*Laboratory of Molecular Genetics,
Laboratory of Gene Regulation and Development,
Unit on Biologic Computation, National Institute of Child Health and Human Development,
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
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Abstract |
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Introduction |
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The RNases H in a variety of organisms have been studied extensively with respect to structure, function, and enzymatic properties on the basis of their specific degradation of the RNA in RNA-DNA hybrids (Crouch and Toulmé 1998
, pp. 1265). The RNases H participate in cellular processes, such as DNA replication, repair, and transcription, as well as in the replication of retroviral genomes. Esherichia coli has two RNases H (HI and HII), each having its own characteristic amino acid sequence (Ohtani et al. 1999a,
1999b
). Bacillus subtilis also has two active RNases H, both related by sequence to E. coli RNase HII (Itaya et al. 1999
; Ohtani et al. 1999a,
1999b
). Despite their similarity at the amino acid sequence level, these two B. subtilis proteins have very different specific activities, specificities of cleavage sites, and strikingly different divalent metal ion preferences and, therefore, have been classified as RNases HII and HIII. Bacillus subtilis has a gene encoding a protein with strong sequence similarity to E. coli RNase HI but lacks a portion of the basic protrusion and has other changes that render the protein nonfunctional as an RNase H (Itaya et al. 1999
; Ohtani et al. 1999a
). The presence of a gene encoding an inactive RNase HI in B. subtilis and the disparate activities of RNases HII and HIII point out the difficulties in assigning a function merely on the basis of amino acid sequence similarity.
Thus far, at least one gene encoding an RNase Hlike protein is present in all prokaryotic and archael genomes (Ohtani et al. 1999b
). Most often there are two genes, either a combination of HI and HII or HII and HIII. Little is known about the number and types of RNases H in eukaryotes. Two proteins from mammalian sources and Saccharomyces cerevisiae (RNase H1 and RNase H2L) (Crouch and Cerritelli 1998
; Frank, Braunshofer-Reiter, and Wintersberger 1998
) have been shown to be related by amino acid sequence to E. coli RNases HI and HII. A third RNase H, RNase H70 (Frank et al. 1999
), also is present in S. cerevisiae that has sequence similarity to several other proteins in S. cerevisiae, including Rex3P (RNA exonuclease), Rex4P, and Pan2P, the last being a subunit of the polyA ribonuclease. All these proteins are related to exonuclease III, an enzyme known for many years to degrade RNA of RNA-DNA hybrids (Keller and Crouch 1972
). At present, there is no clear consensus amino acid sequence that will permit defining a protein related to RNase H70 as an RNase H.
An analysis of the genome sequence and EST data from C. elegans reveals four RNase HIlike genes. This large number of potential RNases H in a single organism is unprecedented, and there are features of the predicted proteins that are unique. In contrast, the human genome has two genes encoding RNase H1 and RNase H2. We characterized cDNAs obtained from the mRNA of C. elegans for different RNases H and identified unique structural features of some proteins. The splicing events for the regulation of the rnh-1.0 gene, their phylogenetic relationship, and the enzymatic properties of different RNases H expressed in E. coli are also described.
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Materials and Methods |
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Computer Analysis
Caenorhabditis elegans BLAST searches (Altschul et al. 1990
) were performed at either the Sanger (http://www.sanger.ac.uk/Projects/C_elegans/blast_server.html) or National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/BLAST/) web sites. Query RNase H protein sequences were E. coli RNase HI and RNases H1 of S. pombe, S. cerevisiae, and human. The human genome sequences at NCBI or at Celera were examined separately. Query sequences for the NCBI site were the four RNases H used to search the C. elegans genome plus the four RNase H1related proteins of C. elegans derived from this work. Accession numbers from each of the eight files were combined. Duplicate accession numbers were eliminated, creating a file used for Batch Entrez (http://www.ncbi.nlm.nih.gov:80/entrez/batchentrez.cgi?db=Nucleotide). The output from the Batch Entrez search is the Unmasked database. The Unmasked database was searched by the hurep.ref and hurep.sub file (http://www.girinst.org/server/RepBase/) (Jurka 2000
), removing repeat sequences and generating the Masked database. The Masked database was searched using local tBLASTn with each of the RNase H queries to determine which sequences were selected by all or only some of the protein queries. The Celera database (http://publication.celera.com) was searched using the human RNase H1 protein and mRNA (cDNA) sequence as queries using BLASTp and BLASTn, respectively. Analyses of DNA and protein sequences were done using Wisconsin Package Version 10.0 (Genetics Computer Group [GCG], Madison, Wisc.).
Phylogenetic Analysis
The phylogenetic analysis was performed on the multiple sequence alignment shown in figure 1A
using the maximum likelihood method implemented in version 3.6a2.1 of the PHYLIP package (Felsenstein 2001
). The alignment was generated using PILEUP of the GCG package, and adjustments were made, taking into account the structural data for E. coli RNase HI. The sequence alignment was converted to the PHYLIP format, and SEQBOOT was used to generate 100 data replicates. The data were subsequently analyzed with PROML (constant rate of change), followed by NEIGHBOR, and finally with CONSENSE to generate the bootstrapped tree. The phylogram was rooted with E. coli RNase HI, and the tree was visualized with TreeViewPPC version 1.5.3 (Page 1996
).
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Transcript Analysis
The 5' and 3'rapid amplification of cDNA end (RACE) reactions were carried out to analyze transcripts of different RNase H genes. The 3', 5'-RACE primers (Life Technologies) with gene-specific primers were used in PCR reactions to characterize the 3'- and 5'-ends of the messages. In some cases, primers specific for splice leaders (Huang and Hirsh 1989
) were used with the gene-specific primers to identify the 5'-end of messages. To confirm that the PCR reaction products were indeed derived from the target mRNA when splice leader and gene-specific primers were used in PCR reactions, Southern analysis of the PCR products was carried out using the appropriate probes. Two different mRNA preparations were used.
Expression in E. coli
Once the complete cDNA sequence was obtained and the coding region determined, primers were synthesized to amplify the coding regions such that an NdeI or NcoI site was at the first Met codon and the downstream primer included a BamHI or XhoI restriction enzyme site. The PCR products were cloned into pCRII-TOPO and digested with the appropriate enzymes, and the fragment was cloned into the pET15b expression vector (Novagen) and transformed into the BL21(DE3) pLysS E. coli strain (Novagen). Cells were grown at 32°C to mid-log phase, and expression was induced by the addition of IPTG (final concentration 1 mM), followed by incubation for 3 h. HIS-tagged proteins were purified from HIS-bind columns, as described by the manufacturer (Novagen, Clontech).
RNase H Activity
In Gel Assay
Renaturation gel assays were carried out with the partially purified HIS-tagged proteins (Han, Ma, and Crouch 1997
; Cazenave, Mizrahi, and Crouch 1998
). Renaturation was carried out either with Mg2+ or Mn2+ ions in the buffer. Autoradiograms were developed upon exposing gels to films.
Complementation Assay
The pET15b vectors harboring different rnh-like cDNAs derived from mRNAs of C. elegans were transformed into the MIC1066 E. coli strain (Cazenave, Mizrahi, and Crouch 1998
). Transformants were plated on LB-amp plates at 32 and 42°C. Growth at 42°C indicates that a functional RNase H is present in MIC1066. The pET15b vector was used as a control in this study.
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Results |
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Human Genome
Having found several RNase H1 proteins in C. elegans, we searched the human genomic sequence for genes encoding RNase H1like proteins. Although gaps are still present in the genomic sequence of the human DNA, we were unable to uncover any RNase H1encoding genes except for the RNASEH1 gene on chromosome 2 (our modification of AC108488see supplementary material at MBE web site: http://www.molbiolevol.org) and two pseudogenes; one located on chromosome 17p11.2 (AC022596.9) and one on chromosome 1q32.1-4 (AL035414.30) (supplementary material). Unlike the search of C. elegans, the human DNA has numerous retroviral sequences that yield very low (significant) expect scores (supplementary material). One class of these is shown in figure 2
and is a member of the human endogenous retrovirus L (HERVL) family. Elimination of these sequences for further examination using RepMask sequences reduced the total number of sequences to about 47 (supplementary material). These sequences score well in the BLAST searches because of the three highly conserved Trp residues because the substitution matrix Blosum62 (Henikoff S. and Henikoff J. G. 1992
) credits 11 points to Trp residues compared with 4 points for Leu-Leu matches. Interestingly, the conserved Trp residues are in the
B-
C-
Dregion (fig. 1A
), where HIV-1 RT has a deletion when compared with cellular RNases H1. Thus, the RNase H domain of HERVL elements are more similar to cellular RNase H1 than to the RNase H domain of HIV-1.
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In Gel Assay
Results obtained from the gel renaturation assays with labeled RNA-DNA hybrids are presented in figure 3
. RNase H1 (F59A6.6) and RNase H1A (ZK1290.6) exhibited enzymatic activity in this assay with activity detected in two bands for both samples. The major activity of RNase H1 (marked with an arrow) is coincident with the stained protein with some minor activity migrating at a position of a dimer of the 33-kDa protein. We occasionally see dimer bands in this gel assay (Han, Ma, and Crouch 1997
). RNase H1A activity marked by the arrow corresponds to the full-length protein, whereas the band at about 35 kDa most likely represents activity derived from a proteolytic product of RNase H1A. Proteins containing only the RNase H domain of RNase H1 or RNase H1A have significant RNase H activity in the gel assay (data not shown). Retention of the substrate in the region in the Activity Gel (fig. 3B
H1Aarrow at about 66 kDa) indicates the binding of the enzyme to the substrate without substantial degradation. This phenomenon is related to the N-terminal portion of RNase H1A to bind to some types of nucleic acids independent of the RNase H domain (A. Arudchandran and R. J. Crouch, unpublished data).
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Complementation Assay
The inability of the E. coli strain MIC1066 [rnhA-339::cat recB270(Ts)] to grow at 42°C can be overcome if an active RNase H protein is expressed. The strain has a T7 RNA polymerase gene to drive transcription of the RNase H genes cloned into the pET15b expression vector. In many instances, the RNase H produced by basal levels of transcription is sufficient to permit growth at 42°C. Of the five C. elegans RNase Hlike genes, only rnh-1.0 and rnh-1.1 were able to complement the temperature-sensitive phenotype of MIC1066 (data not shown). For complementation of the ts-growth defect by these RNase Hlike proteins, the polypeptide needs to exhibit RNase H activity. Thus, in agreement with the gel assay, RNase H1 and RNase H1A can express RNase H activity and require no C. elegansspecific modification to be active.
Evolutionary Relationships of RNase H1
The four RNases H1 of C. elegans share the several conserved amino acids present in RNases H of this class yet differ in significant ways. For example, RNase H1 and RNase H1A (fig. 1A and B
) have the RNase H domain attached to an N-terminal nonRNase H sequence, whereas RNase H1B and RNase H1C consist of only an RNase H domain. To assess their evolutionary relationship, we generated the maximum likelihood phylogram shown in figure 4
. The phylogram was generated using only the RNase H region seen in figure 1A.
The tree is shown rooted with E. coli RNase HI. Indeed, these results support, as suggested above, that the C. elegans RNases H1B and H1C are more related to one another than either is to any other RNase H used in this analysis. Interestingly, C. elegans RNase H1A groups with the S. cerevisiae and S. pombe RNases H1, albeit quite weakly. Also, the phylogram reveals that C. elegans RNase H1 does appear to be an orthologue of the human RNase H1. One interpretation of this phylogram suggests that the ancestral state may have consisted of multiple RNases H and that fungi and animals have both independently lost different members of this group. A more probable interpretation discussed later is that the RNase H of C. elegans has duplicated several times since its separation from the common ancestor of fungi and mammals.
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Discussion |
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We have taken advantage of the wealth of information about the DNA sequences of C. elegans and humans to examine potential genes coding for RNases H. To know what the number and types of RNases H might be in animals on the basis of the genomic DNA sequence, we used several RNases H as query sequences in a BLAST search (tBLASTn). Use of multiple proteins helps to overcome the poor conservation of sequences of RNases H1, including the spacing between those regions that are most indicative of RNases H. Even when employing these searches, no retrotransposon element of C. elegans was detected. The alignment shown in figure 1A
helps explain why we failed to detect C. elegans retrotransposons in our searches. Only 17 of 155 amino acids are identical in the alignment, and three important conserved regions are missing (the C region is missing as well as the region between ß5 and
E, including the important His124-E. coli numbering). Even though Cer1-1 contains a Glu48 residue (fig. 1A
), the context in which it resides bears little resemblance to the other RNases H1. In contrast, numerous human endogenous retrovirus (HERV) elements are readily detected in the human genome by using almost any of the RNase H proteins as query sequences. This indicates that many HERV RNase H domains are more recently derived from cellular RNases H or vice versa, whereas the separation between the origin of retroelements in C. elegans and cellular enzymes is much greater. This conclusion supports that of Malik and Eickbush (2001)
.
Five different genes yielding proteins related to RNases H were found in the C. elegans genome. In organisms such as E. coli and S. cerevisiae, cells deleted for both RNase Hencoding genes are viable (Frank et al. 1999
; Itaya et al. 1999
; Arudchandran et al. 2000
; A. Arudchandran and R. J. Crouch, unpublished data). We found that RNAi (Fire et al. 1998
) inactivation of any or several of the RNase H mRNAs also produced no easily detectable phenotype (data not shown). Thus far, only in Drosophila melanogaster is there a serious defect related to an RNase H mutation (Filippov, Filippova, and Gill 2001
).
Of the five RNase Hrelated genes, one is similar to RNase H2 or RNase HII. When expressed in E. coli or when assayed in a gel renaturation assay, no enzymatic activity is detected (fig. 3
). We have expressed RNase H2 from S. cerevisae, human, and mouse and uniformly find no enzymatic activity (Crouch and Cerritelli 1998
). A similar observation has been reported for the human protein (Lima, Wu, and Crooke 2001
), but others have observed very weak activity after refolding of the S. cerevisiae RNase H2 in E. coli (Qiu et al. 1999
). The RNase H2 may be composed of two subunits (Frank et al. 1998
) or may require modification to exhibit RNase H activity.
Two C. elegans proteins, similar to RNase HI of E. coli in amino acid sequence, exhibit RNase H activity when expressed in E. coli. Caenorhabditis elegans RNase H1 is similar to most RNases H1 of eukaryotes having a duplex RNAbinding domain at its N-terminus and the RNase H domain at the C-terminus. RNase H1A is unique. The N-terminal region is not found in any of the other RNase H sequences and contains a large number of the Arg-Ser repeats (fig. 1B
), typical of SR proteins involved in splicing (Graveley 2000
). The Arg-Ser repeats are important for protein-protein interactions and may direct these proteins to the spliceosome (Yuryev et al. 1996
). There seems to be no obvious direct role for RNase H in splicing, and we are unaware of any report indicating a requirement for RNase H in splicing. The importance of the amino terminal region is unclear, particularly in light of the fact that it is not required for enzymatic activity (data not shown). The RNase H domain does differ from those of other active RNases H1. In particular, the
B-C-D-helices of RNase H1A are more similar in size and content to HIV-1 RNase H than to E. coli RNase HI, suggesting that additional amino acids are important for the binding of the protein to nucleic acid substrates. It may be that the C-terminal extension seen in RNase H1A supplies the binding function through the many basic amino acid residues present there. It should be pointed out that several of the Arg residues are followed by Ser, similar to what is found near the N-terminus of the protein. We are currently examining RNase H1A for determinants of RNase H activity and inquiring into the role of the nonRNase H domain.
The RNases H1B and H1C are inactive, as expressed from the cDNAs we have cloned. The genes encoding RNases H1B and H1C contain introns and, therefore, are probably not pseudogenes. RNase H1C does not have the C-terminal E-helix whose presence is necessary for enzymatic activity (Haruki et al. 1994
; Goedken, Raschke, and Marqusee 1997
). If a splice were to occur near the end of the gene, an
-helix could possibly be attached. We have examined four independently derived cDNAs and have found no example of a transcript encoding the putative
E-helix. The defect in RNase H1B is most likely due to the unusual nature of the
B-
C-
Dregion. In RNase H1B, there are numerous Thr and Ser residues rather than the typical basic and Trp residues at conserved locations (fig. 1A
). We have obtained three independent clones of rnh-1.2, all of which have the same sequence. The RNases H1 and H1C contain introns between the coding sequences for
A and ß4 (fig. 1A
reverse letters indicate splice site). RNase H1B has no equivalent splice site. Translation of the mRNA in the
B-
C-
Dregion of the rnh-1.2 in all three reading frames reveals the presence of an out-of-frame coding sequence that yields a very good
B-
C-
Dregion, thereby suggesting that the formation of an active RNase H1B is possible (data not shown). The RNases H1B and H1C may have a function(s) other than providing RNase H activity, but it is also possible that their expression in an RNase H active form may be limited to specialized situations.
The abundance and diversity of alternatively spliced mRNAs of C. elegans RNase H1 is striking (fig. 5B and supplementary material) and makes it clear that synthesis of RNase H1 is regulated by splicing. The primary transcript is differentially spliced to produce two types of dicistronic messages, one of which is processed by the usual pathway for the generation of two mRNAs encoding two different proteins. Because the intercistronic region having the poly(A) addition signal is deleted when splicing occurs in the alternatively spliced mRNA (fig. 5B exon 3 to exon 4), the two coding regions remain on a single message and would require internal initiation of translation to produce RNase H1. The alternative splice joining exon 5 to exon 6 (fig. 5B ) does not permit the synthesis of an active RNase H1.
Generality of Multiple Genes in C. elegans
In contrast to the C. elegans genome, we have been unable to find evidence for multiple RNase H1like proteins in the human genome. This disparity in the numbers of proteins of a particular type between C. elegans and other genomes is not unique to RNases H (Combes et al. 2000
; Keiper et al. 2000
; Robertson 2000
; Hodgkin 2001
). Sternberg (2001)
has suggested that each of the small number of cells comprising C. elegans may be more complex or may respond in more complex ways due to increased molecular diversity within each cell. One prime example is in olfactory neuronal cells where one cell possesses multiple receptors and yet can sense different odors (Bargmann 1998
). In simpler cell types, a single protein may perform many functions but would be limited in its role in any cell by the presence of one or only a few substrates. For example, RNase H1 in human cells may have multiple functions, but within a given cell type these functions may be limited by environmental factors such as substrates. In C. elegans, RNases H of several types may be present within a single cell type but may of necessity be limited to one function or one cell organelle, and the apparent regulation of RNase H1 levels by splicing may indicate that this protein may be able to recognize all the cellular substrates and, therefore, needs to be kept under tight control so that it does not subsume another enzyme's role. Alternatively, RNase H1A may have a requirement uniquely present in C. elegans for splicing or some splice-related event, as indicated by the N-terminal SR character (fig. 1B
).
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Acknowledgements |
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Footnotes |
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Keywords: ribonuclease H
splicing
multiple genes
Caenorhabditis elegans
human
genome
double-stranded RNA
RNA-DNA hybrids
Address for correspondence and reprints: Robert J. Crouch, Building 6B Room 2B-231, 6 Center Drive MSC 2790, National Institutes of Health, Bethesda, MD 20892. robert_crouch{at}nih.gov
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