Human RNase III Is a 160-kDa Protein Involved in Preribosomal RNA Processing
2000; Elsevier BV; Volume: 275; Issue: 47 Linguagem: Inglês
10.1074/jbc.m005494200
ISSN1083-351X
AutoresHongjiang Wu, Hong Xu, Loren Miraglia, Stanley T. Crooke,
Tópico(s)RNA Interference and Gene Delivery
ResumoA human RNase III gene encodes a protein of 160 kDa with multiple domains, a proline-rich, a serine- and arginine-rich, and an RNase III domain. The expressed purified RNase III domain cleaves double-strand RNA and does not cleave single-strand RNA. The gene is ubiquitously expressed in human tissues and cell lines, and the protein is localized in the nucleus of the cell. The levels of transcription and translation of the protein do not change during different phases of the cell cycle. However, a significant fraction of the protein in the nucleus is translocated to the nucleolus during the S phase of the cell cycle. That this human RNase III is involved in processing of pre-rRNA, but might cleave at sites different from those described for yeast RNase III, is shown by antisense inhibition of RNase III expression. Inhibition of human RNase III expression causes cell death, suggesting an essential role for human RNase III in the cell. The antisense inhibition technique used in this study provides an effective method for functional analysis of newly identified human genes. A human RNase III gene encodes a protein of 160 kDa with multiple domains, a proline-rich, a serine- and arginine-rich, and an RNase III domain. The expressed purified RNase III domain cleaves double-strand RNA and does not cleave single-strand RNA. The gene is ubiquitously expressed in human tissues and cell lines, and the protein is localized in the nucleus of the cell. The levels of transcription and translation of the protein do not change during different phases of the cell cycle. However, a significant fraction of the protein in the nucleus is translocated to the nucleolus during the S phase of the cell cycle. That this human RNase III is involved in processing of pre-rRNA, but might cleave at sites different from those described for yeast RNase III, is shown by antisense inhibition of RNase III expression. Inhibition of human RNase III expression causes cell death, suggesting an essential role for human RNase III in the cell. The antisense inhibition technique used in this study provides an effective method for functional analysis of newly identified human genes. double-strand RNA ribonuclease III ribosomal RNA rapid amplification of cDNA ends Dulbecco's phosphate-buffered saline fluorescence-activated cell sorter serine and arginine external transcribed spacer internal transcribed spacer kilobase(s) glutathioneS-transferase RNase III enzymes are highly conserved double-strand RNA (dsRNA)1 endoribonucleases expressed in most, perhaps all, living cells (1Robertson H.D. Methods Enzymol. 1990; 181: 189-202Crossref PubMed Scopus (19) Google Scholar, 2Court D. Belasco J. Brawerman G. Control of Messenger RNA Stability. Academic Press, Inc., New York1993: 71-116Crossref Google Scholar). The COOH-terminal portion of the enzyme contains a dsRNA-binding domain, which has been found exclusively in proteins recognizing dsRNA. NMR methods have suggested that the dsRNA-binding domain has an α-β-β-β-α topology in which a three-stranded anti-parallel β-sheet packs on one side against the two α-helices (3Kharrat A. Macias M.J. Gibson T.J. Nilges M. Pastore A. EMBO J. 1995; 14: 3572-3584Crossref PubMed Scopus (246) Google Scholar). The enzyme also contains a catalytic domain independent from substrate-binding domain, which implies that substrate recognition is not necessarily coupled to catalysis. All RNase III species cloned to date contain a signature sequence (HNERLEFLGDS). In some species like Drosophila andCaenorhabditis elegans the enzyme contains 2 copies of the sequence (4Filippov V. Solovyev V. Filippova M. Gill S.S. Gene ( Amst. ). 2000; 245: 213-221Crossref PubMed Scopus (113) Google Scholar) which may suggest that some RNase IIIs may be capable of forming an active catalytic center as monomers. Deletion and mutation of this sequence abolishes the catalytic activity of the enzyme (5Nashimoto H. Uchida H. Mol. Gen. Genet. 1985; 201: 25-29Crossref PubMed Scopus (49) Google Scholar, 6Nicholson A.W. Prog. Nucleic Acids Res. Mol. Biol. 1996; 52: 1-65Crossref PubMed Google Scholar). In Escherichia coli, RNase III forms a homodimer (7Dunn J.J. J. Biol. Chem. 1976; 251: 3807-3814Abstract Full Text PDF PubMed Google Scholar) and requires a divalent metal ion. The enzyme produces 5′-phosphate, 3′-hydroxy termini and participates in RNA maturation and decay pathways by site specifically cleaving double-helical structures in cellular and viral RNAs. By comparing RNase III substrates, Zhang and Nicholson (8Zhang K. Nicholson A.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13437-13441Crossref PubMed Scopus (89) Google Scholar) showed several specific Watson-Crick base pairs at defined positions relative to the cleavage site. Introducing these disfavored base pairs into a model substrate inhibited efficient cleavage in vitro by interfering with RNase III binding. By comparing sequences of a large number of S. cerevisiae RNase III substrates and introducing mutations in the model substrate, Chanfreau et al. (9Chanfreau G. Buckle M. Jacquier A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3142-3147Crossref PubMed Scopus (78) Google Scholar) suggested that yeast RNase III cleavage specificity may be based on recognizing consensus AGNN tetraloops and cleaving the substrate at a fixed distance from the tatraloop. Multiple functions have been ascribed to RNase III. In E. coli RNase III has been reported to affect the expression of several phage, plasmid, and cellular genes (6Nicholson A.W. Prog. Nucleic Acids Res. Mol. Biol. 1996; 52: 1-65Crossref PubMed Google Scholar, 10Arraiano C.M. World J. Microbiol. Biotechnol. 1993; 9: 421-432Crossref PubMed Scopus (7) Google Scholar). The E. coli enzyme participates in the rRNA maturation process by processing 16 S and 23 S from the primary 30 S precursor (6Nicholson A.W. Prog. Nucleic Acids Res. Mol. Biol. 1996; 52: 1-65Crossref PubMed Google Scholar). RNase III also plays a determinant role in control of the decay of messenger RNA and thus the level of corresponding proteins (2Court D. Belasco J. Brawerman G. Control of Messenger RNA Stability. Academic Press, Inc., New York1993: 71-116Crossref Google Scholar, 11Santos J.M. Drider D. Marujo P.E. Lopez P. Arraiano C.M. FEMS Microbiol. Lett. 1997; 157: 31-38Crossref PubMed Google Scholar). In yeast, it has been suggested that RNase III is a general factor in small nuclear RNA processing since enzyme cleavage sites were identified in several small nuclear RNA species (U1, U2, and U5). As these RNAs are essential components of the mRNA splicing apparatus (12Chanfreau G. Elela S.A. Ares Jr., M. Guthrie C. Genes Dev. 1997; 11: 2741-2751Crossref PubMed Scopus (98) Google Scholar, 13Abou Elela S. Ares Jr., M. EMBO J. 1998; 17: 3738-3746Crossref PubMed Scopus (104) Google Scholar, 14Seipelt R.L. Zheng B. Asuru A. Rymond B.C. Nucleic Acids Res. 1999; 27: 587-595Crossref PubMed Scopus (66) Google Scholar, 15Ares Jr., M. Weiser B. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 131-159Crossref PubMed Scopus (79) Google Scholar, 16Madhani H.D. Guthrie C. Annu. Rev. Genet. 1994; 28: 1-26Crossref PubMed Scopus (319) Google Scholar, 17Staley J.P. Guthrie C. Cell. 1998; 92: 315-326Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar), RNase III is thought to play a role in pre-mRNA processing. Yeast RNase III (RNT1) is also required for the synthesis of several small nucleolar RNAs from large precursors (18Chanfreau G. Rotondo G. Legrain P. Jacquier A. EMBO J. 1998; 17: 3726-3737Crossref PubMed Scopus (140) Google Scholar, 19Qu L.H. Henras A. Lu Y.J. Zhou H. Zhou W.X. Zhu Y.Q. Zhao J. Henry Y. Caizergues-Ferrer M. Bachellerie J.P. Mol. Cell. Biol. 1999; 19: 1144-1158Crossref PubMed Scopus (139) Google Scholar, 20Chanfreau G. Legrain P. Jacquier A. J. Mol. Biol. 1998; 284: 975-988Crossref PubMed Scopus (141) Google Scholar). As most small nucleolar RNAs are implicated in site-specific cleavage of precursor ribosomal RNA (20Chanfreau G. Legrain P. Jacquier A. J. Mol. Biol. 1998; 284: 975-988Crossref PubMed Scopus (141) Google Scholar, 21Tollervey D. Science. 1996; 273: 1056-1057Crossref PubMed Scopus (64) Google Scholar, 22Tollervey D. Kiss T. Curr. Opin. Cell Biol. 1997; 9: 337-342Crossref PubMed Scopus (380) Google Scholar), the yeast enzyme is thought to play a key role in ribosome biogenesis (23Elela S.A. Igel H. Ares Jr., M. Cell. 1996; 85: 115-124Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 24Kufel J. Dichtl B. Tollervey D. RNA. 1999; 5: 909-917Crossref PubMed Scopus (125) Google Scholar). Additionally, RNase III mutants of E. coli accumulate unprocessed pre-rRNA but are still viable because an alternative pathway for the processing of 16 S rRNA exists, and 23 S rRNA retaining unprocessed extensions at its 5′ and 3′ ends can assemble into functional ribosomes (25Gegenheimer P. Watson N. Apirion D. J. Biol. Chem. 1977; 252: 3064-3073Abstract Full Text PDF PubMed Google Scholar). On the other hand, the yeast RNase III (RNT1) is an essential gene in Saccharomyces cerevisiae. To date, human RNase III has not been cloned. We now report the cloning and characterization of a cDNA that expresses a human RNase III. We demonstrate that the human enzyme is distinctly different from the homologues in other species and is involved in pre-rRNA processing. An internet search of the XREF data base in the National Center of Biotechnology Information (NCBI) yielded a 393-base pair human expressed sequenced tag (GeneBank accession numberAA083888) homologous to yeast RNase III (RNT1, GeneBank accession number U27016) and its C. elegans RNase III homolog (accession number Z81070). The first set of three oligonucleotide primers (NIII-2, NIII-4, and NIII-6) corresponding to the human expressed tag sequence sequence was synthesized (Fig. 1 A). By 3′ RACE (rapid amplification of 3′ cDNA), the human RNase III cDNA 3′ from the expressed tag sequence was amplified by polymerase chain reaction, using human liver Marathon ready cDNA (CLONTECH, Polo Alto, CA) as templates, and NIII-2/AP1 (for the first amplification) and NIII-4/AP2 (for the second amplification) as primers (Fig. 1 A). The standard polymerase chain reaction procedure was performed using native pfu DNA polymerase (Stratagene, San Diego, CA) and its reaction buffer. The annealing temperature range was 55–60 °C. The elongation time was approximately 6–8 min. The fragments were subjected to agarose gel electrophoresis in the TAE buffer, denatured in 0.5 m NaOH and then electronically transferred to a nitrocellulose membrane (Bio-Rad) for confirmation by Southern blot. Southern blots were performed using 32P-end labeled NIII-6 oligonucleotide as a probe in hybridization buffer (6 × SSC, 5 × Denhardt's solution) containing 100 μg/ml sheared denatured salmon sperm DNA, 0.5% SDS, 10 mm EDTA at 46 °C for 4 h, then washed twice with 1 × SSC and 0.1% SDS at 42–59 °C for 20 min. The confirmed fragments were excised from the agarose gel and purified by gel extraction (Qiagen, Germany), then subcloned into a zero-blunt vector (Invitrogen, Carlsbad, CA) and subjected to DNA sequencing. A human liver cDNA λ phage Uni-ZAP library (Stratagene, La Jolla, CA) was screened using the RACE products as specific probes. The positive cDNA clones were excised into pBluescript phagemid from λ phage and subjected to DNA sequencing. Sequencing of the positive clones was performed with an automatic DNA sequencer by Retrogen Inc. (San Diego, CA). Since the cDNA clones from the first run of the screening library were not long enough to encompass the full-length of human RNase III sequence, several sets of oligonucleotide primers based on the information from the cloned cDNA fragments were synthesized. Using these primers, several runs of 5′ RACE were performed to clone three overlapping cDNAs, which resulted in full-length of cDNA (Fig. 1, A and B). The overlapping sequences were aligned and combined by the assembling program of MacDNASISv3.0 (Hitachi Software Engineering Co., America, Ltd.). Protein structure and analysis were performed by the program MacVector v6.0 (Oxford Molecular Group, United Kingdom). A homology search was performed on the NCBI data base through the internet. An SR domain peptide (H-CRSDYDRGRTPSRHRSYERS-OH, amino acids 266–284) and an RNase III domain peptide (H-CRWEREHQEREPDETEDIKK-OH, amino acids 1356–1374) (Fig.1 C) were synthesized, purified (>90% pure), and conjugated to diphtheria toxoid with maleimidocaproyl-N-hydroxysuccinimide and used to raise polyclonal antibodies in rabbits. Anti-SR and anti-III peptide IgGs were affinity purified with SR and III peptides coupled to thiopropyl-Sepharose 6B, respectively (26Harlow E. Lane D. Antibodies : A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Nuclear and non-nuclear fractions from HeLa cells were prepared as described (27Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar). Whole cell, non-nuclear, and nuclear fractions were boiled in SDS sample buffer. Then the samples were separated by SDS-polyacrylamide gel electrophoresis using 4–20% Tris glycine gels (NOVEX, San Diego, CA) under reducing conditions. Molecular weight prestained markers were used (NOVEX) to determine the protein sizes. The proteins were electrophoretically transferred to a polyvinylidene difluoride membrane and processed for immunoblotting using affinity purified anti-SR or anti-III peptide antibody at 5 μg/ml. The immunoreactive bands were visualized using the enhanced chemiluminescence method (Amersham Pharmacia Biotech) and analyzed using PhosphorImager Storm 860 (Molecular Dynamics, Sunnyvale, CA). A cDNA fragment encoding the human RNase III-like domain (COOH-terminal 466 amino acids) was amplified by polymerase chain reaction and introduced into a BamHI site upstream andNotI site downstream. This fragment was further subcloned into the sites of the expression vector pGEX-4T-1 (Amersham Pharmacia Biotech) to produce the RNase III fusion protein with glutathioneS-transferase (GST) at its NH2 terminus. The identity of the construct was proven by DNA sequencing. The GST-RNase III fusion protein was expressed in E. coli strain BL21 and purified using glutathione-agarose (Amersham Pharmacia Biotech) under native conditions with B-PER bacterial protein extraction reagent (Pierce, Rockford, IL). Control GST protein was also prepared in parallel from the pGEX-4T-1 plasmid. The purified products were identified by Coomassie staining after 12% SDS-polyacrylamide gel electrophoresis and Western blot analyses with anti-RNase III peptide antibody (see above). The dsRNA substrate was generated by hybridization of two complementary strands of RNA produced with T7 and T3 polymerase transcription of the polylinker region of the pBluescript II KS(−) plasmid (Stratagene, San Diego, CA). The plasmid was digested with either SstI or KpnI and further purified with phenol/chloroform extraction and ethanol precipitation. The SstI- or KpnI-digested plasmids were then transcribed using T7 or T3 RNA polymerase, respectively (Stratagene, San Diego, CA), with or without [α-32P]UTP. The resulting transcribed RNAs (about 100 nucleotides) were purified by electrophoresis on 6% denaturing polyacrylamide gel. The32P-radiolabeled T7 transcript and unlabeled T3 transcript fragments were mixed and heated for 5 min at 90 °C in a buffer containing 20 mm KCl, 50 mm Tris-HCl (pH 7.5), 0.1 mm EDTA. MgCl, bovine serum albumin, and RNase inhibitor were added to the mixture after heating (final concentrations were 10 mm, 100 ng/ml, and 10 units/ml, respectively). The mixture was incubated at 37 °C for 2 h and the duplex RNA was purified on 6% non-denaturing gels. The 32P-labeled T7 transcript was also used as the ssRNA control substrate. To evaluate cleavage, 0.4 μg of GST protein or GST-RNase III (approximately 5–10 pmol of purified GST-RNase III) fusion protein were incubated with labeled dsRNA (250,000 cpm) (approximately 5–10 fmol) and ssRNA (250,000 cpm) at 37 °C in a buffer containing 20 mm KCl, 50 mm Tris-HCl (pH 7.5), 5 mm MgCl, 50 mm NaCl, 0.1 mm dithiothreitol, 0.1 mg/ml yeast tRNA, and 10 units/ml RNase inhibitor in the total volume of 60 μl. The digested samples were quenched at specific times and analyzed using nondenaturing polyacrylamide gel electrophoresis and PhosphorImager analysis. HeLa cells were cultured in chamber slides for immunostaining. Cells were washed once with d-PBS (pH 7.0), and then fixed in 10% neutral-buffered formalin for 10 min followed by washing three times with d-pbs. Fixed cells were then blocked for 30 min with 20% fetal bovine serum plus 0.5% Tween 20. Cells were first stained with anti-III or anti-SR peptide antibody (10 μg/ml) for 1 h at 37 °C, washed three times with d-PBS plus 0.1% Nonidet P-40, and incubated for 1 h at 37 °C with the fluorescein isothiocyanate goat anti-rabbit IgG (Jackson ImmunoResearch Laboratory, Inc., West Grove, PA). The cells were washed with d-PBS three times and mounted in mounting medium (Vector, Burlingame, CA) for examination under a fluorescence microscope. HeLa cells were synchronized at early-S phase using the double thymidine method (28Johnson R.T. Downes C.S. Meyn R.E. Fantes P. Brooks R. The Cell Cycle: A Practical Approach. IRL Press, New York1993: 1-24Google Scholar). Briefly, cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 2 mm thymidine for 17 h. After washing twice with d-pbs, cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum for 9 h followed by a second thymidine treatment for 15 h. Synchronized cells were then washed twice with d-pbs, cultured, and harvested at 0, 2, 4, 6, 8, and 24 h for immunofluorescence staining and FACS analysis. HeLa cells were detached from culture flasks with trypsin-EDTA and washed once with d-pbs containing 5 mm EDTA. Cells were then fixed in 70% ethanol for 1–24 h at 4 °C followed by propidium iodine (50 μg/ml) staining for 1 h at room temperature. Cell counts and propidium iodine contents were determined by FACS analysis (Becton Dickinson and Co., San Jose, CA). 2′-Methoxyethyl chimeric phosphorothioate oligonucleotides (Table I) were synthesized and purified as described previously (29McKay R.A. Miraglia L.J. Cummins L.L. Owens S.R. Sasmor H. Dean N.M. J. Biol. Chem. 1999; 274: 1715-1722Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). They were >85% pure. HeLa cells were transfected with oligonucleotides mixed with Lipofectin (Life Technologies, Inc., Gaithersburg, MD) at various concentrations for 5 h at 37 °C in serum-free Dulbecco's modified Eagle's medium. After removing the oligonucleotide containing medium, cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum for another 0 to 67 h and harvested for analysis.Table ISequences, mRNA target sites and effects of antisense oligonucleotides designed to bind to human RNase III mRNABold letters in sequences represent 2′-MOE nucleotides; others are deoxynucleotides. All oligonucleotides contained phosphorothioate linkages throughout the molecule. Below the table is the chemical structure of 2′-O-methoxyethyl and 2′-deoxynucleotides with a phosphorothioate linkage between them. % inhibition: reduction of RNase III mRNA level by antisense oligonucleotides after treatment with 200 nm for 24 h compared to control (no oligonucleotide treatment). N/A represents not applicable, control oligonucleotide and ND represents not detectable inhibition. Bold letters in sequences represent 2′-MOE nucleotides; others are deoxynucleotides. All oligonucleotides contained phosphorothioate linkages throughout the molecule. Below the table is the chemical structure of 2′-O-methoxyethyl and 2′-deoxynucleotides with a phosphorothioate linkage between them. % inhibition: reduction of RNase III mRNA level by antisense oligonucleotides after treatment with 200 nm for 24 h compared to control (no oligonucleotide treatment). N/A represents not applicable, control oligonucleotide and ND represents not detectable inhibition. Total RNA was isolated from different human cell lines (ATCC, Rockville, MD) using the guanidine isothiocyanate method (30Kingston R.E. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 1. John Wiley & Sons Inc., New York1997: 4.2.3-4.2.5Google Scholar). Fifteen μg of total RNA was separated on a 1.2% agarose/formaldehyde gel and transferred to Hybond-N+ (Amersham Pharmacia Biotech) followed by fixing using UV cross-linker (Strategene, La Jolla, CA). The premade multiple tissue Northern blots were also purchased from CLONTECH (Polo Alto, CA). To detect RNase III mRNA, hybridization was performed by using32P-labeled human RNase III cDNA probe (1.4 kb, clone 3-1) in Quik-Hyb buffer (Strategene, La Jolla, CA) at 68 °C for 2 h. After hybridization, membranes were washed in a final stringency of 0.1 × SSC, 0.1% SDS at 60 °C for 30 min. To detect pre-rRNA and rRNA, hybridization was performed by using32P-end labeled oligo probes 5′ETS-1 (5′-CAAGGCACGCCTCTCAGATCGCTAGAGAAGGCTTTTCTCA-3′), corresponded to 5′ETS and 5.8S-1 (5′-CATTAATTCTCGCAGCTAGCGCTGCGTTCTTCATCGACGC-3′), corresponded to 5.8 S rRNA at 40 °C for 2 h and washed in 2 × SSC, 0.1% SDS at 40 °C for 1 h. Membranes were analyzed using PhosphorImager Storm 860 (Molecular Dynamics, Sunnyvale, CA). The cloning strategy is described in detail under "Materials and Methods" and in the legend to Fig. 1. Using primers based on a search of the XREF data base, an approximate 1.2-kb cDNA (clone U4) corresponding to the COOH-terminal portion of the protein was cloned by 3′ RACE. Eight positive clones were isolated by screening a liver cDNA library with this clone. The two longest clones, 3-1 and 3-4, correspond to the COOH-terminal region, 2636–3912 and 3350–4764 base pairs, respectively, of the full-length cDNA. With primers (3RACE1, 3RACE2, and 3RACE3) based on the NH2-terminal portion of the clone 3-4, 5′ RACE was performed to clone a cDNA (clone L40) of approximately 1 kb, which encodes the middle part of the full-length cDNA. In the same way, a cDNA (clone 25) of the NH2-terminal portion was cloned. Using clone 25 to screen the liver library again, several clones were isolated, but none of these included additional NH2-terminal sequence. The most NH2-terminal clone is 328, which corresponds the sequence 799–2191 base pairs. So the last 5′ RACE was performed with the primers (33G, 33H, and 33 Dec based on clone 25) and the NH2-terminal portion of the cDNA (clone 81) was generated. These overlapping clones were sequenced and assembled to a full-length human RNase III cDNA with the total of 4764 nucleotides (Fig.1 B, GenBank accession number AF189011). The cDNA contained a coding sequence of 4125 (from 246 to 4370) nucleotides that encodes a 1374-amino acid protein. The calculated molecular mass of the protein is 160 kDa based on the prediction of the first methionine as the translation initiation site. The proposed initiation codon is in reasonable agreement with the mammalian translation initiation consensus sequence (Kozak sequence) (31Kozak M. J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2810) Google Scholar). Northern hybridization analyses using clone 3-1 (Fig. 1 B, 3′ portion of cDNA) demonstrated that the human RNase III mRNA was approximately 5 kb. It was ubiquitously expressed in human tissues and cell lines (Fig.2). When clone 25 (Fig. 1, 5 portion of cDNA) was used to probe Northerns, equivalent results were obtained (data not shown). Compared with C. elegans, yeast, and bacterial RNase III, the full-length of human RNase III clone is substantially larger and contains multiple domains (Fig.1 C). The RNase III domain (approximately 426 amino acids) is located at the carboxyl terminus of the protein and is well conserved with other species such as C. elegans, yeast, and bacterial RNase III. This domain shares strong homology with C. elegans RNase III (41% amino acid identity, Fig. 1 D). Both the human RNase III domain and C. elegans RNase III contain two RNase III signature sequences. The human RNase III domain also contains multiple potential phosphorylation sites. The full-length human protein also contains proline-rich (220 amino acids) and SR-rich (250 amino acids) domains near the amino terminus (Fig. 1 C). The SR and RNase III domains are separated by the 478-amino acid region.Figure 2Northern blot analysis of expression of human RNase III. A, top, a Northern blot containing 15 μg of total RNA of human cell lines were hybridized with human RNase III COOH-terminal cDNA (clone 3-1) probe. Bottom, the blot was rehybridized with a glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA probe. B, top, a Northern blot containing 2 μg of poly(A)+ RNA extracted from human tissues was hybridized with cDNA clone 3-1 probe.Bottom, the blot was rehybridized with G3PDH cDNA probe.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To test whether the human RNase III domain can specifically cleave dsRNA, the RNase III domain-coding region was subcloned into a glutathioneS-transferase (GST) expression vector. The GST-RNase III fusion protein and GST alone were expressed, purified using glutathione-agarose, and analyzed by Coomassie Blue staining of the SDS-polyacrylamide gel electrophoresis and Western blot analysis with anti-human RNase III peptide antibody (data not shown). These studies showed that the human RNase III domain was greater than 85% pure. However, there was evidence of slight degradation during expression and purification. When incubated with labeled dsRNA and ssRNA, the GST-RNase III fusion protein preferentially digested the dsRNA without significant cleavage of ssRNA, while GST alone cleaved neither dsRNA nor single-stranded DNA substrate (Fig. 1 E). Thus, the cleavage observed was not due to contamination with ssRNases or dsRNases from E. coli. Ribonucleases V1(dsRNase), and T1 and A (ssRNases) were used as controls to confirm that the cleavage observed was dsRNA cleavage (data not shown). As described under "Materials and Methods," we incubated approximately 5–10 pmol of GST-RNase III domain with 5–10 fmol of dsRNA. Thus, the GST-human RNase III domain was perhaps 5–10-fold less active than the reported activity for yeast RNase III (23Elela S.A. Igel H. Ares Jr., M. Cell. 1996; 85: 115-124Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). However, the purification and reaction conditions and the substrate have not been optimized, and only the GST RNase III domain has been studied. Clearly, much more work is required with the full-length human enzyme before comparisons of the specific activities of the enzymes are possible. To determine the expression of the human RNase III protein, two anti-peptide antibodies were produced. The anti-III peptide antibody was derived from a peptide (amino acids 1356–1374) corresponding to the RNase III domain present in the COOH-terminal portion of the putative protein. The anti-SR peptide antibody was derived from a peptide (amino acids 266–284) corresponding to the SR-rich domain of the putative protein (Fig.1 C). Using these antibodies, Western blot analyses were performed to determine the size and localization of human RNase III. The anti-SR peptide antibody recognized a band in HeLa whole cell lysate with a size of approximately 160 kDa (Fig.3 A) which is near the calculated protein size confirming that the full coding region is expressed in HeLa cells. The anti-RNase III peptide antibody also recognized a 160-kDa protein in HeLa cells and that protein comigrated with the protein identified with the anti-SR domain antibody (data not shown). Similar experiments were performed using different human cell lines e.g. A549, T24, and HL60 with equivalent results (data not shown). To further confirm that the predicted protein is indeed expressed in human cells, the protein was partially purified with an RNase III domain peptide antibody column. The purified protein was then recognized on Western blots by both antibodies (data not shown). To determine the localization of the protein, nuclear and non-nuclear fractions from HeLa cells (Fig. 3 A) and other human cell lines (data not shown) were prepared and equal amounts of proteins (40 μg) were analyzed by Western blots. RNase III was present primarily in the nuclear fractions (Fig. 3 A). Non-nuclear fractions contained only trace amounts of protein, possibly due to the contamination during sample preparation. The anti-III peptide antibody gave results equivalent to those obtained with the anti-SR peptide antibody (data not shown). To better understand the localization of human RNase III, the protein was identified in cells by indirect immunofluorescence microscopy. The nuclei of HeLa cells were stained by both anti-SR (data not shown) and anti-III (Fig. 3 B) antibodies, confirming that human RNase III is present in the nucleus. Fig. 3 B also shows that RNase III is localized extensively in nucleus and occasionally observed in nucleoli. Double thymidine treatment was used to synchronize HeLa cells to early-S phase (Fig. 4) (28Johnson R.T. Downes C.S. Meyn R.E. Fantes P. Brooks R. The Cell Cycle: A Practical Approach. IRL Press, New York1993: 1-24Google Scholar). Two to four hours after releasing the thymidine block, HeLa cells entered S phase. Six to eight hours after releasing, HeLa cells entered the G2/M phase. There were no significant changes in the mRNA or protein levels
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