III. In this regard, sequence database searches and a southern "zoo" blot (data not shown) indicate significant conservation of the RNase III gene among many other vertebrates, including rat, rabbit and cow. However, functional roles for mammalian RNase III have yet to be fully defined. A role in rRNA maturation is suggested by the observation that antisense oligodeoxynucleotide-mediated reduction in human RNase III levels also causes a decrease in the amount of 5.8 S rRNA and a concomitant increase in the 12 S precursor . A similar defect in rRNA maturation is seen upon U8 snoRNA depletion in Xenopus oocytes . As U8 snoRNA participates in rRNA maturation, it is possible that an RNase III-dependent step also involves U8 snoRNA. Alternatively, a role for RNase III in the maturation of U8 snoRNA (as well as other snoRNAs) is a possibility. In this regard, the S. cerevisiae RNase III orthologue Rnt1p not only cleaves the 35 S rRNA precursor within the 3'-ETS [27–29], but also processes snoRNA and snRNA precursors [30–33]. Further biochemical studies are required to identify the RNA targets for mammalian RNase III and to determine its involvement in RNA maturation pathways.
Chemicals and reagents were molecular biology grade or reagent grade and were purchased from Fisher Scientific or Sigma Chemical Company. Restriction enzymes were purchased from New England Biolabs and were used according to the supplied instructions. The radiolabeled nucleotide [α-32P]dCTP (3,000 Ci/mmol) and nick translation kits were purchased from Amersham-Pharmacia. Oligodeoxynucleotides used for DNA sequencing and PCR were synthesized by Invitrogen. The Multiple Tissue Northern (MTN) blot was obtained from Clontech. I.M.A.G.E. Consortium cDNA clones were obtained from Invitrogen/Research Genetics. The cDNA used as template for PCR cloning and sequencing was obtained from Clontech (Marathon-Ready cDNA), or prepared from mouse liver or kidney RNA as previously described . Bacterial plasmids used in DNA sequencing reactions were purified using Qiagen plasmid purification kits. DNA sequencing reactions employed an ABI 3700 automated DNA Analyzer, and Big Dye terminator kits. Sequences were assembled with the ABI DNA sequencing analysis software (v3.6).
Determination of the mouse ribonuclease III cDNA sequence
Several avenues were followed to obtain the complete cDNA sequence for mouse RNase III. One strategy was based on a previous study  which identified eukaryotic RNase III orthologues by a BLAST search of the NCBI translated mouse EST database, using E. coli RNase III as query sequence. We carried out a similar search against the NCBI mouse EST database, using the Drosophila and human RNase III [18, 19] as query sequences. Specific clones were identified, obtained and sequenced. The clone positions and accession numbers are given in Figure 2.
In a second approach, mouse kidney cDNA (Clontech Marathon-ready cDNA) served as a template for PCR which used the Advantage-2 Polymerase Mix (Clontech). The PCR reaction (50 μl volume) also included: 10 μM each primer (sequences available by request), and 5 μl of the cDNA preparation. The PCR conditions were: 94° for 30 sec, then 5 cycles of 94° (5 sec) and 72° (4 min.). This was followed by 5 cycles of: 94° (5 sec) and 70° (4 min.). The final steps consisted of 20–25 cycles of 94° (5 sec.) and 68° (4 min.). The products were purified and cloned into plasmid pPICZ-C (Invitrogen) at the SfiI and SnaB1 sites. The recombinant clone (indicated in Figure 2) was sequenced (Accession Number AF533013). All sequences obtained as described above were subjected to a series of CLUSTALW alignments. Additional clone sequences obtained from the NCBI database were used for further verification of the assembled sequence. The assembled cDNA sequence was subjected to hypothetical translation using the "Translate" program available on the EXPASY website http://www.expasy.ch. Functional domains were determined using CLUSTALW and manual methods. The molecular weight was calculated using the primary structure analysis program available from ExPASY proteomics tools. Potential post-translational modification sites and nuclear localization signals were identified using PROSITE and Psort programs, respectively http://www.expasy.ch.
Total RNA was obtained from mouse embryonic tissue or adult mouse organs and reverse transcription carried out as described . For each tissue analysis, PCR was carried out in a 25 μl reaction volume using 1 μl of the first-strand cDNA reaction, according to the Superscript Preamplification System for First Strand cDNA Synthesis (Invitrogen). Primer sequences were (forward) 5'-GGGGCCATCCCATGCTAGAA-3' and (reverse) 5'-CCACTCCTGCCCTCGTTTACT-3'. PCR conditions were 95°C × 10 min; 25 cycles of 94°C × 1 min; 55°C × 1 min; 72°C × 1 min; with a final extension of 72°C × 5 min. β-actin was used as an internal standard; primer sequences were (forward) 5'-CCCAACTTGATGTATGAAGG-3' and (reverse) 5'-TTGTGTAAGGTAAGGTGTGC-3'. PCR conditions were 95°C × 1 min, 30 cycles of 94°C × 30 sec, 58°C × 30 sec, 72°C × 1 min, with a final extension of 72°C × 3 min. Reactions were analyzed by electrophoresis in 1% agarose gels, and DNA was visualized by ethidium staining.
The authors thank Mike Hagen and Tara Twomey of the Wayne State DNA sequencing facility for carrying out sequence analyses. We also thank SeeDNA Biotech, Inc. (Windsor, Ontario) for the FISH analyses, and Dr. Henry Heng (Wayne State University) for advice on the FISH analyses. We also thank the other members of the lab for their encouragement and interest in the project. This paper is dedicated to the memory of William A. Lepeak. This research is supported in part by NIH grant GM56457.
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