PCR mutagenesis of DBR1.
DBR1 is required for debranching of intron RNA lariats and for full levels of transposition of the yeast LTR retrotransposon Ty1. Comparison of DBR1 genes from different organisms shows strong conservation of coding sequences. However, the importance of specific amino acid residues for Dbr1p function has not been demonstrated. Furthermore, the relationship between Dbr1p's roles in debranching and Ty1 transposition is unknown. If the 2′-5′ phosphodiesterase activity of Dbr1p is required for both processes, it would suggest that Ty1 transposition involves an RNA with this type of linkage, for which there is no evidence at present. Alternatively, Dbr1p may act differently in Ty1 transposition and debranching. In this case, dbr1 mutant alleles could be identified that affect one process but not the other.
In order to study how Dbr1p functions in Ty1 transposition, a library of dbr1
alleles was generated by mutagenic PCR. The method used (GeneMorph; Stratagene) was chosen because it can produce all possible transition and transversion mutants with minimal bias. Mutations were introduced in the DBR1
ORF present in pTM235. This plasmid contains the 1,218-nt DBR1
ORF fused at its 5′ end to the yeast GAL1
promoter and at its 3′ end to a V5 epitope tag followed by a six-His tag (1
) (Invitrogen) (Fig. ). Mutagenic PCR was performed with two different sets of primers: one set (primers 99 and 100) for the 5′ portion of the DBR1
ORF (nt 1 to 679) and the other set (primers 101 and 102) for the 3′ portion of the ORF (nt 528 to 1218). PCR conditions were adjusted to limit multiple mutations in the DBR1
gene in order to generate a collection of single-point mutations (see Materials and Methods). The PCR products were digested either with Eco
RI and Hin
dIII (5′ region of DBR1
) or with Hind
III and Bst
EII (3′ region of DBR1
), and purified fragments were used to replace the corresponding portions of pTM235. Because the mutation rate in the PCR is low, many plasmids in the resulting collection of pTM235 derivatives contain a wild-type DBR1
allele. Therefore, the collection of plasmids was screened to identify those containing dbr1
mutant alleles. Yeast strain TMY60 (dbr1
Δ) was cotransformed with the collection of pTM235 derivatives and pDG784 (pGAL1
AI). Individual transformants were screened for the ability to support Ty1 transposition with a replica-plating assay (see Materials and Methods). In this assay, a pTM235 derivative is the only source of Dbr1p in the cell and expression of both Dbr1p and Ty1 is activated by d
-(+)-galactose. Positive and negative controls were patched on each plate containing transformants being screened. The positive control (wild-type Ty1 transposition phenotype) was a TMY60 transformant containing pDG784 and the original (unmutagenized) pTM235 plasmid (pGAL1
-V5-6xHis). The negative control (dbr1
mutant Ty1 transposition phenotype) was a TMY60 transformant containing pDG784 and a vector plasmid (pYES2.1) containing no DBR1
sequences. Patches of cells unable to support wild-type levels of Ty1 transposition were single colony purified and retested.
The pTM235 derivatives from those TMY60 transformants that retained a mutant phenotype for Ty1 transposition were rescued into E. coli. Purified plasmids were subjected to restriction enzyme analysis to eliminate those with apparent deletions from further studies. Full-length plasmids were reintroduced into TMY60 and retested for Ty1 transposition. Only transformants with a Ty1 transposition defect were analyzed further.
Transformants were subjected to Western blot analysis (1
) with α-V5 antibody (Invitrogen). For structure-function studies, we are interested in identifying missense mutants that express wild-type levels of full-length Dbr1p. The expression of the V5 epitope on an ~50 kDa protein indicates Dbr1p-V5-6xHis expression because V5 is expressed as a C-terminal tag on the plasmid-encoded Dbr1p proteins. Only those mutants that express Dbr1p-V5-6xHis were analyzed further.
A total of 1,098 pTM235 derivatives created by replacement of the 5′ portion of the DBR1 ORF with a mutagenic PCR fragment were screened by the process described above. From this collection, 15 plasmids were identified that contain dbr1 missense alleles resulting in a Ty1 transposition deficiency and of these, 7 plasmids encoded single amino acid substitutions. A total of 955 pTM235 derivatives created by replacement of the 3′ portion of the DBR1 ORF with a mutagenic PCR fragment were screened. From this collection, seven plasmids were identified that contain dbr1 missense alleles resulting in a Ty1 transposition deficiency, and of these, one plasmid encoded a single amino acid substitution. The transposition phenotypes of the eight single amino acid substitution alleles are shown in Fig. along with the phenotypes of control strains that include mutant dbr1-C30, a nonsense allele that produces a truncated form of Dbr1p.
FIG. 2. Ty1 transposition phenotypes of dbr1 mutants. Transposition of Ty1 was measured with the pGAL1-Ty1::his3AI element as described in Materials and Methods. Individual transformants containing pDG784 and a derivative of pTM235 (with mutagenized dbr1 DNA) (more ...)
All of the mutations that encode single amino acid changes in Dbr1p were assessed for dominance or recessiveness. Yeast strain TMY30 (DBR1) was cotransformed with plasmids bearing the dbr1 point mutations and pDG784 (pGAL1-Ty1::his3AI). Individual transformants were screened for the ability to support Ty1 transposition by the replica-plating assay used in the initial mutant screen. Because the TMY30 strain expresses wild-type Dbr1p, recessive dbr1 mutations on the plasmid will not alter the wild-type phenotype of the strain, but dominant DBR1 mutations will produce a mutant phenotype. All of the mutations encoding single amino acid changes in Dbr1p are recessive (data not shown).
Mutations in DBR1 lie in conserved regions.
Amino acid changes in each of the dbr1
missense mutants are shown in Table . Eight of the missense mutations result in single amino acid changes in Dbr1p. The positions of these single amino acid changes are shown in Fig. , in which the S. cerevisiae
Dbr1p sequence is shown aligned with amino acid residues conserved in at least 10 of the 12 Dbr1p sequences available in the GenBank database (Conserved in Fig. ). Importantly, two mutations (dbr1
) map to a GD/GNH phosphoesterase signature motif (19
) (Phos in Fig. ). This motif has been identified in all enzymes that catalyze phosphate bond cleavage. Two independent mutations (dbr1
) were found to reside in a potential tyrosine phosphorylation site at Y68 (pattern [RK]-X[2,3]-[DE]-X[2,3]-Y) (8
dbr1 missense mutants used in this study
FIG. 3. Single-point mutations in dbr1 lie mostly in conserved residues of Dbr1p. The amino acid sequence of S. cerevisiae Dbr1p is shown on the top line of each sequence block. Positions of amino acid changes in the point mutations are indicated by asterisks (more ...) Dbr1p is a phosphoprotein.
Identification of two mutations that produce changes in the Y68 residue prompted us to investigate the possibility that Dbr1p is phosphorylated. Dbr1p-V5-6xHis was purified from TMY60 cells containing DBR1 (pTM235), dbr1-N20 (pTM434), and dbr1-N29 (pTM435) as outlined in Materials and Methods. The wild-type and mutant forms of Dbr1p were purified with the ProBond system (Invitrogen) in the presence of phosphatase inhibitors. The wild-type and mutant forms of Dbr1p were detected on Western blots with anti-V5 antibody (Fig. , left side) (data for N29 mutant not shown). Western analysis with antiphosphotyrosine antibody indicates that the wild-type and mutant forms of Dbr1p are phosphorylated (Fig. , right side) (data for N29 mutant not shown). When purified Dbr1p proteins are treated with λ protein phosphatase, they no longer react with antiphosphotyrosine antibody (Fig. ). λ protein phosphatase removes phosphates from tyrosine, serine, and threonine residues. These data indicate that Dbr1p is phosphorylated on at least one tyrosine other than Y68. Although wild-type Dbr1p may also be phosphorylated on Y68, that cannot be determined from this experiment. As expected, when phosphatase inhibitors were absent during purification, antiphosphotyrosine antibody did not react with either wild-type or mutant Dbr1p (data not shown).
FIG. 4. Dbr1p is a phosphoprotein. (A) Western blots of Dbr1p-V5-6xHis following purification of the protein in the presence of phosphatase inhibitors. The left half shows purified wild-type (wt) and N20 mutant Dbr1p probed with anti-V5 antibody (α-V5). (more ...) Intron lariat RNA accumulation within cells.
We have developed an RNase protection assay, partly based on the lariat protection assay of Vogel and coworkers (33
), to measure the accumulation of intron RNA lariats within cells. The assay uses RNA probes that hybridize to ACT1
contains an intron that accumulates in dbr1
strains after it is spliced from ACT1
). Hybridizations of the probes described below with ACT1
RNA are shown in Fig. . The 5′ intron probe hybridizes to 283 nt in ACT1
RNA: 145 nt at the 5′ end of the ACT1
intron and an adjacent 138 nt in the 5′ exon. The 3′ intron probe hybridizes to 244 nt within the ACT1
intron: 30 nt downstream of the lariat branch point and 214 nt upstream of and including the lariat branch point. The lariat probe hybridizes to 206 nt within the ACT1
intron: 69 nt at the 5′ end of the intron, as well as 137 nt starting at the lariat branch point and extending upstream. As a control for the amount of protection observed by intron RNA sequences, a 3′ exon probe was used in some experiments. This probe hybridizes to 333 nt of the ACT1
FIG. 5. RNase protection assay. (A) Diagram of the 5′ intron probe annealing to different ACT1 RNA species. The three different ACT1 RNA species are pre-mRNA, mature mRNA, and the intron lariat. The different RNA species are not drawn to scale. The extents (more ...)
With RNase protection assays, we have measured the accumulation of ACT1 RNA species in both a dbr1Δ mutant (TMY60) and the wild type (TMY30) (Fig. ). Cells of each strain were grown in rich medium (YPD) to mid-log phase. Total RNA was isolated from the cells and used for RNase protection assays. Extensive accumulation of particular ACT1 RNA species occurs in the dbr1 mutant compared to the wild type, evidenced by the extensive probe protection by dbr1 RNA seen in Fig. , lanes 2, 4, and 6, compared to the minimal protection by wild-type RNA seen in lanes 1, 3, and 5. The sizes of the major protected fragments in lanes 2, 4, and 6 correspond to expectations for protection by ACT1 intron lariat RNA (positions of black arrows). The relative amounts of protected fragments indicate that introns accumulate in the dbr1 mutant at levels 50 to 100 times the levels in the wild type (data not shown).
For the lariat probe, the observed protection of ~211 nt (Fig. , lane 6) is expected if the 2′-5′ phosphodiester bond between the 5′ end of the intron and the lariat branch point is intact and the probe hybridization spans the lariat branch point. Also in line with expectations, enhanced protection of 138- and 74-nt fragments, predicted if intron lariats are debranched, is not observed for the dbr1 mutant. Debranched introns do not accumulate in the wild-type strain either, consistent with their rapid degradation.
As mentioned above, the ACT1 intron lariat protects 206 nt of the lariat probe. The extra nucleotides that bring the protected fragment size to 211 nt are due to the positions of the first RNase T1 cut sites on either site of the hybridizing region. Differences for other probes between the extents of probe hybridization with ACT1 RNAs and the size of protected fragments in the RNase protection assays are also due to the positions of the RNase T1 cut sites.
RNase protection assays with the 5′ intron probe indicate that a fragment of ~156 nt is extensively protected by RNA from the dbr1 strain (Fig. , lane 2). This result is expected if the 5′ portion of the probe, the part that hybridizes to the 5′ portion of the ACT1 intron, is protected because of accumulation of ACT1 intron RNA (Fig. ). Mature ACT1 mRNA will protect only the 3′ part of the probe, a fragment of 143 nt. Evidence of such protection by the mature ACT1 mRNA is indicated in lanes 1 and 2 of Fig. by the stippled arrow. Note that this fragment is present in roughly equal amounts in the dbr1 mutant and the wild type. This result indicates that production of mature ACT1 mRNA is not grossly affected in the dbr1 mutant.
RNase protection assays with the 3′ intron probe indicate that a fragment of ~215 nt is extensively protected by RNA from the dbr1 strain (Fig. , lane 4). This result is expected if ~30 nt at the 5′ end of the probe are not protected. The hybridizations diagrammed in Fig. show both the entire intron lariat and a trimmed intron lariat in which the 3′ end is 1 base downstream of the lariat branch point. Our experimental results indicate that the 3′ tail of the intron lariat is degraded to the branch point or just downstream of it.
Accumulation of ACT1 intron lariat RNA in dbr1 mutants.
We have performed RNase protection assays to measure the accumulation of ACT1 intron lariat RNA in the newly generated dbr1 mutants. These RNase protection assays are a means by which to assess the debranching capability encoded by the various dbr1 mutant alleles. The newly generated alleles are carried on plasmids in a dbr1Δ strain (TMY60) and are under the control of the GAL1 promoter. TMY60 cells containing the various plasmids were pregrown on selective raffinose medium to early log phase. The precultures were split into two portions. d-(+)-Galactose was added to one portion to a final concentration of 2% to induce expression of the plasmid-borne dbr1 alleles. d-(+)-Glucose was added to the other portion to a final concentration of 2% to repress expression of dbr1 (negative control).
All of the dbr1 point mutant alleles identified in this study on the basis of their Ty1 transposition deficiencies are also deficient for debranching of intron lariats (Fig. ). The extent of the debranching deficiency of each mutant is consistent with a severe loss of function relative to the wild-type allele. Figure depicts RNase protection assays with both the 5′ intron probe and the 3′ intron probe. The black arrows in each panel indicate the fragments that represent accumulation of intron RNAs.
FIG. 6. Intron lariat accumulation in dbr1 mutant strains. (A) RNase protection assay with the 5′ intron probe and 1.5 μg of total RNA from transformants of dbr1 strain TMY60 carrying different plasmids. Lanes: 2 and 3, pTM431 (N14 mutant); 4 (more ...)
The sensitivity of the RNase protection assay uncovers the fact that the Dbr1p-V5-6xHis fusion protein is not as efficient in debranching intron RNA lariats as native Dbr1p. Two different alleles encoding this fusion protein were tested: one allele has the GAL1 promoter driving expression of the fusion protein (pTM235), and the other allele has the native DBR1 promoter (pTM263). As shown in lanes 12 and 13 of Fig. , the 156-nt fragment of the 5′ intron probe is readily detected in RNA samples from strains containing the two different DBR1-V5-6xHis fusion alleles, indicating protection by ACT1 intron sequences. The pGAL1-driven fusion needs galactose for full expression, and the result in lane 12 of Fig. is for an RNA sample from cells grown for 18 h in 2% galactose. This debranching inefficiency can be contrasted with the absence of the 156-nt fragment in lane 1 of Fig. , for which RNA from a strain bearing native, wild-type DBR1 was used. Interestingly, in Ty1 transposition assays, the wild-type Dbr1p-V5-6xHis fusion protein supports levels of transposition that are not readily distinguishable from levels supported by native, wild-type Dbr1p (data not shown).
Despite the inefficiency of the wild-type Dbr1p-V5-6xHis fusion protein in debranching intron RNA lariats, all of the mutants are clearly much less active. In terms of absolute levels of RNA lariat accumulation, the results for the RNase protection assays fluctuate slightly from one experiment to the next. This is most likely due to slight differences in growth state or other factors that vary from experiment to experiment. The sensitivity of the RNase protection assay then displays this variation. However, relative comparisons of the mutants to the wild type clearly show the correlation between Ty1 transposition and debranching activity.
Integration versus recombination of Ty1 cDNA in dbr1 mutant strains.
Ty1 cDNA produced by reverse transcription of Ty1 RNA can become incorporated into yeast chromosomal DNA by two independent processes. One process, integration, requires the Ty1-encoded integrase protein IN. IN binds the ends of the Ty1 cDNA, cleaves the chromosomal target sequence, and joins the Ty1 cDNA to the target DNA. Cellular repair enzymes complete the integration process (fill in gaps and ligate nicks). Interactions between homologous DNA sequences in the Ty1 cDNA and the chromosomal target are not part of the integration process. Integration sites seem to be chosen by protein-protein interactions, possibly between IN and a protein bound to target DNA. Homologous recombination is the second process for incorporation of Ty1 cDNA into host chromosomes. This process can only occur at sites where Ty1 sequences are already present. Rad52p is an essential component of the homologous recombination machinery, so rad52
mutants exhibit severe recombination defects (32
), including Ty1 cDNA recombination. Previous work has shown that incorporation of Ty1 cDNA into the host chromosomes can occur in a rad52
mutant by the integration process described above (30
). Likewise, incorporation of Ty1 cDNA for an IN mutant element can occur very efficiently by homologous recombination (30
). However, incorporation of Ty1 cDNA for an IN mutant element does not occur in a rad52
mutant yeast strain because both integration and recombination are defective (30
; data not shown). In addition, Ty1 mutants that produce alterations of the sequences at the Ty1 cDNA ends transpose only by homologous recombination. IN can no longer bind the cDNA ends in a way that promotes integration. Therefore, Ty1 mutants that produce altered cDNA ends do not transpose in a rad52
We have compared the transposition rates of dbr1
and dbr1 rad52
strains to infer structural aspects of the Ty1 cDNA produced in a dbr1
mutant background. Strains YPH499 (wild type), TMY60 (dbr1
), TMY176 (rad52
), and TMY344 (dbr1 rad52
) were transformed with the pGAL1
AI plasmid (pBJC35) and, separately, the pGAL1
plasmid (pX3). Three transformants for each strain-plasmid combination were tested for Ty1 transposition. The results for transformants containing pX3 are depicted in Fig. . We observed the expected reduction in Ty1 transposition in the dbr1
mutant strain compared to the wild type. However, the frequency of the residual transposition events occurring in the dbr1
mutant strain is not significantly reduced any further in a dbr1 rad52
strain. If integration of the Ty1 cDNA was defective in a dbr1
mutant, transposition would be reduced at least 25-fold in the dbr1 rad52
strain compared to the dbr1
strain. This conclusion is based on the fact that Ty1 elements defective for integration transpose at wild-type levels in RAD52
yeast strains but at least 100-fold less efficiently in rad52
; data not shown). Therefore, we conclude that chromosomal incorporation of Ty1 cDNA produced in dbr1
strains in the absence of the major homologous recombination machinery must be catalyzed by the Ty1 IN protein. Because IN requires a precisely formed cDNA, these results suggest that the Ty1 cDNA produced in dbr1
strains is full length and contains the normal ends that interact with the Ty1 IN protein.
FIG. 7. Ty1 cDNA from a dbr1 mutant can interact with IN protein. Transposition rates were measured by a dilution-spotting method. Strains YPH499 (wild type), TMY60 (dbr1), TMY176 (rad52), and TMY344 (dbr1 rad52) were spotted onto FOA-Trp (to select for transpositions) (more ...) Loss of DBR1 dependence by Ty1 deletion elements.
We have compared the transposition frequency of a Ty1 element containing an internal deletion (pX100) (36
) to a full-length Ty1 element (pX3) (35
) to determine if Dbr1p acts on Ty1 RNA (Table ). Ty1 bases 620 to 5562 are deleted in pX100, and Ty1 proteins are supplied in trans
by an unmarked, transposition-defective Ty1 element carried on the same plasmid (36
). Transposition was measured in wild-type and dbr1
deletion strains. As expected, the full-length element transposed at a much lower frequency in a dbr1
strain than it did in the wild-type strain (29% of the wild-type strain frequency in this experiment). However, the deletion element transposed at much more similar frequencies in wild-type and dbr1
strains (the dbr1
strain frequency was 72% of the wild-type strain frequency; Table ). These data are consistent with the loss of DBR1
-dependent sequences in the deletion element. It is notable, however, that the Ty1 deletion element transposed at an overall lower frequency in both wild-type and dbr1
Transposition of a Ty1 deletion element