BACKGROUND
with a cumulative incidence by age  <dig> of 14% among caucasian-americans and 25% among african-americans, prostate cancer has become the most common non-cutaneous malignancy in the us  <cit> .
despite its high incidence rate, there is little known about the genetics and etiology of prostate cancer.
we recently identified a candidate prostate cancer susceptibility gene through linkage analysis and positional cloning  <cit> .
this gene, elac <dig>  is located on chromosome 17p and encodes a protein of  <dig> amino acids with trnase z activity   <cit> .
elac <dig> belongs to an incompletely characterized family of proteins that is conserved among eukaryotes, archaea and bacteria.
elac <dig> is ubiquitously expressed in all tissues with the highest expression levels found in testes.
elac2p is detected in several cancer cell lines and binds the γ-tubulin complex  <cit> .
at present the role that sequence variants in elac <dig> may play in the genesis of prostate cancer is unclear.
trz <dig> , the yeast ortholog of elac <dig>  encodes an essential protein of  <dig> amino acids.
like elac <dig> and its other eukaryotic orthologs, trz <dig> can be divided roughly into amino- and carboxy-halves, or domains, which are structurally homologous  <cit> .
the carboxy domain possesses trnase z activity; the amino domain is enzymatically inactive but may modulate the specificity of carboxy domain.
the most strongly conserved sequence element is the histidine motif, located near the front of the carboxy domain.
in the amino domain, the structural homolog of the histidine motif is easily recognizable; however, most of the key zinc coordinating histidines have changed to some other polar amino acid, which is entirely consistent with the observation that this domain lacks trnase z activity.
prosite scans revealed that the vertebrate elac2s contain a sequence element near the center of their amino domain that matches the nucleotide binding p-loop motif  <cit> .
although not exactly matching the canonical p-loop sequence, this sequence element is well conserved in trz <dig> and its structural homolog is well conserved near the center of the carboxy domain of all gene family members.
it has recently been shown that both of the two elac family members  from the animals, plants, fungi, bacteria, and archaea possess trnase z activity  <cit> .
the amino acid and enzymatic conservation across elac family members suggests strongly that they all share some basic cellular functions.
since saccharomyces cerevisiae is a well-characterized genetic system, we initiated a genetic analysis of the functions of the yeast member of this gene family, trz <dig>  in order to better understand those of elac <dig>
rna processing, one of the post-transcriptional controls on gene expression, plays an important role in regulating cell growth and fate.
pre-mrna processing is a multi-step process coupled to transcription through the involvement of the unique c-terminal domain  of the large subunit of rna polymerase ii.
the processing involves rna binding proteins, snrnas , endo- and exo-nucleases, and other specific factors.
the function of these proteins is subject to regulation by different signaling pathways, according to the developmental stage of the cell  <cit> .
transfer rnas  undergo extensive post-transcriptional modifications, including: 5' processing; 3' processing; nucleotide modification; and, in the case of about 20% of yeast trna, intron removal in the nucleus before export to the cytosol.
rnase p , which consists of a rna subunit and nine proteins, is responsible for 5' trna processing .
two proteins, trz1p  <cit>  and lhp1p  <cit> , have been shown to be involved in 3' trna maturation in yeast; trz1p has trnase z activity while lhp1p stabilizes pre-trnas as an rna chaperone  <cit> .
it is known that there are more than  <dig> proteins involved in trna splicing, modification, degradation and export.
ribosomal rnas  are transcribed as large precursors by rna polymerases i and iii in nucleoli  <cit> .
these rnas also undergo processing, including 5' and 3' cleavages, removal of introns, and nucleotide modifications through the action of a series of rna/protein complexes.
the mature rrnas complex with about  <dig> proteins to form ribosomes.
the rnase mrp  complex is responsible for the majority of 5' maturation steps and the exosome complex is responsible for 3' rrna maturation.
many of the modifications and splicing events require small nucleolar rnas  and other specific protein factors.
notably, some proteins are shared between the trna and rrna maturation complexes .
in this paper we show that, although trz1p is required for growth, yeast are largely insensitive to the absolute level of its expression.
elac <dig> cannot functionally replace trz1p, indicating that trz1p has additional or specific functions.
in the carboxy domain of the protein, the conserved histidine motif and the putative p-loop are important for trz1p biological function because mutations of the conserved residues in these motifs render the protein inactive in yeast.
through a high copy suppressor screen, we found that trz <dig> genetically interacts with rex <dig>  suggesting a role for trz <dig> in rna processing and mitochondrial maintenance.
RESULTS
effect of altered trz <dig> expression on yeast cell growth
we generated three yeast strain types in order to study the consequences of altered trz <dig> expression in yeast.
in the first , trz <dig> is expressed from a gal <dig> promoter on a low-copy plasmid in a wild type trz <dig> background.
in the second , the gal <dig> promoter is inserted upstream of trz <dig> between the translational start site and its endogenous 5'utr at the chromosomal locus .
in the third strain type , endogenous trz <dig> is deleted and the gene is expressed from a gal <dig> promoter on a low-copy plasmid.
the latter two strain types were made to investigate the effect of up- and down- regulation of trz <dig> expression on growth.
the genotypes of all strains used in this study are listed in table  <dig>  also listed in table  <dig> is the ρ +/ρ - status of each strain, assayed as the ability of the strain to grow on media containing only non-fermentable carbon sources.
the significance of the ρ +/ρ - status, which represents the wild type  or impaired   mitochondrial respiratory function, will become clear below.
as shown in table  <dig>  when trz <dig> is over-expressed in a wild type background , the yeast doubling time is  <dig>  hours , not significantly longer than that of yph <dig>  the parental yeast control  or that of wild type yeast that over-express β-galactosidase .
therefore, over-expression of trz <dig> in wild type yeast does not significantly slow yeast growth.
overexpression of flag-trz <dig> in yl09- <dig> was verified by western blot with anti-flag antibody.
as shown in figure 1a, flag -trz <dig> is rapidly induced in yl09- <dig> grown on galactose-containing medium.
notably, both yl09- <dig> and yl09- <dig> are ρ+.
we then investigated the consequence of over-expression of trz <dig> from the chromosomal locus.
in yl10- <dig> a gal <dig> promoter is inserted immediately upstream of the translational start codon of flag-trz <dig>  as shown in figure 1b, flag-trz <dig> is promptly induced after addition of galactose in the yl10- <dig> strain.
liquid growth assays in a variety of different media were done to determine the doubling time of strains expressing varying levels of trz1p .
no growth was observed for yl10- <dig> and yl10- <dig> in galactose medium, respectively, compared to  <dig>  hr doubling time foryph <dig>  however, these gal <dig> promoter insertion strains also grew more slowly in non-inducing medium.
the doubling time for yl10- <dig> and yl10- <dig> in glucose medium is  <dig> and  <dig> times longer than that of parental yph <dig> cells .
we experienced difficulties when trying to isolate gal <dig> promoter insertion strains and found that the isolated strains were petite mutants; therefore, we decided not to study these strains further.
we observed the growth characteristics of trz <dig> strains with trz <dig> expressed from a gal <dig> promoter on a low-copy plasmid .
as shown in table  <dig>  when trz <dig> expression is repressed in glucose medium, yl10- <dig> grows marginally slower  than yph <dig>  the isogenic wild type yeast .
the same is true for yl10- <dig> and yl10- <dig> with respect to yph <dig>  their isogenic wild type strain.
thus, although trz <dig> is an essential gene, even low levels of its transcript are sufficient for near normal growth.
when trz <dig> expression is induced in galactose medium, however, yl10- <dig> and yl10- <dig> fail to grow, while yl10- <dig> grows at about the same rate as wild type yeast.
this is intriguing, particularly when comparing yl10- <dig> and yl10- <dig>  because the strains share the same genetic background.
they differ in two ways: yl10- <dig> is ρ-  while yl10- <dig> is ρ+ ; and yl10- <dig> expresses untagged trz <dig> while yl10- <dig> expresses trz <dig> tagged with a flag epitope.
the phenotypic discrepancy between yl10- <dig> and yl10- <dig> is not likely due to a lack of expression of trz1p or lack of functional trz1p in yl10- <dig>  because we could detect flag•trz1p in yl10- <dig>  and p414/gal1p-flag•trz <dig> could functionally complement the trz <dig> deletion .
the difference between the strains is more likely due to their ρ status.
we studied these strains further by testing the effect of different carbon sources on their growth.
table  <dig> summarizes these results.
when trz <dig> expression from a gal1p-driven trz <dig> plasmid is suppressed on sc-glucose, wild type yeast  and trz <dig> yeast  grow at a rate similar to that of yph <dig> at room temperature, 30°c and 37°c.
we could not detect any significant difference in growth rate among yph <dig>  yl09- <dig> and yl10- <dig> on sucrose, raffinose or galactose at any temperature tested .
this result agrees with the results from liquid growth assays in that over-expression of trz <dig> in these two strains  does not result in any discernable change in growth rate.
when spotted onto glucose media and incubated at room temperature, yl10- <dig> and yl10- <dig> grow at a rate equivalent to that of yph <dig>  as the incubation temperature is increased, however, yl10- <dig> and yl10- <dig> grow significantly slower than yph <dig>  when spotted on sucrose media, these strains grow slower than yph <dig> at all tested temperatures, with the most significant difference in doubling time occurring at 37°c.
yl10- <dig> and yl10- <dig> barely grow on raffinose and do not grow at all on galactose.
as argued above, the phenotypic discrepancies among the strains is not likely due to differences in expression of functional trz1p.
instead, the different phenotypes resulting from galactose-induced expression of trz1p is more likely attributable to the ρ+/ρ- status of the strains, consistent with the known inability of ρ- cells to grow on galactose-containing media  <cit> .
thus, the ρ- stains  show sugar and temperature sensitivity; the ρ+ strains  do not.
in summary, yeast are largely insensitive to the level of trz1p, provided there is some minimal level of the protein; the apparent sensitivity of some strains to galactose-induced trz1p expression is attributable to the non-specific galactose-sensitivity of ρ- yeast.
elac <dig> does not functionally substitute for trz1; motifs required for trz1p function
since trz <dig> is the yeast homolog of elac <dig>  we tested the ability of elac <dig> to functionally complement deletion of trz <dig> in yeast.
yl03- <dig> was transformed with an array of elac <dig> constructs  .
transformants were plated onto foa to select against the trz <dig> expression plasmid and to determine if any construct could complement trz <dig>  as shown in table  <dig>  neither wild type nor prostate cancer-associated elac <dig> mutants could substitute for wild type trz <dig> function in yl03- <dig>  the human paralog of elac <dig>  elac <dig>  likewise failed to rescue trz <dig> .
we next incorporated three specific mutations into the histidine motif region of trz1:  m535t, which corresponds to the missense substitution a541t in elac <dig> that is associated with human prostate cancer;  h542ter, which corresponds to the frameshift 1641insg associated with human prostate cancer; and  g548r, which corresponds to a ts mutant, g256r, that was isolated in the homologous yeast gene pso <dig>  <cit>   .
notably, in no case did the fusion of two copies of the flag epitope to trz <dig> alleles influence results .
the missense mutant trz <dig> allele  complements trz <dig> as well as the wild type gene.
however, neither trz <dig> g548r in which a conserved glycine in the histidine motif is altered, nor the mutant trz <dig> truncated after his <dig> in the same motif  could complement trz <dig> in yeast.
motif searches identified a putative p-loop  at amino acids 276– <dig> of human elac <dig> .
this sequence element is located near the center of the n-domain and is clearly conserved among the eukaryotic orthologs.
the corresponding sequence in trz1p  falls within the range of variation for the structural p-loop recently explored by brakoulias et al  <cit> .
in the homology between the n- and c-domains of the protein, this candidate p-loop corresponds to a strongly conserved element near the middle of the c-domain , which also has features reminiscent of the structural p-loop  <cit> .
for simplicity, we will refer to these as the first and second putative p-loops.
in so doing, however, we recognize that they may be examples of some other conserved structural element.
we mutated the key residues in both putative p-loops individually and tested the mutants' abilities to complement trz <dig>  the first putative p-loop is dispensable for survival because mutants trz <dig> g229d and trz <dig> q230a, which should destroy its function if it is a p-loop  <cit>  , can functionally substitute for the essential function of trz1p.
however, the second putative p-loop is required for yeast survival because mutants here  cannot complement trz <dig> deletion.
sequence analysis shows that trz <dig> may be the result of ancient tandem duplication/fusion that produced a gene with homologous amino and carboxyl halves.
we therefore tested whether either the n- or c- domain of trz <dig> alone is capable of rescuing trz <dig>  neither half could rescue , even when fused to the complementary domain of elac <dig>  .
screen for and characterization of temperature sensitive  trz <dig> mutants
we screened for ts mutants of trz1p with pcr-based random mutagenesis and bacterial mutator approaches; neither of these attempts identified ts mutants.
to circumvent technical difficulties, we used a targeted approach instead.
we designed degenerate oligos targeting sequence encoding amino acids  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> in the trz1p histidine motif .
these amino acids were selected for mutagenesis because, with the exception of residue  <dig>  they are less conserved across species than the canonical residues of the histidine motif; therefore, mutation may be less likely to completely inactivate protein function .
g <dig> was targeted because, as mentioned before, a ts mutant had been identified at the corresponding amino acid in s. cerevisiae pso <dig>  we generated  <dig> mini-libraries, each of which targets one of these residues in yl03- <dig>  and isolated the clones that appeared at 30°c but that grew slowly or not at all at 37°c.
to confirm the ts phenotype plasmids from these isolates were purified and used to transform naïve yl03- <dig>  the screen identified two ts mutants of trz1p, y537l and l538k .
we also found mutations at position  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> that are lethal at both 30°c and 37°c.
observation of lethal missense substitutions at m <dig> is intrinsically interesting because it is consistent with the idea that the corresponding residue a <dig> of human elac <dig> is under some functional constraint.
more than 60% of the lethal mutations were concentrated at g <dig>  indicating that this residue is more functionally constrained than the corresponding g <dig> of s. cerevisiae pso <dig> .
we integrated trz <dig>  trz1- <dig>  and trz1- <dig>  into the leu <dig> locus in a trz <dig> background to yield yl13- <dig>  yl13- <dig>  and yl13- <dig>  respectively.
as shown in figure  <dig>  the ts mutants grew more slowly on glucose medium than did the controls at all temperatures tested.
the inhibition of growth was more obvious at higher temperature.
growth of the ts mutants was further slowed on sucrose.
when plated on raffinose, ts mutants grow only at 30°c.
yl13- <dig> and yl13- <dig> are also petite mutants  while yl13- <dig> is not .
the ability of yl13- <dig> but not yl13- <dig> or yl13- <dig> to grow on galactose can therefore be explained in terms of the known inability of ρ- yeast to grow on galactose  <cit> .
table  <dig> summarizes the doubling time for each ts mutant at different temperatures in sc-leu medium containing glucose.
at 30°c yl13- <dig> and yl13- <dig> grow at about the same rate in liquid medium containing glucose as does yl13- <dig>  however, at 37°c the ts strains grow significantly more slowly in glucose  than does yl13- <dig>
we integrated the trz1- <dig> allele at the trz <dig> locus to yield yl12- <dig>  yl12- <dig> exhibits a slow-growth phenotype like that of the leu <dig> integrants .
yl12- <dig> grows slower on glucose media compared with the isogenic wild type yph <dig> at all temperatures tested and this growth difference increases at 37°c .
the trz1- <dig> integrants exhibit severe ts growth on sucrose containing medium and they do not grow on raffinose and galactose containing medium .
the doubling time of these mutants in glucose-supplemented liquid medium is in agreement with results from the plate assay .
to verify that the growth phenotype and sugar sensitivity observed with trz1- <dig> integrants do not result from a cloning artifact, we did the following.
yph <dig> and yl12- <dig> were transformed with empty vector  or p416/trz <dig> and plated onto sc-ura containing glucose.
once grown, colonies were replica-plated onto sc-ura supplemented with glucose, sucrose, raffinose or galactose.
providing a wild type trz <dig> on a cen vector restored the growth of yl12- <dig> to the level of yph <dig> on all media tested .
yl12- <dig> transformed with an empty vector grew more slowly than yph <dig> on all media tested, with the decrease in growth rate in the order of glucose, sucrose, raffinose and galactose media, in agreement with the previous plate assay.
in addition, we checked the ρ status for these transformants.
providing a copy of wild type trz <dig> on a cen vector  converted yl12- <dig> from ρ- to ρ+ , indicating that the petite phenotype of yl12- <dig> may not be due to the loss of mitochondrial dna.
instead, it may result from the interference of important mitochondrial functions, suggesting that trz1p also plays a role inside mitochondria.
however, normal growth on a non-fermentable carbon source was not restored in diploid strains derived from crosses between ts mutant strains and a haploid ρ <dig> tester strain .
given the fact that the tester strain should have a wild type copy of trz <dig> and ts mutant haploid strains should have mitochondrial dna, we speculate that ts alleles of trz <dig> may have pleiotropic effects on certain important activities in mitochondria.
the effect on some of these activities may be dominant, and mating with a wild type haploid strain lacking mitochondrial dna may not fully restore the mitochondrial functions.
to determine if the trz1- <dig> allele causes any cell cycle arrest phenotype, we analyzed yl12- <dig>  with yph <dig> as control, by facs; both strains were grown at rt or 37°c for this study.
allele trz1- <dig> caused a  <dig> hour delay in cells entering 2n  at 37°c.
when yeast reached stationary phase, yl12- <dig> had more cells in 2n  than yph <dig> grown under the same conditions .
when viewed under a microscope the trz1- <dig> integrants were observed to aggregate when grown at 37°c while parental yph <dig> cells did not.
with yl12- <dig> we also observed enlarged cells and cells with elongated buds; the percentage of these abnormally shaped cells increased with the length of time that the cells were grown at 37°c .
therefore, trz1- <dig> allele does not cause any cell cycle arrest phenotype and the abnormal shape we observed is most likely the result of cell death in yeast.
high copy suppressor screen
during the characterization of yl12- <dig>  we identified a window of two days wherein it would be possible to screen for high copy suppressors of trz1- <dig>  as shown in figure  <dig>  at 30°c, the appearance of yl12-01/p416gal <dig> transformants takes  <dig> days on sc-ura.
however, colonies appear after only  <dig> days when the same strain is transformed with p416/trz <dig> .
yph <dig> transformed with vector or trz <dig> resulted in observable colonies after  <dig> days of incubation at 30°c on sc-ura.
therefore, we screened for high copy suppressors of trz1- <dig> by using yeast genomic fragments to transform yl12- <dig> and selecting transformants that had grown out in 2- <dig> days.
we screened approximately  <dig>  genomic fragments and isolated  <dig> suppressor clones.
two of the four clones yielded an identical sequence from a  <dig> bp genomic region on chromosome  <dig>  that contains four genes .
one of the remaining clones includes a 2594-bp genomic sequence from chromosome  <dig> that contains the full-length coding region of trz <dig> plus  <dig> bp of 5'utr and  <dig> bp of 3'utr.
the 4th clone contains chromosome  <dig> sequences at one end  and ty <dig> ltr at the other.
this last clone was not pursued further.
we cloned each of the four genes on the  <dig> bp region of chromosome  <dig> and inserted these independently into cen and  <dig> μ expression vectors containing the cyc <dig> promoter.
we tested each gene for an ability to rescue the ts phenotype of trz1- <dig> yeast.
only rex <dig> rescued the ts phenotype and rescue was observed with both cen and  <dig> μ vectors.
rex <dig> encodes a rna 3' processing enzyme that localizes to mitochondria  <cit> .
discussion
in this paper, we report investigation of trz <dig>  the yeast homolog of elac <dig>  altered expression of trz <dig> in yeast does not cause any altered growth phenotype.
conservation of the histidine motif and the c-domain putative p-loop is important for the essential function of trz1p in yeast.
trz1p , a ts mutant, exhibits a slow growth phenotype on a variety of yeast media.
additional copies of rex <dig> rescue the slow growth phenotype associated with trz1p , providing genetic confirmation that the product of trz <dig> plays a role in rna processing.
<dig>  altered expression of trz <dig> is not detrimental to yeast growth
although trz <dig> is an essential gene, yeast can tolerate altered expression of trz <dig>  we down-regulated trz <dig> message by deleting the endogenous gene and expressing trz <dig> from a gal <dig> promoter on a cen plasmid.
we could not detect any apparent growth defect when trz <dig> expression was suppressed on glucose-containing medium.
we also over-expressed exogenous trz <dig> message and protein from a gal <dig> promoter on backgrounds of either an intact or deleted endogenous trz <dig> locus.
in neither of these backgrounds does over-expression of trz <dig> cause any significant alteration of yeast doubling time.
we also attempted to decrease trz <dig> mrna by integration of a thermal labile cassette  or by insertion of a gal <dig> promoter just upstream of the endogenous trz <dig> coding sequence.
in these attempts, however, we did not obtain any integrants that were not petite mutants.
in conclusion, under or overexpression of trz <dig> does not grossly affect growth or cell cycle; however, the tendency for alteration in this gene to cause a petite phenotype confounds our ability to detect small perturbations.
<dig>  elac <dig> cannot substitute for trz <dig> function and the c-domain putative p-loop is important for trz <dig> essential function
human elac <dig> cannot rescue trz <dig> deletion in yeast.
we observed this with both full-length elac <dig> and with trz1/elac <dig> and elac2/trz <dig> fusions.
the same lack of complementation was observed with elac <dig>  the paralog of elac <dig>  these results could be explained if elac <dig> and elac <dig> have overlapping but not totally redundant functions.
trz <dig> is more similar to elac <dig> than to elac <dig>  but trz1p may accomplish functions in yeast performed by both elac <dig> and elac <dig> in humans.
therefore, substituting trz <dig> with either one of these genes may not replace all of the essential functions of trz1p in yeast.
it would be interesting to see whether supplying both human elac <dig> and elac <dig> could rescue trz <dig> deletion in yeast.
a motif search found two putative nucleotide-binding motifs  in trz <dig> and elac <dig>  one in the n-domain and one in the c-domain of each gene  <cit> .
the finding that yeast tolerates mutation of the n-domain but not the c-domain p-loop of trz <dig> suggests the putative c-domain nucleotide-binding motif plays an important role in trz1p function.
the fact that the c-domains of trz <dig> and elac <dig> contain the most conserved sequence across other organisms also supports the notion that the vital functions of trz <dig> require that this sequence element is intact.
however, the n-domain of trz1p is also necessary for function because expression of an n-terminally deleted trz <dig> does not rescue trz <dig> yeast, nor did the expression of the fusion protein between the n-domain of human elac <dig> and the c-domain of yeast trz <dig>  this observation is consistent with the results from nashimoto's lab  <cit> .
their data suggest that the n-terminal half of elac <dig> , to which family yeast trz <dig> also belongs, is responsible for substrate specificity and confers rnase  <dig> activity that is not present in elac <dig> .
and the activities of trnase z and rnase  <dig> toward a set of substrates between human elac <dig> and yeast trz <dig> are different, suggesting the n-domain of trnase zls is essential for certain functions in the cell and they may not functionally equivalent among different species  <cit> .
<dig>  potential role of trz <dig> in rrna biogenesis, mrna splicing and mitochondrion maintenance in addition to its role in trna processing
the majority of the rna in growing yeast is noncoding  <cit>  including rrna, trna, snrna, snorna as well as the rna components of rnase p and rnase mrp.
these rnas are involved in processes that are vital to cell survival.
the biogenesis of rrna is a complicated process that involves a large number of proteins and rnas that catalyze or modulate transcription, translocation, and various processing reactions, including exonucleolytic and endonucleolytic cleavages, ligations, terminal additions , and nucleoside modifications.
the details of yeast rrna processing and some of the complexes involved have been described  <cit> .
rex proteins  belong to a family of well-conserved rna 3' → 5' exonucleases  <cit> .
rex1p is required for 5s and trna-arg <dig> maturation.
deletion of rex2p results in a defect in the processing of the u <dig> snrna that is involved in mrna splicing.
rex3p is required for the maturation of mrp rna, an important endonucleolytic complex involved in processing both rrna and trna.
rex1p and rex2p have been shown to be involved in the final maturation of  <dig> s rrna.
rex1p, rex2p and rex3p are also involved in the maturation of u <dig> snrna and the rna subunit of rnase p, the rnase that is involved in both trna 5' and rrna maturation.
in addition to participating in trna and rrna processing, rex2p has been implicated in spliceosome assembly by association with the snrnp u <dig> component prp <dig>  in dna repair by association with rad50p, and in cytoskeleton reorganization by association with the actin cortical patch component cof1p  <cit> .
rex2p localizes to the mitochondria and interacts genetically with yme <dig> and yme <dig>  components of the inner mitochondrial membrane that participate in mitochondrion organization and biogenesis  <cit> .
in summary, the rex proteins function in both rna maturation and mitochondrial maintenance.
the finding that rex <dig> rescues the slow growth phenotype of yeast expressing trz1p , suggests that trz <dig> may also participate in those same processes.
further support for this hypothesis comes from the finding that trz <dig> exists in a complex with nuc <dig>  <cit> , an rnase and dna endo/exonuclease that localizes to the mitochondria and is involved in mitochondrial dna recombination.
in addition, we observed that supplying an extra copy of wild type trz <dig> on a cen plasmid alters yl12- <dig>  converting the ts mutant trz1p  from ρ- to ρ+ .
the mitochondrial localization of trz1p  <cit>  together with the suppression of the trz1p  phenotype by rex <dig> and the effect of expression of wt trz <dig> on the petite mutant status of yl12- <dig> suggest that trz1p may participate in the activities of mitochondria.
the human homolog of trz <dig>  elac <dig>  was identified as a candidate prostate cancer susceptibility gene  <cit> .
schiffer et al.
<cit>  purified a nuclear trnase z from wheat germ and found two homologs of trnase z in a. thaliana, nuz  and cpz .
both arabidopsis homologs have trnase z activity in vitro.
these authors also showed that nuz is a homolog of elac <dig> and represents a family of proteins that is conserved from bacteria to humans.
subsequently, takaku et al.
showed that human elac <dig>  its paralog elac <dig>  and the yeast homolog trz <dig> have trnase z activity in vitro  <cit> .
the finding in this study that extra copies of rex <dig> rescue a ts allele of trz <dig> confirms a role for trz1p in the processing of rna, placing trz <dig> in particular in rrna and trna maturation pathways.
this is consistent with previous results.
first, rrna biogenesis involves processes both in the nucleolus and cytoplasm, and trz <dig> protein localizes to both of these cellular compartments  <cit> .
second, many proteins and some complexes, for example, mrp, rnase p and the rna polymerase iii complex, participate both in trna and rrna processing and synthesis.
therefore it is possible that trz <dig> participates in both trna and rrna processing.
third, using a tet-promoter trz <dig> allele and a custom dna oligo array for noncoding rna biogenesis, peng et al.
demonstrated that trz <dig> has an undefined role in the processing of 35s rrna  <cit> .
the high-copy suppressor result could also be explained more straightforwardly if we assume that trz1p  has diminished or abolished trnase z activity, which could result in low level of mature trnas, and rex2p could remove the 3' trailers from pre-trnas exonucleolytically, that could result in restored mature trna levels.
since elac <dig> cannot functionally complement trz <dig> deletion, we cannot directly test in yeast the functional effect of the human elac <dig> missense substitution a541t, which appears to confer modest risk of prostate cancer  <cit> .
although this variant does not seem to affect the trnase z activity of human elac <dig> in vitro  <cit> , we did find that some missense substitutions at the corresponding yeast residue, m <dig>  are lethal, demonstrating that this position is under functional constraint.
recent demonstrations that hoe- <dig>  the c. elegans homolog of elac <dig>  plays a role in germline proliferation  <cit> , and human elac <dig> maybe involved in cell cycle regulation  <cit> , may help better illuminate the contribution of elac <dig> to the etiology of human prostate cancer.
CONCLUSIONS
we have undertaken a genetic study of yeast trz <dig> gene, the homolog of prostate cancer susceptibility gene elac <dig>  our data suggest that the absolute level of trz <dig> transcript is not important for the fitness of yeast, the vital functions of trz <dig> require an intact c-terminal p-loop and some conserved residues in histidine motif.
more importantly, our high copy suppressor screen suggests that trz <dig> is involved in rna processing, especially rrna processing and mitochondrial biogenesis.

 with a cumulative incidence by age of 14% among caucasian-americans and 25% among african-americans, prostate cancer has become the most common non-cutaneous malignancy in the us. despite its high incidence rate, there is little known about the genetics and etiology of prostate cancer. we recently identified a candidate prostate cancer susceptibility gene through linkage analysis and positional cloning. this gene, elac is located on chromosome 17p and encodes a protein of amino acids with trnase z activity. elac belongs to an incompletely characterized family of proteins that is conserved among eukaryotes, archaea and bacteria. elac is ubiquitously expressed in all tissues with the highest expression levels found in testes. elac2p is detected in several cancer cell lines and binds the γ-tubulin complex. at present the role that sequence variants in elac may play in the genesis of prostate cancer is unclear. trz, the yeast ortholog of elac encodes an essential protein of amino acids. like elac and its other eukaryotic orthologs, trz can be divided roughly into amino- and carboxy-halves, or domains, which are structurally homologous. the carboxy domain possesses trnase z activity; the amino domain is enzymatically inactive but may modulate the specificity of carboxy domain. the most strongly conserved sequence element is the histidine motif, located near the front of the carboxy domain. in the amino domain, the structural homolog of the histidine motif is easily recognizable; however, most of the key zinc coordinating histidines have changed to some other polar amino acid, which is entirely consistent with the observation that this domain lacks trnase z activity. prosite scans revealed that the vertebrate elac2s contain a sequence element near the center of their amino domain that matches the nucleotide binding p-loop motif. although not exactly matching the canonical p-loop sequence, this sequence element is well conserved in trz and its structural homolog is well conserved near the center of the carboxy domain of all gene family members. it has recently been shown that both of the two elac family members from the animals, plants, fungi, bacteria, and archaea possess trnase z activity. the amino acid and enzymatic conservation across elac family members suggests strongly that they all share some basic cellular functions. since saccharomyces cerevisiae is a well-characterized genetic system, we initiated a genetic analysis of the functions of the yeast member of this gene family, trz in order to better understand those of elac rna processing, one of the post-transcriptional controls on gene expression, plays an important role in regulating cell growth and fate. pre-mrna processing is a multi-step process coupled to transcription through the involvement of the unique c-terminal domain of the large subunit of rna polymerase ii. the processing involves rna binding proteins, snrnas, endo- and exo-nucleases, and other specific factors. the function of these proteins is subject to regulation by different signaling pathways, according to the developmental stage of the cell. transfer rnas undergo extensive post-transcriptional modifications, including: 5' processing; 3' processing; nucleotide modification; and, in the case of about 20% of yeast trna, intron removal in the nucleus before export to the cytosol. rnase p, which consists of a rna subunit and nine proteins, is responsible for 5' trna processing. two proteins, trz1p and lhp1p, have been shown to be involved in 3' trna maturation in yeast; trz1p has trnase z activity while lhp1p stabilizes pre-trnas as an rna chaperone. it is known that there are more than proteins involved in trna splicing, modification, degradation and export. ribosomal rnas are transcribed as large precursors by rna polymerases i and iii in nucleoli. these rnas also undergo processing, including 5' and 3' cleavages, removal of introns, and nucleotide modifications through the action of a series of rna/protein complexes. the mature rrnas complex with about proteins to form ribosomes. the rnase mrp complex is responsible for the majority of 5' maturation steps and the exosome complex is responsible for 3' rrna maturation. many of the modifications and splicing events require small nucleolar rnas and other specific protein factors. notably, some proteins are shared between the trna and rrna maturation complexes. in this paper we show that, although trz1p is required for growth, yeast are largely insensitive to the absolute level of its expression. elac cannot functionally replace trz1p, indicating that trz1p has additional or specific functions. in the carboxy domain of the protein, the conserved histidine motif and the putative p-loop are important for trz1p biological function because mutations of the conserved residues in these motifs render the protein inactive in yeast. through a high copy suppressor screen, we found that trz genetically interacts with rex suggesting a role for trz in rna processing and mitochondrial maintenance.  effect of altered trz expression on yeast cell growth we generated three yeast strain types in order to study the consequences of altered trz expression in yeast. in the first, trz is expressed from a gal promoter on a low-copy plasmid in a wild type trz background. in the second, the gal promoter is inserted upstream of trz between the translational start site and its endogenous 5'utr at the chromosomal locus. in the third strain type, endogenous trz is deleted and the gene is expressed from a gal promoter on a low-copy plasmid. the latter two strain types were made to investigate the effect of up- and down- regulation of trz expression on growth. the genotypes of all strains used in this study are listed in table also listed in table is the ρ +/ρ - status of each strain, assayed as the ability of the strain to grow on media containing only non-fermentable carbon sources. the significance of the ρ +/ρ - status, which represents the wild type or impaired mitochondrial respiratory function, will become clear below. as shown in table when trz is over-expressed in a wild type background, the yeast doubling time is hours, not significantly longer than that of yph the parental yeast control or that of wild type yeast that over-express β-galactosidase. therefore, over-expression of trz in wild type yeast does not significantly slow yeast growth. overexpression of flag-trz in yl09- was verified by western blot with anti-flag antibody. as shown in figure 1a, flag -trz is rapidly induced in yl09- grown on galactose-containing medium. notably, both yl09- and yl09- are ρ+. we then investigated the consequence of over-expression of trz from the chromosomal locus. in yl10- a gal promoter is inserted immediately upstream of the translational start codon of flag-trz as shown in figure 1b, flag-trz is promptly induced after addition of galactose in the yl10- strain. liquid growth assays in a variety of different media were done to determine the doubling time of strains expressing varying levels of trz1p. no growth was observed for yl10- and yl10- in galactose medium, respectively, compared to hr doubling time foryph however, these gal promoter insertion strains also grew more slowly in non-inducing medium. the doubling time for yl10- and yl10- in glucose medium is and times longer than that of parental yph cells. we experienced difficulties when trying to isolate gal promoter insertion strains and found that the isolated strains were petite mutants; therefore, we decided not to study these strains further. we observed the growth characteristics of trz strains with trz expressed from a gal promoter on a low-copy plasmid. as shown in table when trz expression is repressed in glucose medium, yl10- grows marginally slower than yph the isogenic wild type yeast. the same is true for yl10- and yl10- with respect to yph their isogenic wild type strain. thus, although trz is an essential gene, even low levels of its transcript are sufficient for near normal growth. when trz expression is induced in galactose medium, however, yl10- and yl10- fail to grow, while yl10- grows at about the same rate as wild type yeast. this is intriguing, particularly when comparing yl10- and yl10- because the strains share the same genetic background. they differ in two ways: yl10- is ρ- while yl10- is ρ+; and yl10- expresses untagged trz while yl10- expresses trz tagged with a flag epitope. the phenotypic discrepancy between yl10- and yl10- is not likely due to a lack of expression of trz1p or lack of functional trz1p in yl10- because we could detect flag•trz1p in yl10- and p414/gal1p-flag•trz could functionally complement the trz deletion. the difference between the strains is more likely due to their ρ status. we studied these strains further by testing the effect of different carbon sources on their growth. table summarizes these results. when trz expression from a gal1p-driven trz plasmid is suppressed on sc-glucose, wild type yeast and trz yeast grow at a rate similar to that of yph at room temperature, 30°c and 37°c. we could not detect any significant difference in growth rate among yph yl09- and yl10- on sucrose, raffinose or galactose at any temperature tested. this result agrees with the results from liquid growth assays in that over-expression of trz in these two strains does not result in any discernable change in growth rate. when spotted onto glucose media and incubated at room temperature, yl10- and yl10- grow at a rate equivalent to that of yph as the incubation temperature is increased, however, yl10- and yl10- grow significantly slower than yph when spotted on sucrose media, these strains grow slower than yph at all tested temperatures, with the most significant difference in doubling time occurring at 37°c. yl10- and yl10- barely grow on raffinose and do not grow at all on galactose. as argued above, the phenotypic discrepancies among the strains is not likely due to differences in expression of functional trz1p. instead, the different phenotypes resulting from galactose-induced expression of trz1p is more likely attributable to the ρ+/ρ- status of the strains, consistent with the known inability of ρ- cells to grow on galactose-containing media. thus, the ρ- stains show sugar and temperature sensitivity; the ρ+ strains do not. in summary, yeast are largely insensitive to the level of trz1p, provided there is some minimal level of the protein; the apparent sensitivity of some strains to galactose-induced trz1p expression is attributable to the non-specific galactose-sensitivity of ρ- yeast. elac does not functionally substitute for trz1; motifs required for trz1p function since trz is the yeast homolog of elac we tested the ability of elac to functionally complement deletion of trz in yeast. yl03- was transformed with an array of elac constructs. transformants were plated onto foa to select against the trz expression plasmid and to determine if any construct could complement trz as shown in table neither wild type nor prostate cancer-associated elac mutants could substitute for wild type trz function in yl03- the human paralog of elac elac likewise failed to rescue trz. we next incorporated three specific mutations into the histidine motif region of trz1: m535t, which corresponds to the missense substitution a541t in elac that is associated with human prostate cancer; h542ter, which corresponds to the frameshift 1641insg associated with human prostate cancer; and g548r, which corresponds to a ts mutant, g256r, that was isolated in the homologous yeast gene pso. notably, in no case did the fusion of two copies of the flag epitope to trz alleles influence results. the missense mutant trz allele complements trz as well as the wild type gene. however, neither trz g548r in which a conserved glycine in the histidine motif is altered, nor the mutant trz truncated after his in the same motif could complement trz in yeast. motif searches identified a putative p-loop at amino acids 276– of human elac. this sequence element is located near the center of the n-domain and is clearly conserved among the eukaryotic orthologs. the corresponding sequence in trz1p falls within the range of variation for the structural p-loop recently explored by brakoulias et al. in the homology between the n- and c-domains of the protein, this candidate p-loop corresponds to a strongly conserved element near the middle of the c-domain, which also has features reminiscent of the structural p-loop. for simplicity, we will refer to these as the first and second putative p-loops. in so doing, however, we recognize that they may be examples of some other conserved structural element. we mutated the key residues in both putative p-loops individually and tested the mutants' abilities to complement trz the first putative p-loop is dispensable for survival because mutants trz g229d and trz q230a, which should destroy its function if it is a p-loop, can functionally substitute for the essential function of trz1p. however, the second putative p-loop is required for yeast survival because mutants here cannot complement trz deletion. sequence analysis shows that trz may be the result of ancient tandem duplication/fusion that produced a gene with homologous amino and carboxyl halves. we therefore tested whether either the n- or c- domain of trz alone is capable of rescuing trz neither half could rescue, even when fused to the complementary domain of elac. screen for and characterization of temperature sensitive trz mutants we screened for ts mutants of trz1p with pcr-based random mutagenesis and bacterial mutator approaches; neither of these attempts identified ts mutants. to circumvent technical difficulties, we used a targeted approach instead. we designed degenerate oligos targeting sequence encoding amino acids and in the trz1p histidine motif. these amino acids were selected for mutagenesis because, with the exception of residue they are less conserved across species than the canonical residues of the histidine motif; therefore, mutation may be less likely to completely inactivate protein function. g was targeted because, as mentioned before, a ts mutant had been identified at the corresponding amino acid in s. cerevisiae pso we generated mini-libraries, each of which targets one of these residues in yl03- and isolated the clones that appeared at 30°c but that grew slowly or not at all at 37°c. to confirm the ts phenotype plasmids from these isolates were purified and used to transform naïve yl03- the screen identified two ts mutants of trz1p, y537l and l538k. we also found mutations at position and that are lethal at both 30°c and 37°c. observation of lethal missense substitutions at m is intrinsically interesting because it is consistent with the idea that the corresponding residue a of human elac is under some functional constraint. more than 60% of the lethal mutations were concentrated at g indicating that this residue is more functionally constrained than the corresponding g of s. cerevisiae pso. we integrated trz trz1- and trz1- into the leu locus in a trz background to yield yl13- yl13- and yl13- respectively. as shown in figure the ts mutants grew more slowly on glucose medium than did the controls at all temperatures tested. the inhibition of growth was more obvious at higher temperature. growth of the ts mutants was further slowed on sucrose. when plated on raffinose, ts mutants grow only at 30°c. yl13- and yl13- are also petite mutants while yl13- is not. the ability of yl13- but not yl13- or yl13- to grow on galactose can therefore be explained in terms of the known inability of ρ- yeast to grow on galactose. table summarizes the doubling time for each ts mutant at different temperatures in sc-leu medium containing glucose. at 30°c yl13- and yl13- grow at about the same rate in liquid medium containing glucose as does yl13- however, at 37°c the ts strains grow significantly more slowly in glucose than does yl13- we integrated the trz1- allele at the trz locus to yield yl12- yl12- exhibits a slow-growth phenotype like that of the leu integrants. yl12- grows slower on glucose media compared with the isogenic wild type yph at all temperatures tested and this growth difference increases at 37°c. the trz1- integrants exhibit severe ts growth on sucrose containing medium and they do not grow on raffinose and galactose containing medium. the doubling time of these mutants in glucose-supplemented liquid medium is in agreement with results from the plate assay. to verify that the growth phenotype and sugar sensitivity observed with trz1- integrants do not result from a cloning artifact, we did the following. yph and yl12- were transformed with empty vector or p416/trz and plated onto sc-ura containing glucose. once grown, colonies were replica-plated onto sc-ura supplemented with glucose, sucrose, raffinose or galactose. providing a wild type trz on a cen vector restored the growth of yl12- to the level of yph on all media tested. yl12- transformed with an empty vector grew more slowly than yph on all media tested, with the decrease in growth rate in the order of glucose, sucrose, raffinose and galactose media, in agreement with the previous plate assay. in addition, we checked the ρ status for these transformants. providing a copy of wild type trz on a cen vector converted yl12- from ρ- to ρ+, indicating that the petite phenotype of yl12- may not be due to the loss of mitochondrial dna. instead, it may result from the interference of important mitochondrial functions, suggesting that trz1p also plays a role inside mitochondria. however, normal growth on a non-fermentable carbon source was not restored in diploid strains derived from crosses between ts mutant strains and a haploid ρ tester strain. given the fact that the tester strain should have a wild type copy of trz and ts mutant haploid strains should have mitochondrial dna, we speculate that ts alleles of trz may have pleiotropic effects on certain important activities in mitochondria. the effect on some of these activities may be dominant, and mating with a wild type haploid strain lacking mitochondrial dna may not fully restore the mitochondrial functions. to determine if the trz1- allele causes any cell cycle arrest phenotype, we analyzed yl12- with yph as control, by facs; both strains were grown at rt or 37°c for this study. allele trz1- caused a hour delay in cells entering 2n at 37°c. when yeast reached stationary phase, yl12- had more cells in 2n than yph grown under the same conditions. when viewed under a microscope the trz1- integrants were observed to aggregate when grown at 37°c while parental yph cells did not. with yl12- we also observed enlarged cells and cells with elongated buds; the percentage of these abnormally shaped cells increased with the length of time that the cells were grown at 37°c. therefore, trz1- allele does not cause any cell cycle arrest phenotype and the abnormal shape we observed is most likely the result of cell death in yeast. high copy suppressor screen during the characterization of yl12- we identified a window of two days wherein it would be possible to screen for high copy suppressors of trz1- as shown in figure at 30°c, the appearance of yl12-01/p416gal transformants takes days on sc-ura. however, colonies appear after only days when the same strain is transformed with p416/trz. yph transformed with vector or trz resulted in observable colonies after days of incubation at 30°c on sc-ura. therefore, we screened for high copy suppressors of trz1- by using yeast genomic fragments to transform yl12- and selecting transformants that had grown out in 2- days. we screened approximately genomic fragments and isolated suppressor clones. two of the four clones yielded an identical sequence from a bp genomic region on chromosome that contains four genes. one of the remaining clones includes a 2594-bp genomic sequence from chromosome that contains the full-length coding region of trz plus bp of 5'utr and bp of 3'utr. the 4th clone contains chromosome sequences at one end and ty ltr at the other. this last clone was not pursued further. we cloned each of the four genes on the bp region of chromosome and inserted these independently into cen and μ expression vectors containing the cyc promoter. we tested each gene for an ability to rescue the ts phenotype of trz1- yeast. only rex rescued the ts phenotype and rescue was observed with both cen and μ vectors. rex encodes a rna 3' processing enzyme that localizes to mitochondria. discussion in this paper, we report investigation of trz the yeast homolog of elac altered expression of trz in yeast does not cause any altered growth phenotype. conservation of the histidine motif and the c-domain putative p-loop is important for the essential function of trz1p in yeast. trz1p, a ts mutant, exhibits a slow growth phenotype on a variety of yeast media. additional copies of rex rescue the slow growth phenotype associated with trz1p, providing genetic confirmation that the product of trz plays a role in rna processing. altered expression of trz is not detrimental to yeast growth although trz is an essential gene, yeast can tolerate altered expression of trz we down-regulated trz message by deleting the endogenous gene and expressing trz from a gal promoter on a cen plasmid. we could not detect any apparent growth defect when trz expression was suppressed on glucose-containing medium. we also over-expressed exogenous trz message and protein from a gal promoter on backgrounds of either an intact or deleted endogenous trz locus. in neither of these backgrounds does over-expression of trz cause any significant alteration of yeast doubling time. we also attempted to decrease trz mrna by integration of a thermal labile cassette or by insertion of a gal promoter just upstream of the endogenous trz coding sequence. in these attempts, however, we did not obtain any integrants that were not petite mutants. in conclusion, under or overexpression of trz does not grossly affect growth or cell cycle; however, the tendency for alteration in this gene to cause a petite phenotype confounds our ability to detect small perturbations. elac cannot substitute for trz function and the c-domain putative p-loop is important for trz essential function human elac cannot rescue trz deletion in yeast. we observed this with both full-length elac and with trz1/elac and elac2/trz fusions. the same lack of complementation was observed with elac the paralog of elac these results could be explained if elac and elac have overlapping but not totally redundant functions. trz is more similar to elac than to elac but trz1p may accomplish functions in yeast performed by both elac and elac in humans. therefore, substituting trz with either one of these genes may not replace all of the essential functions of trz1p in yeast. it would be interesting to see whether supplying both human elac and elac could rescue trz deletion in yeast. a motif search found two putative nucleotide-binding motifs in trz and elac one in the n-domain and one in the c-domain of each gene. the finding that yeast tolerates mutation of the n-domain but not the c-domain p-loop of trz suggests the putative c-domain nucleotide-binding motif plays an important role in trz1p function. the fact that the c-domains of trz and elac contain the most conserved sequence across other organisms also supports the notion that the vital functions of trz require that this sequence element is intact. however, the n-domain of trz1p is also necessary for function because expression of an n-terminally deleted trz does not rescue trz yeast, nor did the expression of the fusion protein between the n-domain of human elac and the c-domain of yeast trz this observation is consistent with the results from nashimoto's lab. their data suggest that the n-terminal half of elac, to which family yeast trz also belongs, is responsible for substrate specificity and confers rnase activity that is not present in elac. and the activities of trnase z and rnase toward a set of substrates between human elac and yeast trz are different, suggesting the n-domain of trnase zls is essential for certain functions in the cell and they may not functionally equivalent among different species. potential role of trz in rrna biogenesis, mrna splicing and mitochondrion maintenance in addition to its role in trna processing the majority of the rna in growing yeast is noncoding including rrna, trna, snrna, snorna as well as the rna components of rnase p and rnase mrp. these rnas are involved in processes that are vital to cell survival. the biogenesis of rrna is a complicated process that involves a large number of proteins and rnas that catalyze or modulate transcription, translocation, and various processing reactions, including exonucleolytic and endonucleolytic cleavages, ligations, terminal additions, and nucleoside modifications. the details of yeast rrna processing and some of the complexes involved have been described. rex proteins belong to a family of well-conserved rna 3' → 5' exonucleases. rex1p is required for 5s and trna-arg maturation. deletion of rex2p results in a defect in the processing of the u snrna that is involved in mrna splicing. rex3p is required for the maturation of mrp rna, an important endonucleolytic complex involved in processing both rrna and trna. rex1p and rex2p have been shown to be involved in the final maturation of s rrna. rex1p, rex2p and rex3p are also involved in the maturation of u snrna and the rna subunit of rnase p, the rnase that is involved in both trna 5' and rrna maturation. in addition to participating in trna and rrna processing, rex2p has been implicated in spliceosome assembly by association with the snrnp u component prp in dna repair by association with rad50p, and in cytoskeleton reorganization by association with the actin cortical patch component cof1p. rex2p localizes to the mitochondria and interacts genetically with yme and yme components of the inner mitochondrial membrane that participate in mitochondrion organization and biogenesis. in summary, the rex proteins function in both rna maturation and mitochondrial maintenance. the finding that rex rescues the slow growth phenotype of yeast expressing trz1p, suggests that trz may also participate in those same processes. further support for this hypothesis comes from the finding that trz exists in a complex with nuc, an rnase and dna endo/exonuclease that localizes to the mitochondria and is involved in mitochondrial dna recombination. in addition, we observed that supplying an extra copy of wild type trz on a cen plasmid alters yl12- converting the ts mutant trz1p from ρ- to ρ+. the mitochondrial localization of trz1p together with the suppression of the trz1p phenotype by rex and the effect of expression of wt trz on the petite mutant status of yl12- suggest that trz1p may participate in the activities of mitochondria. the human homolog of trz elac was identified as a candidate prostate cancer susceptibility gene. schiffer et al. purified a nuclear trnase z from wheat germ and found two homologs of trnase z in a. thaliana, nuz and cpz. both arabidopsis homologs have trnase z activity in vitro. these authors also showed that nuz is a homolog of elac and represents a family of proteins that is conserved from bacteria to humans. subsequently, takaku et al. showed that human elac its paralog elac and the yeast homolog trz have trnase z activity in vitro. the finding in this study that extra copies of rex rescue a ts allele of trz confirms a role for trz1p in the processing of rna, placing trz in particular in rrna and trna maturation pathways. this is consistent with previous results. first, rrna biogenesis involves processes both in the nucleolus and cytoplasm, and trz protein localizes to both of these cellular compartments. second, many proteins and some complexes, for example, mrp, rnase p and the rna polymerase iii complex, participate both in trna and rrna processing and synthesis. therefore it is possible that trz participates in both trna and rrna processing. third, using a tet-promoter trz allele and a custom dna oligo array for noncoding rna biogenesis, peng et al. demonstrated that trz has an undefined role in the processing of 35s rrna. the high-copy suppressor result could also be explained more straightforwardly if we assume that trz1p has diminished or abolished trnase z activity, which could result in low level of mature trnas, and rex2p could remove the 3' trailers from pre-trnas exonucleolytically, that could result in restored mature trna levels. since elac cannot functionally complement trz deletion, we cannot directly test in yeast the functional effect of the human elac missense substitution a541t, which appears to confer modest risk of prostate cancer. although this variant does not seem to affect the trnase z activity of human elac in vitro, we did find that some missense substitutions at the corresponding yeast residue, m are lethal, demonstrating that this position is under functional constraint. recent demonstrations that hoe- the c. elegans homolog of elac plays a role in germline proliferation, and human elac maybe involved in cell cycle regulation, may help better illuminate the contribution of elac to the etiology of human prostate cancer.  we have undertaken a genetic study of yeast trz gene, the homolog of prostate cancer susceptibility gene elac our data suggest that the absolute level of trz transcript is not important for the fitness of yeast, the vital functions of trz require an intact c-terminal p-loop and some conserved residues in histidine motif. more importantly, our high copy suppressor screen suggests that trz is involved in rna processing, especially rrna processing and mitochondrial biogenesis. 
the temperature sensitivity of a trz mutant can be rescued by multiple copies of rex which encodes a protein with rna 3' processing activity, suggesting a role of trz1p in rna processing in vivo. in humans, mutation of elac is associated with an increased risk of prostate cancer. our results provide genetic evidence that prostate cancer susceptibility gene elac may be involved in rna processing, especially rrna processing and mitochondrial function. trz1p has two putative nucleotide triphosphate-binding motifs and a conserved histidine motif. the histidine motif and the putative nucleotide binding motif at the c-domain are important for trz1p function because mutant proteins bearing changes to the critical residues in these motifs are unable to rescue deletion of trz the growth defect exhibited by trz yeast is not complemented by the heterologous elac suggesting that trz1p may have additional functions in yeast. in this work, we show that the yeast homolog of elac encoded by trz, is involved genetically in rna processing. elac has been shown to have trnase z activity and is associated with the γ-tubulin complex.
 in humans, mutation of elac is associated with an increased risk of prostate cancer. elac has been shown to have trnase z activity and is associated with the γ-tubulin complex.  in this work, we show that the yeast homolog of elac encoded by trz, is involved genetically in rna processing. the temperature sensitivity of a trz mutant can be rescued by multiple copies of rex which encodes a protein with rna 3' processing activity, suggesting a role of trz1p in rna processing in vivo. trz1p has two putative nucleotide triphosphate-binding motifs and a conserved histidine motif. the histidine motif and the putative nucleotide binding motif at the c-domain are important for trz1p function because mutant proteins bearing changes to the critical residues in these motifs are unable to rescue deletion of trz the growth defect exhibited by trz yeast is not complemented by the heterologous elac suggesting that trz1p may have additional functions in yeast.  our results provide genetic evidence that prostate cancer susceptibility gene elac may be involved in rna processing, especially rrna processing and mitochondrial function. 